Phase I Study of SYNB1891, an Engineered E. coli Nissle Strain Expressing STING Agonist, with and without Atezolizumab in Advanced Malignancies.

PURPOSE: SYNB1891 is a live, modified strain of the probiotic Escherichia coli Nissle 1917 (EcN) engineered to produce cyclic dinucleotides under hypoxia, leading to STimulator of INterferon Genes (STING) activation in phagocytic antigen-presenting cells in tumors and activating complementary innate immune pathways. PATIENTS AND METHODS: This first-in-human study (NCT04167137) enrolled participants with refractory advanced cancers to receive repeat intratumoral injections of SYNB1891 either alone or in combination with atezolizumab, with the primary objective of evaluating the safety and tolerability of both regimens. RESULTS: Twenty-four participants received monotherapy across six cohorts, and 8 participants received combination therapy in two cohorts. Five cytokine release syndrome events occurred with monotherapy, including one that met the criteria for dose-limiting toxicity at the highest dose; no other SYNB1891-related serious adverse events occurred, and no SYNB1891-related infections were observed. SYNB1891 was not detected in the blood at 6 or 24 hours after the first intratumoral dose or in tumor tissue 7 days following the first dose. Treatment with SYNB1891 resulted in activation of the STING pathway and target engagement as assessed by upregulation of IFN-stimulated genes, chemokines/cytokines, and T-cell response genes in core biopsies obtained predose and 7 days following the third weekly dose. In addition, a dose-related increase in serum cytokines was observed, as well as stable disease in 4 participants refractory to prior PD-1/L1 antibodies. CONCLUSIONS: Repeat intratumoral injection of SYNB1891 as monotherapy and in combination with atezolizumab was safe and well tolerated, and evidence of STING pathway target engagement was observed.

Safety and pharmacodynamics of an engineered E. coli Nissle for the treatment of phenylketonuria: a first-in-human phase 1/2a study.

Phenylketonuria (PKU) is a rare disease caused by biallelic mutations in the PAH gene that result in an inability to convert phenylalanine (Phe) to tyrosine, elevated blood Phe levels and severe neurological complications if untreated. Most patients are unable to adhere to the protein-restricted diet, and thus do not achieve target blood Phe levels. We engineered a strain of E. coli Nissle 1917, designated SYNB1618, through insertion of the genes encoding phenylalanine ammonia lyase and L-amino acid deaminase into the genome, which allow for bacterial consumption of Phe within the gastrointestinal tract. SYNB1618 was studied in a phase 1/2a randomized, placebo-controlled, double-blind, multi-centre, in-patient study ( NCT03516487 ) in adult healthy volunteers (n = 56) and patients with PKU and blood Phe level ≥600 mmol l-1 (n = 14). Participants were randomized to receive a single dose of SYNB1618 or placebo (part 1) or up to three times per day for up to 7 days (part 2). The primary outcome of this study was safety and tolerability, and the secondary outcome was microbial kinetics. A D5-Phe tracer (15 mg kg-1) was used to study exploratory pharmacodynamic effects. SYNB1618 was safe and well tolerated with a maximum tolerated dose of 2 × 1011 colony-forming units. Adverse events were mostly gastrointestinal and of mild to moderate severity. All participants cleared the bacteria within 4 days of the last dose. Dose-responsive increases in strain-specific Phe metabolites in plasma (trans-cinnamic acid) and urine (hippuric acid) were observed, providing a proof of mechanism for the potential to use engineered bacteria in the treatment of rare metabolic disorders.

Efficacy and safety of tofacitinib in US and non-US rheumatoid arthritis patients: pooled analyses of phase II and III.

OBJECTIVES: Tofacitinib is an oral Janus kinase inhibitor for the treatment of rheumatoid arthritis. This post-hoc pooled analysis assessed commonalities and differences in tofacitinib efficacy and safety for US versus rest of the world (ROW) populations. METHODS: Pooled phase (P) III data from patients receiving tofacitinib 5 or 10 mg twice daily (BID) or placebo were assessed for efficacy at Month 3 and for safety outcomes over 12 months. For adverse events of special interest, data on tofacitinib 5 or 10 mg BID or placebo were pooled from six PII and five PIII randomised studies. RESULTS: PIII data were available for 664 vs. 2447 and PII/PIII data for 943 vs. 3567 US vs. ROW patients, respectively. The US population had a higher proportion of Caucasians (81.5% vs. 54.4%), lower proportion of Asians (1.0% vs. 34.6%), and higher mean body weight (85.7 vs. 66.2 kg) and body mass index (31.5 vs. 25.6 kg/m2) compared with ROW. At Month 3, PIII efficacy was similar between US and ROW as assessed by ACR 20/50/70 response rates, remission rates (DAS 28-4[ESR]<2.6), and HAQ-DI scores. Diarrhoea, peripheral oedema, and upper respiratory tract infection occurred in >5% of PIII patients in the US population. Incidence rates for adverse events of special interest were similar between the US and ROW PII/PIII populations. CONCLUSIONS: Patients in the US achieved similar efficacy and safety with tofacitinib 5 and 10 mg BID compared with patients in ROW.

Safety and efficacy of tofacitinib, an oral janus kinase inhibitor, for the treatment of rheumatoid arthritis in open-label, longterm extension studies.

OBJECTIVE: To describe the longterm safety and efficacy profile of tofacitinib in patients with moderate to severe active rheumatoid arthritis (RA). METHODS: Data were pooled from 2 open-label studies (NCT00413699, NCT00661661) involving patients who had participated in qualifying phase I, II, or III index studies of tofacitinib. Safety data included over 60 months of observation; efficacy data are reported up to Month 48. Treatment was initiated with tofacitinib 5 or 10 mg twice daily. Primary endpoints were adverse events (AE) and laboratory safety data. Secondary endpoints included American College of Rheumatology (ACR) response rates, and Disease Activity Score (28 joints) (DAS28)-4[erythrocyte sedimentation rate (ESR)] and Health Assessment Questionnaire-Disability Index (HAQ-DI) assessments. RESULTS: Overall, 4102 patients were treated for 5963 patient-years; mean (maximum) treatment duration was 531 (1844) days; 20.8% of patients discontinued treatment over 60 months. The most common AE were nasopharyngitis (12.7%) and upper respiratory tract infection (10.5%). Serious AE were reported in 15.4% of patients with an exposure-estimated incidence rate of 11.1 events/100 patient-years. Serious infections were reported in 4.5% of patients with an exposure-estimated incidence rate of 3.1 events/100 patient-years (95% CI: 2.66-3.55). Mean values for laboratory variables were stable over time and consistent with phase II and III studies. Persistent efficacy was demonstrated through Month 48, as measured by ACR response rate (ACR20/50/70) DAS28-4-ESR, and HAQ-DI. Safety and efficacy were similar for patients receiving tofacitinib as monotherapy or with background nonbiologic disease-modifying antirheumatic drugs. CONCLUSION: Tofacitinib demonstrated consistent safety and persistent efficacy over 48 months in patients with RA.

Lack of effect of tofacitinib (CP-690,550) on the pharmacokinetics of the CYP3A4 substrate midazolam in healthy volunteers: confirmation of in vitro data.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: • Tofacitinib (CP-690,550) is a novel, oral Janus kinase inhibitor being investigated as a targeted immunomodulator and disease-modifying therapy in rheumatoid arthritis. • Non-renal elimination accounts for 70% of the total clearance of tofacitinib and the metabolism is primarily mediated by cytochrome P450 (CYP) 3A4. • This study was required to determine the effect of tofacitinib on the in vivo pharmacokinetics of a sensitive CYP3A4 substrate. WHAT THIS STUDY ADDS: • The pharmacokinetics of midazolam, a sensitive CYP3A4 substrate, are not altered when co-administered with tofacitinib in healthy subjects. • Tofacitinib is unlikely to affect the clearance of drugs metabolized by CYP enzymes. • There is no need for dose adjustments of CYP substrates when co-administered with tofacitinib. AIMS: To investigate inhibitive and inductive effects of tofacitinib (CP-690,550), a Janus kinase inhibitor, on CYP3A4 function via in vitro and in vivo studies. METHODS: In vitro experiments were conducted to assess the inhibition and induction potential of tofacitinib for major drug metabolizing enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4). A phase 1, randomized, open-label, two-way crossover study (NCT00902460) was conducted to confirm the lack of inhibitive/inductive effect on a sensitive CYP3A4 substrate, midazolam, in healthy subjects. Midazolam pharmacokinetics were assessed over 24 h following single dose 2 mg administration prior to administering tofacitinib and after twice daily dosing of tofacitinib 30 mg for 6 days. The primary endpoint was midazolam area under the concentration-time profile, from time 0 to infinity (AUC(0,∞)). RESULTS: In vitro studies demonstrated low potential for CYP inhibition (IC(50) estimates tofacitinib > 30 µm), CYP3A4 mRNA induction (observed at tofacitinib concentrations ≥ 25 µm) and no effect on enzymatic activity of CYP substrates. In the human study, AUC(0,∞) adjusted geometric mean ratio for midazolam plus tofacitinib to midazolam alone was 103.97% [90% confidence interval (CI) 95.57, 113.12], wholly within the pre-specified acceptance region (80, 125). The 90% CI for the ratio of adjusted geometric means of maximum plasma concentration (C(max) ) (95.98, 108.87) was also wholly within this acceptance region. CONCLUSIONS: These data confirm a lack of an inhibitive or inductive effect of tofacitinib on CYP3A activity in humans and, in conjunction with in vitro data, support the conclusion that tofacitinib is unlikely to influence the CYP enzyme system as a whole.

Long-term intersession variability for single-breath diffusing capacity.

BACKGROUND: Characterizing long-term diffusing capacity (DL(CO)) variability is important in assessing quality control for DL(CO) equipment and patient management. Long-term DL(CO) variability has not been reported. OBJECTIVES: It was the aim of this study to characterize long-term variability of DL(CO) in a cohort of biocontrols and to compare different methods of selecting a target value. METHODS: Longitudinal DL(CO) monitoring of biocontrols was performed as part of the inhaled insulin development program; 288 biocontrols were tested twice monthly for up to 5 years using a standardized technique. Variability, expressed either as percent change or DL(CO) units, was assessed using three different target values. RESULTS: The 90th percentile for mean intersession change in DL(CO) was between 10.9 and 15.8% (2.6-4.1 units) depending on the target value. Variability was lowest when the mean of all DL(CO) tests was used as the target value and highest when the baseline DL(CO) was used. The average of the first six DL(CO) tests provided an accurate estimate of the mean DL(CO) value. Using this target, the 90th percentile for mean intersession change was 12.3% and 3.0 units. Variability was stable over time and there were no meaningful associations between variability and demographic factors. CONCLUSIONS: DL(CO) biocontrol deviations >12% or >3.0 units, from the average of the first six tests, indicate that the instrument is not within quality control limits and should be carefully evaluated before further patient testing.

Trough insulin levels in bronchoalveolar lavage following inhaled human insulin (Exubera) in patients with diabetes mellitus.

BACKGROUND/AIM: There are few data regarding insulin levels in the lungs during diabetes therapy with inhaled insulin. We examined the disposition of inhaled human insulin (Exubera(®) [EXU] human insulin [recombinant DNA origin], Pfizer, New York, NY) in the lungs by measuring trough insulin levels in bronchoalveolar lavage (BAL) fluid after 12 weeks of EXU treatment. METHODS: After a 4-week run-in period of subcutaneous insulin therapy, 24 subjects with type 1 diabetes mellitus (T1DM) and 26 with type 2 diabetes mellitus (T2DM) continued their basal insulin regimen and received premeal subcutaneous (SC) insulin for 13 weeks, followed by 12 weeks of premeal EXU. BAL was performed approximately 12 h after the last insulin dose at (1) baseline, (2) following SC insulin, and (3) following EXU. RESULTS: Twenty patients with T1DM and 24 patients with T2DM completed all three bronchoscopies. BAL trough insulin levels were undetectable at baseline or following SC insulin. After EXU therapy, they increased to a median of 4.5 nM (1.6-9.0 nM) and 2.3 nM (0.5-9.4 nM) in T1DM and T2DM, respectively. BAL trough insulin levels did not correlate with treatment efficacy, adverse effects, plasma insulin levels, or changes in pulmonary function. A larger proportion of previous EXU doses was present in the BAL in patients with T1DM. We found no correlation between average daily insulin doses and BAL trough insulin levels. CONCLUSIONS: BAL trough insulin increased following EXU therapy, but this increase did not correlate with other clinical or laboratory parameters, suggesting no significant biological action. Further studies are warranted to better understand inhaled insulin deposition and clearance and possible effects of increased insulin levels on the lungs.

Inhibition of JAK kinases in patients with rheumatoid arthritis: scientific rationale and clinical outcomes.

CP-690,550 is an orally active and selective inhibitor of the janus kinase (JAK) molecules. The molecular pathways through which the JAK moieties function are described along with the clinical mechanisms associated with their inhibition. Animal models of JAK inhibition are reviewed as a background for the possible inhibition of JAK in humans. The pharmacokinetics of CP-690,550 in humans is described, and the Phase IIA and IIB trials are reviewed in some detail. These trials were dose-ranging and showed a general dose response with relatively robust American College of Rheumatology 20 (ACR20) responses. A proof-of-concept 6-week trial in which CP-690,550 was given as monotherapy was associated with highly efficacious responses at the mid and higher twice-daily dose ranges employed. A subsequent 24 week dose-ranging trial in which CP-690,550 was administered in combination with methotrexate showed ACR20 responses, which were also statistically significant versus placebo interventions. CP-690,550 treatment was associated with side effects, which included headache and nausea. Infections were more common versus placebo as were elevations in transaminase enzymes when administered in combination with methotrexate, and increases in low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol. Decreases in haemoglobin and white blood cell (WBC) counts were also observed along with small increases in serum creatinine. Occasional significant decreases of haemoglobin (>2 g dl(-1)) were observed, although decreases of WBC to less than 1000 per mm(3) were not seen. Plans for long-term follow-up of the described trials are described along with the features of five presently ongoing Phase III trials of the CP-690,550 janus kinase (JAK) inhibitor. Future directions include completion and publication of these trials along with study of JAK inhibition for other indications.

Safety and efficacy of inhaled human insulin (Exubera) during discontinuation and readministration of therapy in adults with type 1 diabetes: A 3-year randomized controlled trial.

OBJECTIVE: To assess pulmonary safety during discontinuation and readministration of inhaled human insulin (EXU; Exubera((R)) insulin human [rDNA origin]) Inhalation Powder) therapy in adults with type 1 diabetes. METHODS: Patients were randomized to receive basal insulin plus either pre-meal EXU (n=290) or a short-acting subcutaneous (SC) insulin (n=290) for 2 years (comparative phase), followed by 6 months of SC insulin (washout) and 6 months of their original therapy (readministration). Highly standardized lung function tests were performed throughout. RESULTS: Small treatment group differences favoring SC insulin in change from baseline forced expiratory volume in 1s (FEV(1)) and carbon monoxide diffusing capacity (DL(CO)) occurred early and were non-progressive. These differences resolved during washout and recurred at the same magnitude during readministration. Both groups maintained glycemic control, and hypoglycemic event rates were similar. In the EXU group, insulin antibody (IAb) levels plateaued at 12 months, declined to near baseline levels during washout and increased during readministration to levels observed in the comparative phase. CONCLUSIONS: FEV(1) and DL(CO) changes observed during discontinuation and readministration of EXU therapy are consistent with a reversible, non-progressive and non-pathological effect on lung function. EXU readministration is not associated with an augmented IAb response.

Intersession variability in single-breath diffusing capacity in diabetics without overt lung disease.

RATIONALE: American Thoracic Society guidelines state that a 10% or greater intersession change in diffusing capacity of the lung (DL(CO)) should be considered clinically significant. However, little is known about the short-term intersession variability in DL(CO) in untrained subjects or how variability is affected by rigorous external quality control. OBJECTIVES: To characterize the intersession variability of DL(CO) and the effect of different quality control methods in untrained individuals without significant lung disease. METHODS: Data were pooled from the comparator arms of 14 preregistration trials of inhaled insulin that included nonsmoking diabetic patients without significant lung disease. A total of 699 participants performed repeated DL(CO) measurements using a highly standardized technique. A total of 948 participants performed repeated measurements using routine clinical testing. MEASUREMENTS AND MAIN RESULTS: The mean intersession absolute change in DL(CO) using the highly standardized method was 1.45 ml/minute/mm Hg (5.64%) compared with 2.49 ml/minute/mm Hg (9.52%) in the routine testing group (P < 0.0001 for both absolute and percent difference). The variability in absolute intersession change in DL(CO) increased with increasing baseline DL(CO) values, whereas the absolute percentage of intersession change was stable across baseline values. Depending on the method, 15.5 to 35.5% of participants had an intersession change of 10% or greater. A 20% or greater threshold would reduce this percentage of patients to 1 to 10%. CONCLUSIONS: Intersession variability in DL(CO) measurement is dependent on the method of testing used and baseline DL(CO). Using a more liberal threshold to define meaningful intersession change may reduce the misclassification of normal variation as abnormal change.

Standardization of the single-breath diffusing capacity in a multicenter clinical trial.

BACKGROUND: Standardization of the measurement of single-breath diffusing capacity of the lung for carbon monoxide (DLCO) is difficult to implement in multicenter trials as differences in equipment, training, and performance guidelines have led to high variability between and within centers. The safety assessment of inhalable insulin required the standardization of measurement of single-breath DLCO in multicenter clinical trials to optimize test precision. METHODS: This was an open-label, 24-week, parallel-group, outpatient study of inhaled human insulin in participants with type 1 diabetes who were randomly assigned to receive treatment with daily premeal inhaled or subcutaneous (SC) insulin for 12 weeks, followed by SC insulin for 12 weeks. Monitoring of single-breath DLCO using standardized methodology was performed. Standardization included uniform instrumentation, centrally trained study coordinators, and centralized data monitoring and review of quality control. Sites received feedback within 24 h for any tests of unacceptable quality with recommendations for improvement. RESULTS: A total of 226 study participants at 33 sites completed 11,335 DLCO efforts during 4,797 test sessions; 3,607 (75.2%) and 4,581 (95.5%) of all testing sessions yielded two American Thoracic Society-acceptable efforts that varied by < 1 and 2 mL/min/mm Hg, respectively. Only 65 sessions produced one or fewer acceptable efforts. The root mean square intrasubject coefficient of variation in DLCO at the end of the comparative dosing phase was 6.01%. CONCLUSIONS: The standardized methodology employed in this study demonstrates the feasibility of collecting high-quality single-breath DLCO data in the setting of a multicenter clinical trial with reliability that is comparable to spirometry.

Dissociation of lung function changes with humoral immunity during inhaled human insulin therapy.

RATIONALE: Inhaled human insulin (INH; Exubera [human insulin (recombinant DNA origin) Inhalation Powder]) causes small changes in pulmonary function and increases in insulin antibodies compared with subcutaneous (SC) insulin. OBJECTIVES: To investigate the relationship between changes in pulmonary function and insulin antibodies and acute effects of INH on lung function. METHODS: In a 24-wk multicenter study, 226 patients with type 1 diabetes were randomized to receive daily premeal INH or SC insulin for 12 wk (comparative phase), followed by SC insulin for 12 wk (washout phase). MEASUREMENTS: Spirometry tests were conducted and insulin antibody levels were measured throughout the study. Acute insulin-induced changes in lung function were calculated as the difference between FEV1 before, and 10 and 60 min after, insulin. MAIN RESULTS: There was a temporal dissociation between pulmonary function changes and insulin antibody generation. Small treatment group differences in changes in FEV1 from baseline, favoring SC insulin, were fully manifest by 2 wk of INH therapy, did not increase during the remainder of the comparative phase, and resolved within 2 wk of INH discontinuation. By contrast, insulin antibody levels remained low for the first 2 wk with INH, increased during Weeks 2 to 12, and gradually declined during washout. There was no evidence of acute insulin-induced alterations in lung function 10 and 60 min postinhalation. CONCLUSION: The small lung function changes observed with INH therapy are not mediated by the humoral immune response, or associated with acute decrements in lung function immediately after insulin inhalation.

Imbalance between cysteine proteases and inhibitors in a baboon model of bronchopulmonary dysplasia.

RATIONALE: Bronchopulmonary dysplasia (BPD) continues to be a major morbidity in preterm infants. The lung pathology in BPD is characterized by impaired alveolar and capillary development. An imbalance between proteases and protease inhibitors in association with changes in lung elastic fibers has been implicated in the pathogenesis of BPD. OBJECTIVE: To investigate the expression and activity levels of papain-like lysosomal cysteine proteases, cathepsins B, H, K, L, S, and their inhibitors, cystatins B and C, in a baboon model of BPD. METHODS: Real-time reverse transcriptase-polymerase chain reaction, immunohistochemistry, immunoblotting, active site labeling of cysteine proteases, and in situ hybridization were performed. MEASUREMENTS AND MAIN RESULTS: The steady-state mRNA and protein levels of all cathepsins were significantly increased in the lung tissue of baboons with BPD. In contrast, the steady-state mRNA and protein levels of two major cysteine protease inhibitors, cystatin B and C, were unchanged. Correlating with these alterations, the activity of cysteine proteases in lung tissue homogenates and bronchoalveolar lavage fluid was significantly higher in the BPD group. The levels of cathepsin B, H, and S increased and cathepsin K decreased with advancing gestation. All cathepsins, except for cat K, were immunolocalized to macrophages in BPD. In addition, cathepsin H and cystatin B were colocalized in type 2 alveolar epithelial cells. Cathepsin L was detected in some bronchial epithelial, endothelial, and interstitial cells. Cathepsin K was localized to some perivascular cells by in situ hybridization. CONCLUSIONS: Cumulatively, these findings demonstrate an imbalance between cysteine proteases and their inhibitors in BPD.

PU.1 regulates cathepsin S expression in professional APCs.

Cathepsin S (CTSS) is a cysteine protease that is constitutively expressed in APCs and mediates processing of MHC class II-associated invariant chain. CTSS and the Ets family transcription factor PU.1 are highly expressed in cells of both myeloid (macrophages and dendritic cells) and lymphoid (B lymphocytes) lineages. Therefore, we hypothesized that PU.1 participates in the transcriptional regulation of CTSS in these cells. In A549 cells (a human epithelial cell line that does not express either CTSS or PU.1), the expression of PU.1 enhances CTSS promoter activity approximately 5- to 10-fold. In RAW cells (a murine macrophage-like cell line that constitutively expresses both CTSS and PU.1), the expression of a dominant-negative PU.1 protein and a short-interfering RNA PU.1 construct attenuates basal CTSS promoter activity, mRNA levels, and protein expression. EMSAs show binding of PU.1 to oligonucleotides derived from the CTSS promoter at two different Ets consensus binding elements. Mutation of these sites decreases the baseline CTSS activity in RAW cells that constitutively express PU.1. Chromatin immunoprecipitation experiments show binding of PU.1 with the CTSS promoter in this same region. Finally, the expression of PU.1, in concert with several members of the IFN regulatory factor family, enhances CTSS promoter activity beyond that achieved by PU.1 alone. These data indicate that PU.1 participates in the regulation of CTSS transcription in APCs. Thus, manipulation of PU.1 expression may directly alter the endosomal proteolytic environment in these cells.

MHC class II-associated invariant chain isoforms regulate pulmonary immune responses.

Asthma, a chronic inflammatory disease of the lung, is characterized by reversible airway obstruction and airway hyperresponsiveness (AHR), and is associated with increased production of IgE and Th2-type cytokines (IL-4, IL-5, and IL-13). Development of inflammation within the asthmatic lung depends on MHC class II-restricted Ag presentation, leading to stimulation of CD4(+) T cells and cytokine generation. Conventional MHC class II pathways require both MHC-associated invariant chain (Ii) and HLA-DM (H2-M in mice) chaperone activities, but alternative modes of Ag presentation may also promote in vivo immunity. In this study, we demonstrate that Ii(-/-) and H2-M(-/-) mice fail to develop lung inflammation or AHR following sensitization and challenge with OVA in a mouse model of allergic inflammation. To assess potentially distinct contributions by Ii chain isoforms to lung immunity, we also compared allergen-induced lung inflammation, eosinophilia, IgE production, and AHR in mice genetically altered to express either p31 Ii or p41 Ii isoform alone. Sole expression of either Ii isoform alone facilitates development of allergen-induced lung inflammation and eosinophilia. However, animals expressing only the p31 Ii isoform exhibit abrogated IgE and AHR responses as compared with p41 Ii mice in this model of allergen-induced lung inflammation, suggesting that realization of complete immunity within the lung requires expression of p41 Ii. These findings reveal a crucial role of Ii and H2-M in controlling the immune response within the lung, and suggest that p31 Ii and p41 Ii manifest nonredundant roles in development of immunity.

Constitutive and cytokine-induced expression of the ETS transcription factor ESE-3 in the lung.

Family studies of asthma suggest that the genes ESE-2 and ESE-3 contain polymorphisms that contribute to disease susceptibility. Each gene codes for an ETS transcription factor that is characterized by epithelium-restricted constitutive expression and may function as a context-dependent activator or repressor of transcription; however, nothing is known about the role of these genes in lung homeostasis or the pathogenesis of airway disease. In this study, we show that ESE-3 mRNA and protein are constitutively expressed in bronchial and mucous gland epithelial cells. Consistent with these findings, ESE-3 mRNA is constitutively expressed in human bronchial epithelial cells grown in tissue culture. In contrast, ESE-2 mRNA could not be detected in the lung or cultured human bronchial epithelial cells. Human bronchial smooth muscle cells and fibroblasts do not constitutively express ESE-3; however, after stimulation with interleukin-1beta or tumor necrosis factor-alpha, levels of ESE-3 mRNA and protein increase dramatically by 24 h. This cytokine induction is dose-dependent and abrogated by specific inhibitors of the MEK1/2 (U0126) and p38 (SB03580) signal transduction pathways. Overexpression of ESE-3 protein in 3T3 cells and human bronchial smooth muscle cells inhibits MMP-1 promoter activity, suggesting that ESE-3 may function as a transcriptional repressor.

IFN regulatory factor-1 regulates IFN-gamma-dependent cathepsin S expression.

Cathepsin S is a cysteine protease with potent endoproteolytic activity and a broad pH profile. Cathepsin S activity is essential for complete processing of the MHC class II-associated invariant chain within B cells and dendritic cells, and may also be important in extracellular matrix degradation in atherosclerosis and emphysema. Unique among cysteine proteases, cathepsin S activity is up-regulated by IFN-gamma. Given its importance, we sought to elucidate the pathway by which IFN-gamma increases cathepsin S expression. Our data demonstrate that the cathepsin S promoter contains an IFN-stimulated response element (ISRE) that is critical for IFN-gamma-induced gene transcription in a cell line derived from type II alveolar epithelial (A549) cells. IFN response factor (IRF)-2 derived from A549 nuclear extracts associates with the ISRE oligonucleotide in gel shift assays, but is quickly replaced by IRF-1 following stimulation with IFN-gamma. The time course of IRF-1/ISRE complex formation correlates with increased levels of IRF-1 protein and cathepsin S mRNA. Overexpression of IRF-1, but not IRF-2, markedly augments cathepsin S promoter activity in A549 cells. Furthermore, overexpression of IRF-1 increases endogenous cathepsin S mRNA levels in 293T epithelial cells. Finally, freshly isolated bone marrow cells from IRF-1(-/-) mice fail to up-regulate cathepsin S activity in response to IFN-gamma. Thus, IRF-1 is the critical transcriptional mediator of IFN-gamma-dependent cathepsin S activation. These data elucidate a new pathway by which IRF-1 may affect MHC class II processing and presentation.