Bleomycin Lung Research Articles

P311 KO Mice are Protected from Experimentally Induced Lung Fibrosis

Pulmonary fibrosis (PF) is a fatal chronic interstitial lung disease and one of the most irreversible forms of tissue/organ fibrosis. Idiopathic pulmonary fibrosis is a very progressive disease accompanied by inflammatory damage with a median survival of less than 5 years. Etiology of IPF is still elusive with potential reasons being genetic predisposition and environmental exposures including particulates, radiation, and SARS-CoV-2. Recent studies indicate that about 50% of COVID19 associated acute respiratory distress syndrome survivors develop PF. There are very limited treatment options with no curative therapies available for PF apart from invasive lung transplantation. P311 is an 8 kDa protein previously shown to have a key role in lung regeneration. As P311 overexpression in fibroblasts induced myofibroblast phenotype, we hypothesized that P311 has a role in tissue fibrosis. We induced PF by intra-tracheally instilling bleomycin (BL) in P311 knockout (KO) mice and wild type (WT, C57BL/6) control mice and collected lungs after 2-5 weeks. P311 KO mice showed significantly less fibrosis. After intra-tracheal instillation of BL, the lungs of P311 KO mice (D21) had significantly reduced number of myofibroblasts as indicated by reduced SM α-actin expression and collagen deposition as indicated by Masson trichrome staining compared to their WT counterparts. We further observed reduced levels of TGF-β1-3 and Smad-2 and -3 along with decreased epithelial to mesenchymal transdifferentiation markers in the lungs of bleomycin treated P311 KOs compared to WT controls. Further bronchoalveolar lavage fluid (BALF) was collected (D14) and utilized to determine the pulmonary inflammation by determining the total and different immune cell populations in the BALF and lung samples by FACS analysis. Even though we saw a significant increase in the BALF cellularity, we did not observe significant changes in T-cell, macrophage, and neutrophil numbers between WT and P311KOs. However, lung cell isolates showed significantly increased CD3 and CD4 positive cells indicating increased T-cells and helper T-cells in fibrotic P311KO lungs compared to WT lungs. In conclusion, bleomycin administration resulted in lung fibrosis in WT mice, but P311 KO mice are protected. This study was partially supported by research funding to KB, from American Lung Association, DA-196629-N. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Protective effects of microbial biosurfactants produced by Bacillus halotolerans and Candida parapsilosis on bleomycin-induced pulmonary fibrosis in mice: Impact of antioxidant, anti-inflammatory and anti-fibrotic properties via TGF-β1/Smad-3 pathway and miRNA-326

Idiopathic pulmonary fibrosis (IPF) is an irreversible disease which considered the most fatal pulmonary fibrosis. Pulmonary toxicity including IPF is the most severe adverse effect of bleomycin, the chemotherapeutic agent. Based on the fact that, exogenous surfactants could induce alveolar stabilization in many lung diseases, the aim of this study was to explore the effects of low cost biosurfactants, surfactin (SUR) and sophorolipids (SLs), against bleomycin-induced pulmonary fibrosis in mice due to their antioxidant, and anti-inflammatory properties. Surfactin and sophorolipids were produced by microbial conversion of frying oil and potato peel wastes using Bacillus halotolerans and Candida parapsilosis respectively. These biosurfactants were identified by FTIR, 1H NMR, and LC-MS/MS spectra. C57BL/6 mice were administered the produced biosurfactants daily at oral dose of 200 mg kg−1 one day after the first bleomycin dose (35 U/kg). We evaluated four study groups: Control, Bleomycin, Bleomycin+SUR, Bleomycin+SLs. After 30 days, lungs from each mouse were sampled for oxidative stress, ELISA, Western blot, histopathological, immunohistochemical analyses. Our results showed that the produced SUR and SLs reduced pulmonary oxidative stress and inflammatory response in the lungs of bleomycin induced mice as they suppressed SOD, CAT, and GST activities also reduced NF-κβ, TNF-α, and CD68 levels. Furthermore, biosurfactants suppressed the expression of TGF-β1, Smad-3, and p-JNK fibrotic signaling pathway in pulmonary tissues. Histologically, SUR and SLs protected against lung ECM deposition caused by bleomycin administration. Biosurfactants produced from microbial sources can inhibit the induced inflammatory and fibrotic responses in bleomycin-induced pulmonary fibrosis.

Local, Quantitative Morphometry of Fibroproliferative Lung Injury Using Laminin.

Investigations into the mechanisms of injury and repair in fibroproliferative disease require consideration of the spatial heterogeneity inherent in the disease. Most scoring of fibrotic remodeling in preclinical animal models relies on the modified Ashcroft score, which is an ordinal rubric of macroscopic resolution. The obvious limitations of manual histopathologic scoring have generated an unmet need for unbiased, repeatable scoring of fibroproliferative burden in tissue. Using computer vision approaches on immunofluorescence imaging of the extracellular matrix component laminin, we generated a robust and repeatable quantitative remodeling scorer. In the bleomycin lung injury model, the quantitative remodeling scorer shows significant agreement with the modified Ashcroft scale. This antibody-based approach is easily integrated into larger multiplex immunofluorescence experiments, which we demonstrate by testing the spatial apposition of tertiary lymphoid structures to fibroproliferative tissue, a poorly characterized phenomenon observed in both human interstitial lung diseases and preclinical models of lung fibrosis. The tool reported in this article is available as a stand-alone application that is usable without programming knowledge.

Contrast enhanced longitudinal changes observed in an experimental bleomycin-induced lung fibrosis rat model by radial DCE-MRI at 9.4T.

Identifying biomarkers in fibrotic lung disease is key for early anti-fibrotic intervention. Dynamic contrast-enhanced (DCE) MRI offers valuable perfusion-related insights in fibrosis but adapting human MRI methods to rodents poses challenges. Here, we explored these translational challenges for the inflammatory and fibrotic phase of a bleomycin lung injury model in rats. Eleven male Sprague-Dawley rats received a single intratracheal dose of bleomycin (1000iU), four control rats received saline. Imaging was performed on days 7 and 28 post-induction. Ultra-short echo time imaging was used to image the lung for 7 minutes after which Clariscan was injected intravenously. Lung signal changes were measured for an additional 21 minutes. Images were reconstructed with a sliding-window approach, providing a temporal resolution of 10 seconds per image. After imaging on day 28, animals were euthanized, and lungs were collected for histology. Bleomycin-exposed rats initially exhibited reduced body weight, recovering to control levels after 20 days. Lung volume increased in bleomycin animals from 4.4±0.9 ml in controls to 5.5±0.5 ml and 6.5±1.2 ml on day 7 and 28. DCE-MRI showed no change of initial gradient of relative enhancement in the curves between controls and bleomycin animals on day 7 and 28 post-induction. On day 7, the DCE-MRI washout phase in bleomycin animals had higher signals than the saline group and than observed at a later time point. Lung pixels were binned in 7 enhancement classes. On day 28, the size of low relative enhancement bins almost doubled in volume compared to controls and animals on day 7 post-induction. Histology on day 28 suggests that findings could be explained by changes in lung tissue density due to lung volume increase. Adapting this clinical MRI method to rodents at 9.4T remains a challenge. Future studies may benefit from lower field strength MRI combined with higher temporal resolution DCE-MRI.

Modeling Molecular Pathogenesis of Idiopathic Pulmonary Fibrosis-Associated Lung Cancer in Mice.

This characterization of tumors associated with lung diseases provides new therapeutic targets that may aid in the development of treatment paradigms for lung cancer patients with preexisting pulmonary diseases.

Hodgkin Lymphoma - Mitigating Toxicity While Preserving Cure - Eight Year Data from MIOT International, Chennai, India

Background One of the most treatable cancers, Hodgkin's lymphoma (HL), has a 5-year survival rate of approximately 85%.We still see relapse and refractoriness in a tiny percentage of patients despite the fact that our disease is largely curable, and we also have additional issues including secondary neoplasia and long-term consequences on the heart. Our Hodgkin lymphoma statistics from 2011 to 2019 have been given in this paper. Methodology Between 2011 and 2019, data on patients with newly diagnosed classical HL (CHL) who received treatment were gathered. Analysis was done on the presenting characteristics, IPS risk classification, treatment procedures, treatment response, and 3-year outcome data. Results At presentation, there were 97 patients with newly diagnosed CHL, with a median age of 24 years (range, 11-72). The ratio of men to women was 1.62:1. Mixed cellularity, lymphocyte-rich, and lymphocyte-depleted CHL (LD, CHL) were histological subtypes that were present in 80.40%, 12.40%, and 7.20% of cases, respectively. At presentation, 52% of patients had advanced illness, and 80% had B symptoms. The Hodgkin IPI score was high in 58% of individuals with advanced stages. ABVD was given as the first line of therapy to 92% of patients. According to interim PET-CT scan findings, around 7 advanced stage Hodgkin patients were started with EscBEACOPP and then switched to ABVD prior to 2017. The front line treatment's overall response rate was 94.8%. Complete response rates for early-stage favourable and unfavourable risk groups were 94% and 86%, respectively, whereas they were 81% and 67% for advanced-stage low- and high-risk groups. The median duration of event free survival was 36 months. The 3-year EFS showed a significant difference between the early- and advanced-stage diseases, 93% and 81%, respectively (p 0.04). Relapse was found in eight patients, all of whom had BEAM Autograft post platinum based salvage treatment. Following interim PET negativity, we discontinued Bleomycin according to RATHL protocol. Three of our patients had bleomycin lung injury. Three patients had post-autograft recurrence, one of whom passed away due to a subsequent non-Hodgkin lymphoma. With the Brentuximab Bendamustine regimen, two patients were successfully salvaged and they had Haplo-identical transplant as third line salvage. Conclusions In early and late stage Hodgkin lymphoma, the three year EFS of 93% and 81% is equivalent to most western literature. In light of our little experience with Indian patients, the effect of Brentuximab is overwhelmingly favorable. At our facility, post-Autograft Brentuximab maintenance is being tested. There is no information on how Nivolumab will be useful in our context. Although there is little evidence to draw a conclusion that is statistically significant, frontline ABVD protocol treatment across risk categories and discontinuing Bleomycin after interim PET negative are helpful in our patients to enhance prognosis and decrease toxicity. Reference: Adapted Treatment Guided by Interim PET-CT Scan in Advanced Hodgkin's Lymphoma N Engl J Med 2016; 374:2419-2429 DOI: 10.1056/NEJMoa1510093Mohty, R., Dulery, R., Bazarbachi, A.H. et al. Latest advances in the management of classical Hodgkin lymphoma: the era of novel therapies. Blood Cancer J. 11, 126 (2021). https://doi.org/10.1038/s41408-021-00518-z

Growth hormone-releasing hormone receptor antagonist MIA-602 attenuates cardiopulmonary injury induced by BSL-2 rVSV-SARS-CoV-2 in hACE2 mice.

COVID-19 pneumonia causes acute lung injury and acute respiratory distress syndrome (ALI/ARDS) characterized by early pulmonary endothelial and epithelial injuries with altered pulmonary diffusing capacity and obstructive or restrictive physiology. Growth hormone-releasing hormone receptor (GHRH-R) is expressed in the lung and heart. GHRH-R antagonist, MIA-602, has been reported to modulate immune responses to bleomycin lung injury and inflammation in granulomatous sarcoidosis. We hypothesized that MIA-602 would attenuate rVSV-SARS-CoV-2-induced pulmonary dysfunction and heart injury in a BSL-2 mouse model. Male and female K18-hACE2tg mice were inoculated with SARS-CoV-2/USA-WA1/2020, BSL-2-compliant recombinant VSV-eGFP-SARS-CoV-2-Spike (rVSV-SARS-CoV-2), or PBS, and lung viral load, weight loss, histopathology, and gene expression were compared. K18-hACE2tg mice infected with rVSV-SARS-CoV-2 were treated daily with subcutaneous MIA-602 or vehicle and conscious, unrestrained plethysmography performed on days 0, 3, and 5 (n = 7 to 8). Five days after infection mice were killed, and blood and tissues collected for histopathology and protein/gene expression. Both native SARS-CoV-2 and rVSV-SARS-CoV-2 presented similar patterns of weight loss, infectivity (~60%), and histopathologic changes. Daily treatment with MIA-602 conferred weight recovery, reduced lung perivascular inflammation/pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued altered respiratory rate, increased expiratory parameters (Te, PEF, EEP), and normalized airflow parameters (Penh and Rpef) compared to vehicle, consistent with decreased airway inflammation. RNASeq followed by protein analysis revealed heightened levels of inflammation and end-stage necroptosis markers, including ZBP1 and pMLKL induced by rVSV-SARS-CoV-2, that were normalized by MIA-602 treatment, consistent with an anti-inflammatory and pro-survival mechanism of action in this preclinical model of COVID-19 pneumonia.

Abstract 15709: Antagonist of Growth Hormone-Releasing Hormone Receptor Protects Against Cardiopulmonary Injury Induced by rVSV-SARS-CoV-2-S Virus Through Rag2 Regulation

Background: SARS-CoV-2 (COVID-19) transmits a multi-systemic disease that can lead to acute respiratory distress syndrome. Growth hormone-releasing hormone receptor (GHRH-R) and its splice variant are expressed in murine and human lung and heart. GHRH-R antagonist, MIA-602, has been shown to regulate inflammation in animal models and immune cell responses to bleomycin lung injury. Using a BSL2-compatible recombinant VSV-eGFP-SARS-CoV-2-S virus (rVSV-SARS-CoV-2-S) which mimics native SARS-CoV-2 infection in K18 hACE2tg mice, we tested our hypothesis that MIA-602 attenuates COVID-19-induced cardiopulmonary injury by reducing inflammation. Methods: Male and female K18-hACE2tg mice were infected with SARS-CoV-2/USA-WA1/2020, rVSV-SARS-CoV-2-S, or PBS and lung viral load, weight-loss and histopathology were compared (N=8). Mice infected with rVSV-SARS-CoV-2-S were subject to daily subcutaneous injections of 10 μg MIA-602 or vehicle (control) starting at 24h post-infection. Pulmonary function was measured via whole-body plethysmography on day 0, day 3, and day 5 (n=7). Five days after viral infection mice were sacrificed, and blood and tissues collected for histopathological analyses, H&E staining, RNA and protein work. Heart and lung tissues were used for RNASeq (n=3 per group). T-test or One-way ANOVA-test was used for statistical analysis. Results: SARS-CoV-2 and rVSV-SARS-CoV-2-S presented similar pathology for weight loss, infectivity (~60%) and histopathologic changes. Daily treatment with MIA-602 ameliorated weight loss, reduced lung inflammation, pneumonia and pulmonary dysfunction evidenced by rescued respiratory rate, expiratory parameters, and dysregulated airway parameters (p<.05). MIA-602 normalized the high expression of the inflammatory protein ICAM-1 in heart and lung (p<0.01), and master immune modulator Rag2 in lung (RNA:10-FC; Protein: 2-FC; p<.001). Conclusions: The results indicate a possible role for pulmonary Rag2 in protecting against pulmonary dysfunction and heart/lung inflammation by peptide GHRH-R antagonist MIA-602 in a novel animal model of COVID-19 pneumonia.

Abstract P2192: Growth Hormone-releasing Hormone Receptor Antagonist Attenuates Cardiopulmonary Injury Induced By A Bsl2 Rvsv-sars-cov-2-s Virus Mimic Of Sars-cov-2 Infectivity

Background: COVID-19 causes severe pulmonary injury that can lead to acute respiratory distress syndrome. Growth hormone-releasing hormone receptor (GHRH-R) and its splice variant are expressed in murine and human lung and heart. GHRH-R antagonist, MIA-602, has been shown to modulate cellular immune responses to bleomycin lung injury and decrease inflammation in models of sarcoid granuloma. Using the BSL2-friendly rVSV-SARS-CoV-2-S of K18 hACE2tg mice to mimic native SARS-CoV-2 infection, we tested our hypothesis that MIA-602 attenuates cardiopulmonary injury in this COVID-19 model. Methods: Male and female K18 hACE2tg mice were inoculated with SARS-CoV-2 Washington (WA-1) native strain, recombinant VSV-SARS-CoV-2-Spike virus (rVSV-SARS-CoV-2-S), or PBS and lung viral load, weight loss and histopathology compared between groups (N=5-8). K18 h ACE2 tg mice infected with rVSV-SARS-CoV-2-S were subject to daily subcutaneous injections of 10 μg MIA-602 or vehicle starting at 24h post-infection. Pulmonary function was measured via whole-body plethysmography on day 0, day 3, and day 5 (n=7). Five days after viral infection mice were sacrificed; and blood and tissues collected for histopathological analyses, H&E staining and ICAM-1 immunohistochemistry. T-test or One-way ANOVA-test was used for statistical analysis. Results: Native SARS-CoV-2 and rVSV-SARS-CoV-2-S presented with similar patterns of weight loss, infectivity (~60%) and histopathologic changes. Daily treatment with MIA-602 ameliorated weight loss, reduced lung perivascular inflammation and pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued respiratory rate, increased expiratory parameters (Te, PEF, EEP) and mitigated dysregulated measures of airway obstruction (Penh and Rpef) compared to vehicle. Conclusions: rVSV-SARS-CoV-2-S is an accurate and safe alternative to native SARS-CoV-2 for preclinical studies. Daily treatment with the synthetic peptide GHRH-R antagonist MIA-602 attenuates pulmonary dysfunction and heart inflammation in this new preclinical mouse model of COVID-19 pneumonia. We hypothesize that the molecular mechanism involves anti-inflammatory actions of MIA-602.

Overexpression of fatty acid synthase attenuates bleomycin induced lung fibrosis by restoring mitochondrial dysfunction in mice

Proper lipid metabolism is crucial to maintain alveolar epithelial cell (AEC) function, and excessive AEC death plays a role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The mRNA expression of fatty acid synthase (FASN), a key enzyme in the production of palmitate and other fatty acids, is downregulated in the lungs of IPF patients. However, the precise role of FASN in IPF and its mechanism of action remain unclear. In this study, we showed that FASN expression is significantly reduced in the lungs of IPF patients and bleomycin (BLM)-treated mice. Overexpression of FASN significantly inhibited BLM-induced AEC death, which was significantly potentiated by FASN knockdown. Moreover, FASN overexpression reduced BLM-induced loss of mitochondrial membrane potential and the production of mitochondrial reactive oxygen species (ROS). Oleic acid, a fatty acid component increased by FASN overexpression, inhibited BLM-induced cell death in primary murine AECs and rescue BLM induced mouse lung injury/fibrosis. FASN transgenic mice exposed to BLM exhibited attenuated lung inflammation and collagen deposition compared to controls. Our findings suggest that defects in FASN production may be associated with the pathogenesis of IPF, especially mitochondrial dysfunction, and augmentation of FASN in the lung may have therapeutic potential in preventing lung fibrosis.

Brentuximab vedotin plus doxorubicin and dacarbazine innonbulky limited-stage classical Hodgkin lymphoma.

ABVD (Adriamycin, bleomycin, vinblastine, dacarbazine) with or without radiation has been the standard treatment for limited-stage Hodgkin lymphoma (HL) but carries risks of bleomycin lung injury and radiation toxicity. Brentuximab vedotin (BV) is approved with AVD for stage III-IV HL, but carries increased risks of peripheral neuropathy (PN) and neutropenic fever, likely due to overlapping toxicity between BV and vinblastine. We therefore evaluated BV in combination with AD for 4 or 6 cycles based on interim positron emission tomography response. Thirty-four patients with nonbulky stage I-II HL were enrolled. Risk was early favorable in 53% and unfavorable in 47%. The overall and complete response rates (CRRs) were 100% and 97%, respectively, with a 5-year progression-free survival (PFS) of 91%. No differences in outcome were observed based on stage (I vs II) or risk status (early favorable vs unfavorable). The most common adverse events were nausea (85%), peripheral sensory neuropathy (59%), and fatigue (56%). There were no cases of grade-4 neutropenia or neutropenic fever, and no patient received granulocyte-colony stimulating factor. Most cases of PN were grade 1, and no patient experienced grade ≥3 PN. BV-AD produced a high CRR and durable PFS with most patients requiring 4 cycles of therapy. Compared with BV-AVD, the toxicity profile appeared improved, with predominantly grade 1 reversible PN and no case of grade 4 neutropenia or neutropenic fever. This regimen warrants further study in HL and may serve as a backbone for the addition of novel agents. This trial is registered on clinicaltrials.gov (NCT02505269).

POMB/ACE, a cocktail of 7 drugs to treat intermediate to poor prognosis and high volume germ cell cancers: A large retrospective study.

419 Background: Patients presenting with intermediate and poor risk germ cell tumours tend to be young and fit. Although more than half of patients may hope to be cured, a proportion will die because of their cancer. An aggressive strategy is required to maximise the chance of cure. Alternating high dose chemotherapy regimens have been trialled with varying outcomes. We present real world data demonstrating the efficacy and safety of first-line POMB/ACE (Cisplatin, Vincristine, Methotrexate, Bleomycin alternating with Actinomycin D, Cyclophosphamide, Etoposide) for these patients. Methods: 40 metastatic germ cell patients treated with POMB/ACE were identified at our centre. Patients were classified as being in intermediate or poor prognostic groups based on the International germ cell cancer collaborative group classification (IGCCCG). Treatment was administered every 2 weeks for up to 7 cycles. Response to treatment was determined by radiological imaging and serum tumour markers. Toxicities were evaluated using CTCAE version 5.0. Results: Clinical characteristics were as follows: median age at diagnosis 32 years (range 18-69). 36 patients (90%) were male and 4 patients (10%) were female. 29 (73%) presented with poor risk disease and 11 (27%) with intermediate risk. Patients completed between 1 and 7 cycles of POMB/ACE with the median number of cycles being 5. Data on toxicity severity was collected where available. This is summarised in the table. One grade 4 event was noted. 10 patients experienced grade 3 toxicities. Interestingly no patients were reported to develop bleomycin lung toxicity. This may be due to bleomycin being omitted in patients felt to be at higher risk of lung toxicity (smokers, older patients, pre-existing lung conditions). 30 (75%) patients were progression free following POMB/ACE. 9 (22%) patients went on to have subsequent systemic chemotherapy regimens and high dose stem cell transplant (HDSCT), as they had high volume disease, and persistent marker positive disease. 16 (40%) patients went on to have further surgical interventions, most commonly retroperitoneal lymph node dissection (RPLND), no active malignancy was seen in the resected specimen. At a median follow-up of 43 months, 39 (97.5%) of patients are still alive. Conclusions: POMB/ACE is a safe and effective regimen for patients presenting with intermediate to poor risk germ cell tumours. Patients with high volume disease should be considered for subsequent HDSCT to achieve better outcomes. [Table: see text]

Loss of IGFBP2 mediates alveolar type 2 cell senescence and promotes lung fibrosis

Accumulation of senescent cells contributes to age-related diseases including idiopathic pulmonary fibrosis (IPF). Insulin-like growth factor binding proteins (IGFBPs) regulate many biological processes; however, the functional contributions of IGFBP2 in lung fibrosis remain largely unclear. Here, we report that intranasal delivery of recombinant IGFBP2 protects aged mice from weight loss and demonstrated antifibrotic effects after bleomycin lung injury. Notably, aged human-Igfbp2 transgenic mice reveal reduced senescence and senescent-associated secretory phenotype factors in alveolar epithelial type 2 (AEC2) cells and they ameliorated bleomycin-induced lung fibrosis. Finally, we demonstrate that IGFBP2 expression is significantly suppressed in AEC2 cells isolated from fibrotic lung regions of patients with IPF and/or pulmonary hypertension compared with patients with hypersensitivity pneumonitis and/or chronic obstructive pulmonary disease. Altogether, our study provides insights into how IGFBP2 regulates AEC2-cell-specific senescence and that restoring IGFBP2 levels in fibrotic lungs can prove effective for patients with IPF.

Interim PET-Guided ABVD or ABVD/Escalated BEACOPP for Newly Diagnosed Advanced-Stage Classic Hodgkin Lymphoma (JCOG1305, INNOVATE-HL study)

Introduction Interim positron-emission tomography (PET) can predict clinical outcomes in patients with classic Hodgkin lymphoma (cHL). The objective of this single-arm confirmatory study (JCOG1305, INNOVATE-HL) is to establish interim PET-guided treatment strategy for newly diagnosed advanced-stage cHL. Methods Patients aged 16-60 years with cHL received 2 cycles of ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) as an induction chemotherapy, and then underwent an interim PET scan. Images were centrally reviewed with the use of a 5-point scale for PET findings for further response guided treatments. Patients with negative interim PET findings (defined as score 1 to 3) continued additional 4 cycles of ABVD, whereas those with positive interim PET findings (score 4 or 5) switched to 6 cycles of escalated BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisolone). Involved site radiation therapy (ISRT) was set as a protocol treatment if patients with partial response had a single residual lesion after completion of chemotherapy. The primary endpoint were 2-year progression-free survival (PFS) among all eligible patients and among interim PET-positive patients (co-primary endpoints). We had the following hypotheses: 2-year PFS of eligible patients and interim-PET positive patients will exceed 75% and 35%, respectively, compared to a historical control with one-sided alpha of 0.1. Secondary endpoints included overall survival (OS), event-free survival (EFS), in which events were defined as death, progression or not achieving complete response at end of protocol treatment, and incidences of adverse events and secondary malignancy. This study was registered at jRCTs031180218. Results A total of 93 patients were enrolled from 35 institutions between January 2016 and December 2019. One patient was ineligible because of diagnostic error. Baseline characteristics of 92 eligible patients who underwent an interim PET scan were as follows: a median age of 35 years (interquartile range [IQR]: 28-48); 58% were male; most of patients (87 [95%]) had performance status of 0 or 1; Ann Arbor stage III and IV consisted of 40 (43%) and 43 (47%), respectively, the remaining 9 (10%) had stage IIB with bulky (10 cm or more) mediastinal lesion or nodal lesion involving directly to extranodal lesion; 51 (55%) and 15(16%) had B symptoms and bulky lesions, respectively. An interim PET scan showed 19 patients (21%) with positive findings; those patients received escalated BEACOPP; 18 completed 6 cycles of BEACOPP including one who underwent ISRT, the remaining one discontinued BEACOPP. On the other hand, 73 patients (79%) who had interim PET-negative findings continued ABVD; 70 completed additional 4 cycles of ABVD including two who underwent ISRT, the remaining 3 discontinued ABVD. Average relative dose intensity of induction ABVD, additional ABVD and escalated BEACOPP were 98.6% (±4.0), 96.7% (±5.7) and 82.2% (±9.3%), respectively. With a median follow-up of 41.1 months (IQR: 29.7-53.1), the 2-year PFS were 84.8% (80% confidence interval [CI]: 79.2-88.9) and 84.2% (80% CI: 69.7-92.1) among all 92 eligible patients and 19 interim PET-positive patients, respectively (Figure 1). The 2-year EFS were 76.1% and 68.4% among all eligible patients and interim-PET positive patients, respectively. Bleomycin lung toxicity (BLT) occurred in 9 patients, but no patients led to BLT-related death. Twelve and three patients underwent autologous and allogeneic hematopoietic stem cell transplantation (HSCT) as a salvage treatment, respectively, including one received allogeneic HSCT for relapse after autologous HSCT. The 2-year OS was 98.9%; two patients died due to interstitial pneumonia after autologous HSCT and brain hemorrhage without relapsing cHL among the interim PET-positive patients, whereas no patients died among interim PET-negative patients. Diffuse large B-cell lymphoma developed in one patient with interim PET-negative findings as a secondary malignancy 4.6 years after enrollment. Conclusions This INNOVATE-HL study demonstrated that interim PET-guided treatment strategy was useful for younger patients with newly diagnosed advanced-stage cHL. Of note, 6 cycles of escalated BEACOPP is a feasible and effective regimen for interim PET-positive cHL patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SENP1 regulates the transformation of lung resident mesenchymal stem cells and is associated with idiopathic pulmonary fibrosis progression

BackgroundLung resident mesenchymal stem cells (LR-MSCs) play an important role in idiopathic pulmonary fibrosis (IPF) by transforming into myofibroblasts, thereby losing their repair ability. Evidence suggests that key proteins of multiple signaling pathways are involved in myofibroblast differentiation of LR-MSCs, such as β-Catenin and GLI family zinc finger 1 (GLI1). These proteins are regulated by SUMO (small ubiquitin-like modifier) modification, which is a post-translational modification that promotes protein degradation, while Sumo specific protein 1 (SENP1)-mediated deSUMOylation produces the opposite biological effects. Therefore, we speculated that SENP1 might be a potential target for treating pulmonary fibrosis by preventing the myofibroblast differentiation of LR-MSCs.MethodsLR-MSCs were isolated from mice by using immunomagnetic beads. The extracted LR-MSCs were identified by flow cytometric analysis and multilineage differentiation assays. Lentivirus packaged shRNA silenced the expression of SENP1 in vitro and vivo. The silencing efficacy of SENP1 was verified by real-time quantitative PCR. The effect of down-regulated SENP1 on the myofibroblast differentiation of LR-MSCs was assessed by Immunofluorescence and Western blot. Immunoprecipitation was used to clarify that SENP1 was a key target for regulating the activity of multiple signaling pathways in the direction of LR-MSCs differentiation. LR-MSCs resident in the lung was analyzed with in vivo imaging system. HE and Masson staining was used to evaluate the therapeutic effect of LR-MSCs with SENP1 down-regulation on the lung of BLM mice.ResultsIn this study, we found that the myofibroblast differentiation of LR-MSCs in IPF lung tissue was accompanied by enhanced SENP1-mediated deSUMOylation. The expression of SENP1 increased in LR-MSCs transition of bleomycin (BLM)-induced lung fibrosis. Interfering with expression of SENP1 inhibited the transformation of LR-MSCs into myofibroblasts in vitro and in vivo and restored their therapeutic effect in BLM lung fibrosis. In addition, activation of the WNT/β-Catenin and Hedgehog/GLI signaling pathways depends on SENP1-mediated deSUMOylation.ConclusionsSENP1 might be a potential target to restore the repair function of LR-MSCs and treat pulmonary fibrosis.8mnU6TCL-hGPayjm_r7Fe6Video

Profibrotic Role of Inducible Heat Shock Protein 90α Isoform in Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease that affects skin and multiple internal organs. TGF-β, a central trigger of cutaneous fibrosis, activates fibroblasts with the involvement of the stress-inducible chaperone heat shock protein 90 isoform α (Hsp90α). Available evidence supports overexpression and secretion of Hsp90α as a feature in profibrotic pathological conditions. The aim of this work is to investigate the expression and function of Hsp90α in experimental models of skin fibrosis such as human fibroblasts, C57BL/6 mice, and in human SSc. For this purpose, we generated a new experimental model based on doxorubicin administration with improved characteristics with respect to the bleomycin model. We visualized disease progression in vivo by fluorescence imaging. In this work, we obtained Hsp90α mRNA overexpression in human skin fibroblasts, in bleomycin- and doxorubicin-induced mouse fibrotic skin, and in lungs of bleomycin- and doxorubicin-treated mice. Hsp90α-deficient mice showed significantly decreased skin thickness compared with wild-type mice in both animal models. In SSc patients, serum Hsp90α levels were increased in patients with lung involvement and in patients with the diffuse form of SSc (dSSc) compared with patients with the limited form of SSc. The serum Hsp90α levels of patients dSSc were correlated with the Rodnan score and the forced vital capacity variable. These results provide new supportive evidence of the contribution of the Hsp90α isoform in the development of skin fibrosis. In SSc, these results indicated that higher serum levels were associated with dSSc and lung fibrosis.

Aberrant Multiciliogenesis in Idiopathic Pulmonary Fibrosis.

We previously identified a novel molecular subtype of idiopathic pulmonary fibrosis (IPF) defined by increased expression of cilium-associated genes, airway mucin gene MUC5B, and KRT5 marker of basal cell airway progenitors. Here we show the association of MUC5B and cilia gene expression in human IPF airway epithelial cells, providing further rationale for examining the role of cilium genes in the pathogenesis of IPF. We demonstrate increased multiciliogenesis and changes in motile cilia structure of multiciliated cells both in IPF and bleomycin lung fibrosis models. Importantly, conditional deletion of a cilium gene, Ift88 (intraflagellar transport 88), in Krt5 basal cells reduces Krt5 pod formation and lung fibrosis, whereas no changes are observed in Ift88 conditional deletion in club cell progenitors. Our findings indicate that aberrant injury-activated primary ciliogenesis and Hedgehog signaling may play a causative role in Krt5 pod formation, which leads to aberrant multiciliogenesis and lung fibrosis. This implies that modulating cilium gene expression in Krt5 cell progenitors is a potential therapeutic target for IPF.

Inhibition of DNA methylation de‐represses peroxisome proliferator‐activated receptor‐γ and attenuates pulmonary fibrosis

Development of pulmonary fibrosis is associated with altered DNA methylation modifications of fibrogenic gene expression. However, their causal relationships and the underlying mechanisms remain unclear. This study investigates the critical role of DNA methylation aberration-associated suppression of peroxisome proliferator-activated receptor-γ (PPARγ) in pulmonary fibrosis. Expression of PPARγ and bioactive DNA methyltransferases (DNMTs) and PPARγ promoter methylation status were examined in fibrotic lungs of idiopathic pulmonary fibrosis (IPF) patients and bleomycin (Blm)-treated mice. DNA demethylating agent 5-aza-2'-deoxycytidine (5aza) and glycyrrhizic acid (GA) derived from medicinal plant were assessed for their PPARγ de-repression and anti-pulmonary fibrosis activities. PPARγ knockout mice were created to determine the critical role of PPARγ in this protection. Lung PPARγ expression was markedly suppressed in IPF patients and Blm mice, accompanied by increased DNMT 1/DNMT3a and PPARγ promoter hypermethylation. Administration of 5-aza and GA similarly demethylated PPARγ promoter, restored PPARγ loss and alleviated fibrotic lung pathologies, including structural alterations and adverse expression of fibrotic mediators and inflammatory cytokines. In cultured lung fibroblasts and alveolar epithelial cells, GA alleviated PPARγ-mediated suppression of fibrosis in a gain of DNMT-sensitive manner, and in PPARγ knockout mice, the anti-fibrotic effects of 5aza and GA were significantly reduced, suggesting that PPARγ is a critical mediator of epigenetic pulmonary fibrogenesis. Aberrant DNMT1/3a elevations and the resultant PPARγ suppression contribute significantly to the development of pulmonary fibrosis, and strategies targeting DNMT/PPARγ axis with synthetic or natural compounds might benefit patients with pulmonary fibrotic disorders.

Azithromycin Attenuates Bleomycin-Induced Pulmonary Fibrosis Partly by Inhibiting the Expression of LOX and LOXL-2.

Pulmonary fibrosis (PF) is a chronic and progressive process of tissue repair. Azithromycin (AZM) may be beneficial for the treatment of PF because AZM has anti-inflammatory and immune regulatory roles and inhibits remodeling, but the mechanism is not entirely clear. In this study, we established a mouse PF model induced by bleomycin (BLM) and primary mouse lung fibroblasts stimulated by transforming growth factor (TGF)-β1 to explore the possible mechanisms of AZM in PF. Results showed that AZM reduces mortality and lung inflammation and attenuates BLM-induced PF in mice. AZM effectively reduced the expression of α-smooth muscle actin (SMA) and type I collagen. Meanwhile, expression of lysyl oxidase (LOX) and lysyl oxidase-like protein (LOXL)-2 in the lung tissue of mice after AZM treatment was significantly lower than in the BLM group. In addition, this study found that AZM significantly inhibits the TGF-β1/Smad and JNK/c-Jun signaling pathways in vivo, and expression of a-SMA, type I collagen, LOX and LOXL-2 in the lung tissue of mice treated with AZM was significantly lower than that in the BLM group. In vitro, AZM also effectively inhibited type I collagen, LOX, LOXL-2 and JNK-c-Jun signaling pathways in TGF-β1-stimulated primary mouse fibroblasts, and this effect was similar to that of a JNK-specific inhibitor (SP600125). In conclusion, AZM effectively attenuated BLM-induced PF in mice, which may play a role by partially inhibiting the JNK/c-Jun and TGF-β1/Smad signaling pathways and reducing production of LOX and LOXL2.

Selective Myeloid Depletion of Galectin-3 Offers Protection Against Acute and Chronic Lung Injury.

Rationale: Galectin-3 (Gal-3) is an immune regulator and an important driver of fibrosis in chronic lung injury, however, its role in acute lung injury (ALI) remains unknown. Previous work has shown that global deletion of galectin-3 reduces collagen deposition in a bleomycin-induced pulmonary fibrosis model (MacKinnon et al., Am. J. Respir. Crit. Care Med., 2012, 185, 537–46). An inhaled Gal-3 inhibitor, GB0139, is undergoing Phase II clinical development for idiopathic pulmonary fibrosis (IPF). This work aims to elucidate the role of Gal-3 in the myeloid and mesenchymal compartment on the development of acute and chronic lung injury.Methods:LgalS3fl/fl mice were generated and crossed with mice expressing the myeloid (LysM) and mesenchymal (Pdgfrb) cre drivers to yield LysM-cre +/- /LgalS3 fl/fl and Pdgfrb-cre +/- /LgalS3 fl/fl mice. The response to acute (bleomycin or LPS) or chronic (bleomycin) lung injury was compared to globally deficient Gal-3 −/− mice.Results: Myeloid depletion of Gal-3 led to a significant reduction in Gal-3 expression in alveolar macrophages and neutrophils and a reduction in neutrophil recruitment into the interstitium but not into the alveolar space. The reduction in interstitial neutrophils corelated with decreased levels of pulmonary inflammation following acute bleomycin and LPS administration. In addition, myeloid deletion decreased Gal-3 levels in bronchoalveolar lavage (BAL) and reduced lung fibrosis induced by chronic bleomycin. In contrast, no differences in BAL Gal-3 levels or fibrosis were observed in Pdgfrb-cre +/- /LgalS3 fl/fl mice. Conclusions: Myeloid cell derived Galectin-3 drives acute and chronic lung inflammation and supports direct targeting of galectin-3 as an attractive new therapy for lung inflammation.