Quantitative Evaluation of Changes in Three-Dimensional CT Density Distributions in Pulmonary Alveolar Proteinosis after GM-CSF Inhalation.

BACKGROUND: A previous clinical trial for autoimmune pulmonary alveolar proteinosis (APAP) demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) inhalation reduced the mean density of the lung field on computed tomography (CT) across 18 axial slice planes at a two-dimensional level. In contrast, in this study, we challenged three-dimensional analysis for changes in CT density distribution using the same datasets. METHODS: As a sub-study of the trial, CT data of 31 and 27 patients who received GM-CSF and placebo, respectively, were analyzed. To overcome the difference between various shooting conditions, a newly developed automatic lung field segmentation algorithm was applied to CT data to extract the whole lung volume, and the accuracy of the segmentation was evaluated by five pulmonary physicians independently. For normalization, the percent pixel (PP) in a certain density range was calculated as a percentage of the total number of pixels from -1,000 to 0 HU. RESULTS: The automatically segmented images revealed that the lung field was accurately extracted except for 7 patients with minor deletion or addition. Using the change in PP from baseline to week 25 (ΔPP) as the vertical axis, we created a histogram with 143 HU bins set for each patient. The most significant difference in ΔPP between GM-CSF and placebo groups was observed in two ranges: from -1,000 to -857 and -143 to 0 HU. CONCLUSION: Whole lung extraction followed by density histogram analysis of ΔPP may be an appropriate evaluation method for assessing CT improvement in APAP.

Metabolic and Epigenetic Regulation of SMAD7 by STC1 Ameliorates Lung Fibrosis.

As shown in our previous studies, the intratracheal-administration of STC1 (stanniocalcin-1) ameliorates pulmonary fibrosis by reducing oxidative and endoplasmic reticulum stress through the uncoupling of respiration in a bleomycin-treated mouse model. However, the overall effect of STC1 on metabolism was not examined. Therefore, we first conducted a comprehensive metabolomics analysis to screen the overall metabolic changes induced by STC1 in an alveolar epithelial cell line using capillary electrophoresis time-of-flight mass spectrometry. The results were subsequently validated in multiple alveolar epithelial and fibroblast cell lines by performing precise analyses of each substance. STC1 stimulated glycolysis, acetyl-CoA synthesis, and the methionine and cysteine-glutathione pathways, which are closely related to the uncoupling of respiration, modulation of epigenetics, and reduction in oxidative stress. These results are consistent with our previous study. Subsequently, we focused on the inhibitory factor SMAD7, which exerts an antifibrotic effect and is susceptible to epigenetic regulation. STC1 upregulates SMAD7 in an uncoupling protein 2-dependent manner, induces demethylation of the SMAD7 promoter region and acetylation of the SMAD7 protein in human alveolar epithelial and fibroblast cell lines and a bleomycin-treated mouse model, and subsequently attenuates fibrosis. The antifibrotic effects of STC1 may partially depend on the regulation of SMAD7. In the evaluation using lung tissue from patients with idiopathic pulmonary fibrosis, SMAD7 expression and acetylation were high in the alveolar structure-preserving region and low in the fibrotic region. The intratracheal administration of STC1 may prevent the development of pulmonary fibrosis by regulating the metabolism-mediated epigenetic modification of SMAD7 in patients.

Inhaled GM-CSF for Pulmonary Alveolar Proteinosis.

BACKGROUND: Pulmonary alveolar proteinosis is a disease characterized by abnormal accumulation of surfactant in the alveoli. Most cases are autoimmune and are associated with an autoantibody against granulocyte-macrophage colony-stimulating factor (GM-CSF) that prevents clearing of pulmonary surfactant by alveolar macrophages. An open-label, phase 2 study showed some therapeutic efficacy of inhaled recombinant human GM-CSF in patients with severe pulmonary alveolar proteinosis; however, the efficacy in patients with mild-to-moderate disease remains unclear. METHODS: We conducted a double-blind, placebo-controlled trial of daily inhaled recombinant human GM-CSF (sargramostim), at a dose of 125 µg twice daily for 7 days, every other week for 24 weeks, or placebo in 64 patients with autoimmune pulmonary alveolar proteinosis who had a partial pressure of arterial oxygen (Pao2) while breathing ambient air of less than 70 mm Hg (or <75 mm Hg in symptomatic patients). Patients with severe pulmonary alveolar proteinosis (Pao2 <50 mm Hg) were excluded to avoid possible exacerbation of the disease in patients who were assigned to receive placebo. The primary end point was the change in the alveolar-arterial oxygen gradient between baseline and week 25. RESULTS: The change in the mean (±SD) alveolar-arterial oxygen gradient was significantly better in the GM-CSF group (33 patients) than in the placebo group (30 patients) (mean change from baseline, -4.50±9.03 mm Hg vs. 0.17±10.50 mm Hg; P = 0.02). The change between baseline and week 25 in the density of the lung field on computed tomography was also better in the GM-CSF group (between-group difference, -36.08 Hounsfield units; 95% confidence interval, -61.58 to -6.99, calculated with the use of the Mann-Whitney U test and the Hodges-Lehmann estimate of confidence intervals for pseudo-medians). Serious adverse events developed in 6 patients in the GM-CSF group and in 3 patients in the placebo group. CONCLUSIONS: In this randomized, controlled trial, inhaled recombinant human GM-CSF was associated with a modest salutary effect on the laboratory outcome of arterial oxygen tension, and no clinical benefits were noted. (Funded by the Japan Agency for Medical Research and Development and the Ministry of Health, Labor, and Welfare of Japan; PAGE ClinicalTrials.gov number, NCT02835742; Japan Medical Association Center for Clinical Trials number, JMA-IIA00205.).

Plasma Globotriaosylsphingosine and α-Galactosidase A Activity as a Combined Screening Biomarker for Fabry Disease in a Large Japanese Cohort.

Fabry disease is an X-linked disorder of α-galactosidase A (GLA) deficiency. Our previous interim analysis (1 July 2014 to 31 December 2015) revealed plasma globotriaosylsphingosine as a promising primary screening biomarker for Fabry disease probands. Herein, we report the final results, including patients enrolled from 1 January to 31 December 2016 for evaluating the potential of plasma globotriaosylsphingosine and GLA activity as a combined screening marker. We screened 5691 patients (3439 males) referred from 237 Japanese specialty clinics based on clinical findings suggestive of Fabry disease using plasma globotriaosylsphingosine and GLA activity as primary screening markers, and GLA variant status as a secondary screening marker. Of the 14 males who tested positive in the globotriaosylsphingosine screen (≥2.0 ng/mL), 11 with low GLA activity (<4.0 nmol/h/mL) displayed GLA variants (four classic, seven late-onset) and one with normal GLA activity and no pathogenic variant displayed lamellar bodies in affected organs, indicating late-onset biopsy-proven Fabry disease. Of the 19 females who tested positive in the globotriaosylsphingosine screen, eight with low GLA activity displayed GLA variants (six classic, two late-onset) and five with normal GLA activity displayed a GLA variant (one classic) and no pathogenic variant (four late-onset biopsy-proven). The combination of plasma globotriaosylsphingosine and GLA activity can be a primary screening biomarker for classic, late-onset, and late-onset biopsy-proven Fabry disease probands.

Effectiveness of plasma lyso-Gb3 as a biomarker for selecting high-risk patients with Fabry disease from multispecialty clinics for genetic analysis.

PURPOSE: Plasma globotriaosylsphingosine (lyso-Gb3) is a promising secondary screening biomarker for Fabry disease. Here, we examined its applicability as a primary screening biomarker for classic and late-onset Fabry disease in males and females. METHODS: Between 1 July 2014 and 31 December 2015, we screened 2,359 patients (1,324 males) referred from 168 Japanese specialty clinics (cardiology, nephrology, neurology, and pediatrics), based on clinical symptoms suggestive of Fabry disease. We used the plasma lyso-Gb3 concentration, α-galactosidase A (α-Gal A) activity, and analysis of the α-Gal A gene (GLA) for primary and secondary screens, respectively. RESULTS: Of 8 males with elevated lyso-Gb3 levels (≥2.0 ng ml-1) and low α-Gal A activity (≤4.0 nmol h-1 ml-1), 7 presented a GLA mutation (2 classic and 5 late-onset). Of 14 females with elevated lyso-Gb3, 7 displayed low α-Gal A activity (5 with GLA mutations; 4 classic and 1 late-onset) and 7 exhibited normal α-Gal A activity (1 with a classic GLA mutation and 3 with genetic variants of uncertain significance). CONCLUSION: Plasma lyso-Gb3 is a potential primary screening biomarker for classic and late-onset Fabry disease probands.

Correction: Effectiveness of plasma lyso-Gb3 as a biomarker for selecting high-risk patients with Fabry disease from multispecialty clinics for genetic analysis.

The PDF and HTML versions of the article have been updated to include the Creative Commons Attribution 4.0 International License information.

Correction: Effectiveness of plasma lyso-Gb3 as a biomarker for selecting high-risk patients with Fabry disease from multispecialty clinics for genetic analysis.

In the above article, we noticed that one female patient in the positive group (plasma lyso-Gb3 7.6 ng/ml, α-galactosidase A activity 4.9 nmol/h/ml) who presented at the neurology clinic was already diagnosed with Fabry disease before the current study. We excluded patients with a confirmed diagnosis of Fabry disease and those with relatives known to have Fabry disease. To accurately describe the information in the current study, we must exclude this patient from the analysis. We have accurately revised this information as follows.

Correction: Effectiveness of plasma lyso-Gb3 as a biomarker for selecting high-risk patients with Fabry disease from multispecialty clinics for genetic analysis.

The HTML version of this Article contained errors in Supplementary Figure S2 "Flowchart of the lyso-Gb3 screening and gene analysis in female patients", which have been detailed in the associated Correction.

Risk factors for stomatitis in patients with lymphangioleiomyomatosis during treatment with sirolimus: A multicenter investigator-initiated prospective study.

PURPOSE: Lymphangioleiomyomatosis is a rare lung disease caused by proliferation of abnormal smooth muscle-like cells and typically occurs in premenopausal women. Sirolimus is now the first-line drug for the treatment of lymphangioleiomyomatosis. Sirolimus-induced stomatitis is the most frequent adverse event experienced during treatment. To identify risk factors, we investigated the association of stomatitis incidence with patient background data and treatment parameters, using data from the multicenter long-term sirolimus trial. METHODS: Subjects received sirolimus for 2 years at doses adjusted to maintain a trough blood level of 5 to 15 ng/mL. The incidence of stomatitis was correlated with baseline demographics, clinical characteristics, and changes in the longitudinal data. Risk factors at baseline were assessed by using univariate and multivariate analyses. RESULTS: The most frequent adverse event was stomatitis, with the cumulative rate reaching 88.9% by 9 months, higher than that reported in postrenal transplant patients. The repetition, the duration, and the severity of stomatitis events were variable among patients. We found that patients with low hemoglobin (Hb) (<14.5 g/dL) showed significantly higher incidence than those with high Hb (≥14.5 g/dL, P < .01). The cumulative rate for stomatitis incidence was significantly associated with a decrease in the mean corpuscular volume, while the Hb level was constant; thus, red blood cell count in patients increased during the study. CONCLUSIONS: Baseline Hb levels and a decrease in mean corpuscular volume during treatment were correlated with the incidence of stomatitis.

Elderly-onset hereditary pulmonary alveolar proteinosis and its cytokine profile.

BACKGROUND: Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by surfactant accumulation, and is caused by disruption of granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. Abnormalities in CSF2 receptor alpha (CSF2RA) were reported to cause pediatric hereditary PAP. We report here the first case of CSF2RA-mutated, elderly-onset hereditary (h) PAP. CASE PRESENTATION: The patient developed dyspnea on exertion, and was diagnosed with PAP at the age of 77 years, based on findings from chest computed tomography scan and bronchoalveolar lavage. She tested negative for GM-CSF autoantibodies, with no underlying disease. Her serum GM-CSF level was elevated (91.3 pg/mL), indicating GM-CSF signaling impairment and genetic defects in the GM-CSF receptor. GM-CSF-stimulated phosphorylation in signal transducer and activator of transcription 5 (STAT5) was not observed, and GM-CSF-Rα expression was defective in her blood cells. Genetic screening revealed a homozygous, single-base C > T mutation at nt 508-a nonsense mutation that yields a stop codon (Q170X)-in exon 7 of CSF2RA. High-resolution analysis of single nucleotide polymorphism array confirmed a 22.8-Mb loss of heterozygosity region in Xp22.33p22.11, encompassing the CSF2RA gene. She was successfully treated with whole lung lavage (WLL), which reduced the serum levels of interleukin (IL)-2, IL-5, and IL-17, although IL-3 and M-CSF levels remained high. CONCLUSIONS: This is the first known report of elderly-onset hPAP associated with a CSF2RA mutation, which caused defective GM-CSF-Rα expression and impaired signaling. The analyses of serum cytokine levels during WLL suggested that GM-CSF signaling might be compensated by other signaling pathways, leading to elderly-onset PAP.

A mathematical model to predict protein wash out kinetics during whole-lung lavage in autoimmune pulmonary alveolar proteinosis.

Whole-lung lavage (WLL) remains the standard therapy for pulmonary alveolar proteinosis (PAP), a process in which accumulated surfactants are washed out of the lung with 0.5-2.0 l of saline aliquots for 10-30 wash cycles. The method has been established empirically. In contrast, the kinetics of protein transfer into the lavage fluid has not been fully evaluated either theoretically or practically. Seventeen lungs from patients with autoimmune PAP underwent WLL. We made accurate timetables for each stage of WLL, namely, instilling, retaining, draining, and preparing. Subsequently, we measured the volumes of both instilled saline and drained lavage fluid, as well as the concentrations of proteins in the drained lavage fluid. We also proposed a mathematical model of protein transfer into the lavage fluid in which time is a single variable as the protein moves in response to the simple diffusion. The measured concentrations of IgG, transferrin, albumin, and ß2-microglobulin closely matched the corresponding theoretical values calculated through differential equations. Coefficients for transfer of ß2-microglobulin from the blood to the lavage fluid were two orders of magnitude higher than those of IgG, transferrin, and albumin. Simulations using the mathematical model showed that the cumulative amount of eliminated protein was not affected by the duration of each cycle but dependent mostly on the total time of lavage and partially on the volume instilled. Although physicians have paid little attention to the transfer of substances from the lung to lavage fluid, WLL seems to be a procedure that follows a diffusion-based mathematical model.

Outcome of corticosteroid administration in autoimmune pulmonary alveolar proteinosis: a retrospective cohort study.

BACKGROUND: Although no report has demonstrated the efficacy of corticosteroid therapy for autoimmune pulmonary alveolar proteinosis (aPAP), we sometimes encounter patients who have received this therapy for various reasons. However, as corticosteroids can suppress alveolar macrophage function, corticosteroid therapy might worsen disease severity and increase the risk of infections. METHODS: For this retrospective cohort study, we sent a screening form to 165 institutions asking for information on aPAP patients treated with corticosteroids. Of the resulting 45 patients screened, 31 were enrolled in this study. We collected demographic data and information about corticosteroid treatment period, dose, disease severity score (DSS) over the treatment period, and complications. RESULTS: DSS deteriorated during corticosteroid therapy in 23 cases (74.1 %) and the estimated overall cumulative worsening rate was 80.8 % for the total observation period. The worsening rate was significantly higher in patients treated with high-dose prednisolone (>18.9 mg/day, n = 16) than treated with low-dose prednisolone (≤18.9 mg/day, n = 15) divided by median daily dose (p < 0.02). Of patients with worsening, one died of disseminated aspergillosis and another of respiratory failure. Infections newly emerged in 6 cases during corticosteroid therapy (p < 0.05). Median serum granulocyte/macrophage colony-stimulating factor (GM-CSF) autoantibody levels were similar to previously reported data in a large cohort study. CONCLUSION: The results demonstrate that corticosteroid therapy may worsen DSS of aPAP, increasing the risk for infections.

Low concentrations of recombinant granulocyte macrophage-colony stimulating factor derived from Chinese hamster ovary cells augments long-term bioactivity with delayed clearance in vitro.

To date, the biological activity of granulocyte macrophage-colony stimulating factor (GM-CSF) has been investigated by using mostly Escherichia coli- or yeast cell-derived recombinant human GM-CSF (erhGM-CSF and yrhGM-CSF, respectively). However, Chinese hamster ovary cell-derived recombinant human GM-CSF (crhGM-CSF), as well as natural human GM-CSF, is a distinct molecule that includes modifications by complicated oligosaccharide moieties. In the present study, we reevaluated the bioactivity of crhGM-CSF by comparing it with those of erhGM-CSF and yrhGM-CSF. The effect of short-term stimulation (0.5h) on the activation of neutrophils/monocytes or peripheral blood mononuclear cells (PBMCs) by crhGM-CSF was lower than those with erhGM-CSF or yrhGM-CSF at low concentrations (under 60pM). Intermediate-term stimulation (24h) among the different rhGM-CSFs with respect to its effect on the activation of TF-1 cells, a GM-CSF-dependent cell line, or PBMCs was not significantly different. In contrast, the proliferation/survival of TF-1 cells or PBMCs after long-term stimulation (72-168h) was higher at low concentrations of crhGM-CSF (15-30pM) than that of cells treated with other GM-CSFs. The proportion of apoptotic TF-1 cells after incubation with crhGM-CSF for 72h was lower than that of cells incubated with other rhGM-CSFs. These effects were attenuated by desialylation of crhGM-CSF. Clearance of crhGM-CSF but not desialylated-crhGM-CSF by both TF-1 cells and PBMCs was delayed compared with that of erhGM-CSF or yrhGM-CSF. These results suggest that sialylation of oligosaccharide moieties delayed the clearance of GM-CSF, thus eliciting increased long-term bioactivity in vitro.

Runx1 deficiency in CD4+ T cells causes fatal autoimmune inflammatory lung disease due to spontaneous hyperactivation of cells.

The Runx1 transcription factor is abundantly expressed in naive T cells but rapidly downregulated in activated T cells, suggesting that it plays an important role in a naive stage. In the current study, Runx1(-/-)Bcl2(tg) mice harboring Runx1-deleted CD4(+) T cells developed a fatal autoimmune lung disease. CD4(+) T cells from these mice were spontaneously activated, preferentially homed to the lung, and expressed various cytokines, including IL-17 and IL-21. Among these, the deregulation of IL-21 transcription was likely to be associated with Runx binding sites located in an IL-21 intron. IL-17 produced in Runx1-deleted cells mobilized innate immune responses, such as those promoted by neutrophils and monocytes, whereas IL-21 triggered humoral responses, such as plasma cells. Thus, at an initial stage, peribronchovascular regions in the lung were infiltrated by CD4(+) lymphocytes, whereas at a terminal stage, interstitial regions were massively occupied by immune cells, and alveolar spaces were filled with granular exudates that resembled pulmonary alveolar proteinosis in humans. Mice suffered from respiratory failure, as well as systemic inflammatory responses. Our data indicate that Runx1 plays an essential role in repressing the transcription of cytokine genes in naive CD4(+) T cells and, thereby, maintains cell quiescence.

Secondary pulmonary alveolar proteinosis complicating myelodysplastic syndrome results in worsening of prognosis: a retrospective cohort study in Japan.

BACKGROUND: Secondary pulmonary alveolar proteinosis (sPAP) is a very rare lung disorder comprising approximately 10% of cases of acquired PAP. Hematological disorders are the most common underlying conditions of sPAP, of which 74% of cases demonstrate myelodysplastic syndrome (MDS). However, the impact of sPAP on the prognosis of underlying MDS remains unknown. The purpose of this study was to evaluate whether development of sPAP worsens the prognosis of MDS. METHODS: Thirty-one cases of sPAP and underlying MDS were retrospectively classified into mild and severe cases consisting of very low-/low-risk groups and intermediate-/high-/very high-risk groups at the time of diagnosis of MDS, according to the prognostic scoring system based on the World Health Organization classification. Next, we compared the characteristics, disease duration, cumulative survival, and prognostic factors of the groups. RESULTS: In contrast to previous reports on the prognosis of MDS, we found that the cumulative survival probability for mild MDS patients was similar to that in severe MDS patients. This is likely due to the poor prognosis of patients with mild MDS, whose 2-year survival rate was 46.2%. Notably, 75% and 62.5% of patients who died developed fatal infectious diseases and exacerbation of PAP, respectively, suggesting that the progression of PAP per se and/or PAP-associated infection contributed to poor prognosis. The use of corticosteroid therapy and a diffusing capacity of the lung for carbon monoxide of less than 44% were predictive of poor prognosis. CONCLUSION: Development of sPAP during the course of MDS may be an important adverse risk factor in prognosis of patients with mild MDS.

Light chain (κ/λ) ratio of GM-CSF autoantibodies is associated with disease severity in autoimmune pulmonary alveolar proteinosis.

Previous studies demonstrated that antigranulocyte colony-stimulating factor autoantibody (GMAb) was consistently present in patients with autoimmune pulmonary alveolar proteinosis (aPAP), and, thus, represented candidature as a reliable diagnostic marker. However, our large cohort study suggested that the concentration of this antibody was not correlated with disease severity in patients. We found that the κ/λ ratio of GMAb was significantly correlated with the degree of hypoxemia. The proportion of λ-type GMAb per total λ-type IgG was significantly higher in severely affected patients than those in mildly affected patients, but the proportion of κ-type was unchanged. The κ/λ ratio was significantly correlated with both KL-6 and SP-D, which have been previously reported as disease severity markers. Thus, the light chain isotype usage of GMAb may not only be associated with the severity of aPAP, but may also represent a useful disease severity marker.

Short-term inhalation of sargramostim with concomitant high-dose steroids does not hasten recovery in moderate COVID-19 pneumonia: a double-blind, randomised, placebo-controlled trial.

BACKGROUND: Granulocyte-macrophage colony stimulating factor (GM-CSF) inhalation may alleviate pulmonary inflammation caused by viral pneumonia. To investigate this, we evaluated its efficacy on COVID-19 pneumonia. METHODS: This double-blind, randomised, placebo-controlled study (ClinicalTrials.gov: NCT04642950) evaluated patients in the first half of 2021 at seven Japanese hospitals. Hospitalised patients with COVID-19 pneumonia with moderate hypoxaemia inhaled sargramostim or placebo for 5 days. The primary endpoint was days to achieve a ≥ 2-category improvement from baseline on a modified 7-category ordinal scale. Secondary endpoints included degree of oxygenation, defined by amount of oxygen supply, and serum CCL17 level. RESULTS: Seventy-five patients were randomly assigned in a 2:1 ratio to receive sargramostim or placebo, of which 47 and 23 were analysed, respectively. No difference was observed between groups regarding the primary endpoint (8.0 and 7.0 days for sargramostim and placebo, respectively) or in the secondary endpoints, except for CCL17. A post hoc sub-analysis indicated that endpoint assessments were influenced by concomitant corticosteroid therapy. When the cumulative corticosteroid dose was ≤500 mg during Days 1-5, recovery and oxygenation were faster in the sargramostim group than for placebo. Bolus dose corticosteroids were associated with temporarily impaired oxygenation and delayed clinical recovery. The increase in serum CCL17, a candidate prognostic factor, reflected improvement with sargramostim inhalation. The number of adverse events was similar between groups. Two serious adverse events were observed in the sargramostim group without causal relation. CONCLUSIONS: Inhaled sargramostim was likely to be effective for COVID-19 pneumonia unless the concomitant corticosteroid dose was high.

COVID-19 in patients with pulmonary alveolar proteinosis: a European multicentre study.

Adult PAP patients experience similar #COVID19 rates to the general population, and high rates of hospitalisation and deaths, underscoring their vulnerability and the need for measures to prevent infection. The impact of iGM-CSF must be considered. https://bit.ly/3M0wKnZ.