Pilot safety study of intrabronchial instillation of bone marrow-derived mononuclear cells in patients with silicosis

Forty-one patients with silicosis were screened for eligibility. All but one were male. The reasons for exclusion were normal spirometry (n = 19), spirometry parameters outside the study range (n = 5), death during screening (n = 3), loss to follow-up during screening (n = 3), refusal to sign informed consent (n = 3), active tuberculosis found at screening (n = 1), patient outside age range (n = 1), and psychiatric disorder (n = 1). Five males were included in the study, with a mean age at intervention of 41 years (range 37–45 years). Patients met the following inclusion criteria: (1) age between 18 and 50 years; (2) chronic or accelerated silicosis characterized by the presence of new areas of fibrosis in high-resolution computed tomography of the lungs in the last 2 years; (3) pulmonary function tests showing moderate to severe impairment, characterized by forced expiratory volume in 1 s (FEV₁) <60 % predicted and >40 % predicted, and forced vital capacity (FVC) ≥60 % predicted but with arterial oxygen saturation >90 % on room air. Patients were excluded if they met any of the following criteria: (1) unable to undergo bronchoscopy for instillation of cellular material, (2) infectious diseases at the time of the study or 1 month before enrollment, (3) other lung diseases, including active tuberculosis, (4) current or recent smoker, within 12 months of inclusion, characterized by tobacco intake >10 pack-years, (5) intra- or extrathoracic malignant neoplasms, (6) autoimmune disorders, (7) acute or unstable heart failure, (8) acute or uncontrolled coronary insufficiency, (9) primary hematologic disorders, (10) osteopathy reflecting increased risk for bone marrow aspiration, (11) primary or secondary coagulopathies, (12) liver failure, (13) moderate renal failure (creatinine level >2 mg/dL), (14) dependence on respiratory or circulatory support, (15) pregnancy, (16) human immunodeficiency virus seropositivity, and (17) participation in other clinical trials. The primary endpoint focused on safety and included death and respiratory (clinical, radiologic and functional) deterioration.

Bone marrow aspiration and subsequent cell preparation were accomplished on the same day as the infusion of intrabronchial autologous BMDMCs, and lasted 2–4 h. Collection was performed under local anesthesia and light sedation, through puncture and repeated aspirations at the posterior iliac crest region. A total of 80 mL of bone marrow aspirate was collected from each patient, and after removal of bone and fatty residues, mononuclear cells were isolated by a Ficoll-Paque Plus (Amersham Biosciences, São Paulo, Brazil) density gradient, washed three times in saline solution, resuspended in saline with 5 % human albumin, and filtered through a 100-μm nylon filter. After washing, counting, and viability testing, the cells were resuspended in 10 ml saline solution with 5 % autologous serum, and 2 × 10⁷ cells were labeled with 99mTc, as described in previously published protocols [10, 11]. In brief, 500 μl of sterile SnCl₂ solution was added to the cell suspension in 0.9 % NaCl, and the mixture was incubated for 10 min at room temperature. Then, 45 mCi 99mTc was added and incubation was continued for 10 min. After centrifugation (500 × g for 5 min), the supernatant was removed, the cells were washed again in saline solution, and the pellet was resuspended in saline solution. All cell preparation and labeling procedures were performed in a laminar flow hood. Bacteriological analyses and cultures were also carried out to exclude contamination of the material. A sample of the isolated BMDMCs was characterized by flow-cytometry analysis of specific surface antigens as previously described [11]. The viability of labeled cells was assessed by the trypan blue exclusion test, and was estimated to be greater than 90 % in all cases. A 50-mL aliquot of the autologous BMDMC solution was instilled into each lung and distributed through the lobes by a fiber optic bronchoscope over a 30-min period. Following instillation, patients were positioned in left and right lateral decubitus and Trendelenburg position to facilitate dispersion of the instilled cells. After the procedure, patients were monitored for 1 h and sent to the Nuclear Medicine Department for examinations.

Isolated BMDMCs were characterized by flow-cytometry analysis of specific surface antigens. Cells were incubated for 20 min at room temperature with primary antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), peridinin-chlorophyll-protein (PercP), and phycoerythrin/cyanine 7 (PE/Cy7). The markers tested included: pan-leukocyte, CD45 (Immunostep); pan T cell, CD3 (BD Biosciences Pharmingen); cytotoxic T cell, CD8 (BD Biosciences Pharmingen); helper T cell, CD4 (BD Biosciences Pharmingen); pan B cell, CD19 (BD Biosciences Pharmingen); NK cell, CD56 (BD Biosciences Pharmingen); promonocyte, CD64 (Immunotech); monocyte, CD14 (IQP); hematopoietic progenitor cells, CD117 (BD Biosciences Pharmingen) and CD34 (BD Biosciences Pharmingen); mesenchymal cells, CD105 (Immunostep), CD73 (BD Biosciences Pharmingen), and CD90 (BD Biosciences Pharmingen); neutrophils, CD31 (BD Biosciences Pharmingen) and CD33 (BD Biosciences Pharmingen); and anti-HLA-DR (MHC-II, BD Biosciences Pharmingen). After staining, erythrocytes were lysed with B&D Lysis Buffer Solution. Data were acquired on a BD FACS CANTO cytometer (BD Biosciences) and analyzed with BD Paint-a-Gate software.

After bone marrow harvesting, approximately 2 × 10⁷ BMDMCs were labeled with technetium-99 m (99mTc) and instilled in the bronchi through fiber optic bronchoscopy. Whole-body, planar and tomographic scintigraphy was carried out at 2 h and 24 h after cell transplantation. All cell preparation and labeling procedures were performed in a laminar flow. Briefly, 500 μL of sterile SnCl₂ solution was added to the cells and the mixture was incubated at room temperature for 10 min; 45 mCi of 99mTc was then added and incubation continued for another 10 min. After centrifugation (500 × g for 5 min), the supernatant was removed and the cells were washed in saline solution. The pellet was also resuspended in saline solution. Viability of the labeled cells was assessed by the Trypan Blue exclusion test, and was estimated to be greater than 93 % in all cases. Labeling efficiency (%) was calculated by the activity in the pellet divided by the sum of the radioactivity in the pellet plus supernatant and was estimated to be greater than 90 % in all cases. Perfusion scintigraphy with 99mTc-MAA (Tc-99 m macroaggregated albumin) was performed before and 30, 60, 120, 180 and 360 days after BMDMC therapy. For regional analysis in both the anterior (A) and posterior (P) images, rectangular regions of interest, equal in size, were drawn over the whole lung (A-whole lung and P-whole lung). Both lungs were divided into six regions of interest: right upper, right middle, right lower, left upper, left middle, and left lower lung fields. Regional blood flow evaluated by 99mTc-MAA perfusion scintigraphy in each region of interest was calculated as per Ohno et al. [12]: Q_PS (%) = [(A_roi + P_roi/2)/(A_{whole lung} + P_{whole lung} 2)] × 100, where Q_PS is quantitative perfusion scintigraphy, A_roi is the anterior region of interest, P_roi is the posterior region of interest, A_{whole lung} is the anterior whole lung, and P_{whole lung} is the posterior whole lung.