Penicillamine

We retrospectively studied 82 patients diagnosed with IPF at the National Reference Center for Rare Pulmonary Diseases in Lyon, France between January 2006 and July 2015. All patients were followed up for a period of four years, with no censoring. This period was longer than the conventional follow up, three years, in IPF studies. IPF was diagnosed according to the 2011 American Thoracic Society/European Respiratory Society (ATS/ERS) guidelines. Patients diagnosed before 2011 also met the criteria of these guidelines following a consensus by a multidisciplinary team that included respiratory, pathology, and radiology experts in interstitial lung disease [1 (link)]. Lung function parameters were assessed according to the criteria published by the ATS and the ERS [22 (link)].
Age, gender, body mass index (BMI), respiratory function tests, complete cell blood counts, pharmacological treatment (steroids, antiplatelet drugs, and anticoagulants), and disease stage at diagnosis were retrieved from clinical records and stored in a database. Telephone interviews were conducted to ascertain survival status when the latter was not available from records. Exclusion criteria included malignancy, bleeding tendency, severe hepatic or renal disease, lung transplantation, the ongoing use of antifibrotic drugs before the study, and use of immunosuppressants, interferon, D-penicillamine, colchicine, or oral corticosteroids during the preceding three months. The study was approved by the Institutional Ethical Committee of the Hospital Louis Pradel, Lyon, France, and the Institutional Local Ethics Committee of the University Hospital (AOU) of Cagliari, Italy (PG/2018/4426).
The derivative blood cell count inflammation indexes included the neutrophil-to-lymphocyte ratio (NLR: neutrophils/lymphocytes), neutrophil-to-lymphocyte ratio [dNLR: (WBC-lymphocytes)/lymphocytes)], monocyte-to-lymphocyte ratio (MLR: monocytes/lymphocytes), platelet-to-lymphocyte ratio (PLR: platelet/lymphocyte), systemic inflammatory index (SII: neutrophils × platelets/lymphocytes)], systemic inflammatory response index (SIRI: neutrophils × monocytes/lymphocytes), and aggregate index of systemic inflammation (AISI: neutrophils × platelets x monocytes/lymphocytes).
Results were expressed as mean values and standard deviation (SD), or median values and interquartile ranges (IQR). Individual variable distribution was assessed by the Shapiro–Wilk test.
Student’s t-test or a Mann–Whitney rank sum test were used to assess between-group differences. Correlations between variables were estimated using Spearman’s or Pearson’s correlation, as appropriate. For survival analysis, time zero was defined as the time of diagnosis. Survival probability was estimated using the Kaplan–Meier method and the log-rank test, with death being the end point. Cox proportional hazards regression was performed for both univariate and multivariate analyses, with specific focus on the independent effect of AISI on survival by controlling for potential confounders, i.e., age, gender, BMI, smoking status, disease stage, and therapy. Hazard ratios were calculated from the Cox analysis. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using MedCalc for Windows, version 15.4 64 bit (MedCalc Software, Ostend, Belgium).

All patients were followed up for at least 1 year and were evaluated by routine examinations every year. The mean follow-up time was 48.9 (range, 12–72) months. Penicillamine (PCA) and zinc gluconate (ZG) were administrated as initial and maintenance therapies, respectively for all patients. PCA was administered 1–2 h before meals between 750 and 1250 mg daily, divided into 2–4 doses, and ZG was administered after meals between 210 and 420 mg daily, divided into 3 doses. The clinical, laboratory, and imaging data of all the patients were recorded and evaluated during follow-up.

Differentiation of mESCs to the myocardium was conducted, as described elsewhere [31 (link)]. Briefly, to form the embryonic bodies (EBs), one drop had a volume of 20 μL and contained 800 cells. Eighty-four drops were suspended upside down in a petri dish (10090, SPL, Pocheon-si, Gyeonggi-do, South Korea) and filled with D-PBS at the downside to prevent evaporation. The cells were cultured in a 5% CO₂ incubator at 37 °C for four days. After harvesting the EBs, they were placed in 60mm petri dish for stabilization for two days. Subsequently, the EB was transferred for attachment in a 24-well plate with 0.5 mL of a differentiation medium, a component at 15% FBS concentration with no LIF in the growth medium. Sodium arsenite (SA; S7400, Sigma, Burlington, MA, USA) and D-penicillamine (DPA; P4875 Sigma, Burlington, MA, USA) were treated at this time, and the medium was changed every two days for 15 days.
The beating cardiomyocytes were observed using a phase-contrast microscope every two days. The beating ratio was regarded as the ratio of beating wells and non-beating wells; the well with beating cardiomyocytes was one, and the well with non-beating cardiomyocytes was zero. The number of independent experiments was performed with eight wells in three independent experiments.