Ulrich Costabel

Expression of transferrin receptors and intracellular ferritin during terminal differentiation of human monocytes.

SummaryHuman blood monocytes when cultured on hydrophobic Teflon membranes differentiate into mature macrophages. The expression of transferrin receptors was monitored by monoclonal antibody (OKT9) binding as detected by immunperoxidase staining. Whereas monocytes were negative, an increasing percentage of macrophages, starting from day 2 in culture, labelled with the anti-transferrin receptor antibody as these cells undergo differentiation. After completion of maturation more than 90% of macrophages expressed transferrin receptors. While 90–95% of macrophages from broncho-alveolar lavage fluids labelled with the OKT9 antibody, only a minor portion of macrophages obtained from peritoneal and pleural cavities did so. In parallel, intracellular ferritin in cells of the monocyte-macrophage lineage increased from 10ng/106 cells to 350–1,500ng/106 cells during maturation in vitro. Alveolar macrophages proved to have the highest ferritin content which ranged from 355–8,400ng/106.The results may indicate that iron uptake and storage is a function of cells at late stages of macrophage maturation and that the occurrence of surface receptors for transferrin can be regarded as differentiation dependent marker.

Human macrophage maturation and heterogeneity: restricted expression of late differentiation antigens in situ

SummaryTerminal maturation of human macrophages is an important step for creation of cell diversity amongst site-specific subpopulations and their functional competence in situ. As monocytes undergo differentiation in vitro, they start to express lineage-restricted antigens specific for differentiation stages beyond the blood monocyte level as detected by monoclonal antibodies of the MAX series. We have analyzed the expression of MAX.1, MAX.2, MAX.3 and MAX.11 on exudate-type macrophages from pleural and peritoneal cavity and the alveolar space, as well as on resident and activated tissue macrophages in cryostat sections of spleen, lymph node, tonsil, liver, gut mucosa, skin, placenta, kidney and bone. It was found that “free” macrophages in serous cavities expressed MAX antigens in a heterogenous pattern, whereas none of the organ-specific tissue macrophages subsets did so (with the exception being the weak label of MAX.2 on Kupffer cells). Only during allograft rejection were infiltrating macrophages found to express MAX antigens but not at sites of “nonspecific” inflammation or granuloma formation. However, Cyclosporin A treatment seems to suppress the induction of MAX antigen expression on intragraft macrophages. In addition, freshly harvested MAX-negative exudate macrophages converted to the complete Max+ phenotype on further cultivation. Isolated Kupffer cells were able only to express the MAX.2 antigen in culture but still did not react with the MAX.1 and MAX.3 monoclonal antibodies. Some MAX antigens are co-expressed on glomerular mesangial cells, dendritic reticulum cells and placental cells (MAX.1/. 11) as well as on capillary endothelium within tissues of active immune response (MAX.2). These results add to the knowledge of the phenotypic heterogeneity within the macrophage system as a result of site-specific influences and modulation during a cell-mediated immune response. They also give evidence for a major difference between “free” exudate-type macrophages and resident tissue macrophages.

P13 Safety of pirfenidone in patients with idiopathic pulmonary fibrosis (IPF): Integrated analysis of cumulative data from 5 clinical trials

Introduction and objectives IPF is a chronic, progressive and irreversible disease that requires long-term clinical management. To further evaluate the clinical safety of pirfenidone in patients with IPF, we performed a comprehensive integrated analysis of safety data from 5 clinical trials. Methods All patients assigned to receive pirfenidone (2403 mg/d) in the Phase 3 ASCEND (016) and CAPACITY (004/006) studies and all patients receiving ≥1 dose of pirfenidone in either of two ongoing open-label studies (studies 002 and 012) comprised the integrated population. EAP (002) is a compassionate use study in the U.S.; RECAP (012) is evaluating pirfenidone in patients who completed one of the Phase 3 studies. Analyses were based on the January 15, 2014 interim data cut. Results 1299 patients were included in the integrated population. The cumulative total exposure to pirfenidone was 3160 person exposure years (PEY). The median duration of exposure was 1.7 years (range, 1 week–9.9 years); 545 (42%) patients received pirfenidone for ≥2 years and 325 (25%) patients received pirfenidone for ≥4 years. The majority of patients (75.8%) received a mean daily dose of ≥1800 mg. Consistent with prior observations, gastrointestinal and skin-related events were among the most common treatment emergent adverse events (Table 1); these were almost always mild to moderate in severity, reversible with dose modification and rarely led to treatment discontinuation. Cough, dyspnoea and IPF were the most common respiratory adverse events in the integrated population—a finding that is consistent with expectations in patients with a chronic progressive respiratory disease followed over a long period of observation. Aminotransferase (ALT or AST) elevations (>3 × ULN) occurred in 40/1299 (3.0%) patients in the integrated population.Abstract P13 Table 1 Treatment emergent adverse events in the integrated population compared with the pooled pirfenidone 2403 mg/d and placebo groups in the Phase 3 trials* Integrated population (N = 1299)† OE = treatment emergent adverse event ment emergent adverse events d sun exposure during treatment with pirfenidone. the skin du Median (range) duration of exposure, yr 1.7 (>0, 9.9) Treatment emergent adverse event,% Nausea 37.6 Cough 35.1 Dyspnea 30.9 Upper respiratory tract infection 30.6 Idiopathic pulmonary fibrosis 29.3 Fatigue 28.2 Diarrhoea 28.1 Rash 25.0 Bronchitis 23.8 Headache 21.6 Nasopharyngitis 21.3 Dizziness 21.2 Dyspepsia 18.4 Vomiting 15.9 Weight decreased 15.6 Back pain 15.4 Anorexia 15.2 *Occurring in ≥15% of patients in the cumulative clinical database. †Includes 2 patients in Study 002 with a diagnosis of “pulmonary fibrosis.” Conclusions A comprehensive integrated analysis of safety outcomes in a large, well–defined cohort of 1299 patients with IPF who were treated with pirfenidone for up to 9.9 years demonstrated that treatment with pirfenidone is safe and generally well tolerated. These observations provide further evidence to support the long-term clinical safety of pirfenidone in patients with IPF.

Proteases and antiproteases related to the coagulation system in plasma and ascites: Prediction of coagulation disorder in ascites retransfusion

To improve the ability to predict the occurrence of coagulation disorders in ascites retransfusion and, in addition, to better define the nature of the coagulation disorder, several proteases and antiproteases were analyzed in ascites and plasma before ascites retransfusion in 17 patients. Plasminogen, alpha 2-antiplasmin, antithrombin III, and fibrin(ogen) degradation products in ascites were significantly altered in patients who later developed abnormal coagulation as compared to those who did not. Only plasminogen and alpha 2-antiplasmin in ascites achieved a sufficient predictive value for the occurrence of coagulation abnormalities. The pattern of the coagulation abnormalities observed strongly suggests fibrinolysis induced by the infusion of plasminogen activators as the cause of the coagulation disorder in ascites retransfusion procedures.

Bronchoalveolar Lavage in Sarcoidosis

A characteristic feature of BAL in patients with sarcoidosis is an increase of the total cell number, lymphocytes percent, or CD4/CD8 ratio of T lymphocytes. With respect to T lymphocytes, sarcoidosis has been recognized as a granulomatous disease characterized by dominant expression of Th1 cytokines. Recently Th1 cytokine profile in both CD4+ and CD8+ T lymphocytes in BAL fluid were demonstrated at the single-cell level. Furthermore, alveolar macrophages collected from BAL fluid were also investigated. Increased expression of 25-hydroxyvitamin D3 1 alpha-hydroxylase with mRNA level in alveolar macrophage were revealed in active sarcoidosis. Previously we showed that the B allele of the vitamin D receptor gene polymorphism may be regarded as a risk factor in the onset of sarcoidosis. The metabolism of vitamin D may be related to granuloma formation.

S109 Effect of continued treatment with pirfenidone following a clinically meaningful decline in percent predicted forced vital capacity in patients with idiopathic pulmonary fibrosis (IPF)

Introduction and objectives The clinical course in patients with IPF is characterised by substantial inter- and intra-subject variability in the rates of disease progression, thereby confounding clinical assessments of therapeutic responses in individual patients. We pooled data from three Phase 3 trials to assess the potential benefit of continued treatment with pirfenidone in patients who experienced a ≥10% decline in percent predicted forced vital capacity (%FVC) during the first 6 months of treatment. Methods Source data included all patients randomised to treatment with pirfenidone 2403 mg/d or placebo in the Phase 3 ASCEND or CAPACITY studies (N = 1247). We selected patients with a ≥10% absolute decline in%FVC by the month 3 or 6 study visit and compared the proportion of patients in the pirfenidone and placebo groups who experienced any of the following during the subsequent 6-month interval: (1) ≥10% absolute decline in%FVC or death; (2) no further decline in%FVC; or (3) death. Observed data were used in the analysis. Results 34 (5.5%) and 68 (10.9%) patients in the pooled pirfenidone and placebo groups, respectively, experienced a ≥10% absolute decline in%FVC between baseline and month 6 (relative difference, 49.5%). Analysis of outcomes during the subsequent 6-month interval demonstrated that fewer patients in the pirfenidone group compared with placebo experienced a ≥10% decline in%FVC or death (pirfenidone, 2/34 [5.9%] vs. placebo, 19/68 [27.9%]). More patients in the pirfenidone group compared with placebo had no further decline in%FVC (20/34 [58.8%] vs. 26/68 [38.2%]; Table 1). Additionally, there were fewer deaths in the pirfenidone group (1/34 [2.9%]) compared with placebo (14/68 [20.6%]).Abstract S109 Table 1 Outcomes during the 6-month period following an initial decline in percent predicted FVC ≥10% during the first 6 months of treatment Outcome, n (%) Pirfenidone(n = 34) Placebo(n = 68) Relative difference* P value† ≥10% decline in FVC or death 2 (5.9) 19 (27.9) –78.9% 0.009 Death 1 (2.9) 14 (20.6) –85.7% 0.018 >0% to <10% decline in FVC 12 (35.3) 23 (33.8) 4.3% ND No further decline in FVC‡ 20 (58.8) 26 (38.2) 53.8% 0.059 FVC = forced vital capacity. *Relative difference calculated using the following formula: 100 × [pirfenidone – placebo]/[placebo]. †Fisher’s exact test. ‡Either no decline or an increase in FVC. Conclusions Among patients who experienced a ≥10% decline in%FVC during the first 6 months of treatment, continued treatment with pirfenidone resulted in a lower risk of%FVC decline or death during the subsequent 6 months. These findings suggest a potential benefit to continued treatment with pirfenidone despite an initial decline in FVC.