An intronic variant in TBX4 in a single family with variable and severe pulmonary manifestations.

A male infant presented at term with neonatal respiratory failure and pulmonary hypertension. His respiratory symptoms improved initially, but he exhibited a biphasic clinical course, re-presenting at 15 months of age with tachypnea, interstitial lung disease, and progressive pulmonary hypertension. We identified an intronic TBX4 gene variant in close proximity to the canonical donor splice site of exon 3 (hg 19; chr17:59543302; c.401 + 3 A > T), also carried by his father who had a typical TBX4-associated skeletal phenotype and mild pulmonary hypertension, and by his deceased sister who died shortly after birth of acinar dysplasia. Analysis of patient-derived cells demonstrated a significant reduction in TBX4 expression resulting from this intronic variant. Our study illustrates the variable expressivity in cardiopulmonary phenotype conferred by TBX4 mutation and the utility of genetic diagnostics in enabling accurate identification and classification of more subtly affected family members.

The effect of elexacaftor/tezacaftor/ivacaftor (ETI) on glycemia in adults with cystic fibrosis.

BACKGROUND: Cystic fibrosis related diabetes (CFRD) is associated with pulmonary decline and compromised nutritional status. Emerging data suggest that CFTR dysfunction may play a direct role in the pathogenesis of CFRD; however, studies investigating the effect of CFTR modulators on glycemic outcomes in patients with cystic fibrosis (CF) have shown mixed results. The impact of elexacaftor-tezacaftor-ivacaftor (ETI) on glycemic control is currently unknown. Our objective was to investigate the effect of ETI initiation on glycemia in adults with CF using continuous glucose monitoring (CGM). METHODS: In this prospective observational study, 34 adults with CF and at least one F508del CFTR mutation wore CGM sensors for 14 days prior to starting ETI and again 3-12 months after ETI initiation. Hypoglycemia symptoms were queried at each visit, and most recent anthropometric measures and spirometry data were obtained by chart review. RESULTS: Twenty-three participants completed the study. Compared to baseline, average glucose (AG), standard deviation (SD), % time >200 mg/dL, and peak sensor glucose decreased with ETI treatment, and % time in target range 70-180 mg/dL increased. Improvements in glycemic parameters were most notable in individuals with CFRD. There was no significant change in CGM-measured or self-reported hypoglycemia before and after ETI initiation. CONCLUSION: Initiation of ETI in adults with CF was associated with improvement CGM-derived measures of hyperglycemia and glycemic variability with no effect on hypoglycemia. Further studies are needed to investigate underlying etiology of these changes and the long-term impact of ETI on glycemic control in patients with CF.

1-year outcomes for lung transplantation recipients with non-alcoholic fatty liver disease.

Advanced hepatic fibrosis and cirrhosis are absolute contraindications to lung transplantation. [ 1] However, whether fatty liver disease with mild-moderate fibrosis contributes to increased adverse outcomes post-lung transplantation remains unknown. We present a retrospective analysis of patients transplanted at Brigham and Women's Hospital between 2015 and 2017 to identify whether patients with mild-moderate non-alcoholic fatty liver disease (NAFLD) experience increased short-term complications compared to patients with normal liver architecture. Patients with advanced (F3-F4) fibrosis and/or cirrhosis were considered non-suitable transplant candidates, a priori. This study was powered for a difference in index hospital-free days within the first 30 days of 25% (α=0.05, ß=0.8). Secondary outcomes included index intensive care unit (ICU)-free days within the first 10 days post-transplant, perioperative blood product transfusion, incidence of index hospitalisation arrhythmias and delirium, need for insulin on discharge post-transplant, tacrolimus dose required to maintain a trough of 8-12 ng·mL-1 at index hospital discharge, and 1-year post-transplant incidence of insulin-dependent diabetes, acute kidney injury, acute cellular rejection, unplanned hospital readmissions and infection. 150 patients underwent lung transplantation between 2015 and 2017 and were included in the analysis; of these patients 40 (27%) had evidence of NAFLD. Median index hospital-free days for patients with NAFLD were non-inferior to those without (16 days, IQR 10.5-19.5 versus 12 days, IQR 0-18.0, p=0.03). Regarding secondary outcomes, both index hospitalisation and 1-year outcomes were non-inferior between patients with NAFLD and those with normal liver architecture. This study demonstrates that mild-moderate severity NAFLD may not be a contraindication to lung transplantation.

A homozygous stop-gain variant in ARHGAP42 is associated with childhood interstitial lung disease, systemic hypertension, and immunological findings.

ARHGAP42 encodes Rho GTPase activating protein 42 that belongs to a member of the GTPase Regulator Associated with Focal Adhesion Kinase (GRAF) family. ARHGAP42 is involved in blood pressure control by regulating vascular tone. Despite these findings, disorders of human variants in the coding part of ARHGAP42 have not been reported. Here, we describe an 8-year-old girl with childhood interstitial lung disease (chILD), systemic hypertension, and immunological findings who carries a homozygous stop-gain variant (c.469G>T, p.(Glu157Ter)) in the ARHGAP42 gene. The family history is notable for both parents with hypertension. Histopathological examination of the proband lung biopsy showed increased mural smooth muscle in small airways and alveolar septa, and concentric medial hypertrophy in pulmonary arteries. ARHGAP42 stop-gain variant in the proband leads to exon 5 skipping, and reduced ARHGAP42 levels, which was associated with enhanced RhoA and Cdc42 expression. This is the first report linking a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in human patient. Evidence of smooth muscle hypertrophy on lung biopsy and an increase in RhoA/ROCK signaling in patient cells suggests the potential mechanistic link between ARHGAP42 deficiency and the development of chILD disorder.

Alemtuzumab as a Therapy for Chronic Lung Allograft Dysfunction in Lung Transplant Recipients With Short Telomeres.

Alemtuzumab, a monoclonal antibody targeting CD52 that causes lymphocyte apoptosis, is a form of advanced immunosuppression that is currently used as a therapy for refractory acute cellular rejection and chronic lung allograft dysfunction in lung transplant recipients (1-3). Side effects of alemtuzumab include bone marrow suppression, infection, and malignancy. Whether alemtuzumab can be safely used in allograft recipients that have an increased propensity for bone marrow suppression due to telomeropathies is unknown. In a retrospective case series, we report outcomes associated with alemtuzumab in three lung allograft recipients with short telomere lengths, comparing endpoints such as leukopenia, transfusion needs, infection, hospitalization and survival to those of 17 patients without known telomeropathies that received alemtuzumab. We show that the use of alemtuzumab in lung transplant recipients with short telomeres is safe, though is associated with an increased incidence of neutropenia, thrombocytopenia and anemia requiring packed red blood cell transfusions. Alemtuzumab appears to be an acceptable advanced immunosuppressive therapy in patients with telomeropathies, though given the design and scope of this study, the actual clinical effect needs further evaluation in larger trials.

Mesenchymal folliculin is required for alveolar development: implications for cystic lung disease in Birt-Hogg-Dubé syndrome.

BACKGROUND: Pulmonary cysts and spontaneous pneumothorax are presented in most patients with Birt-Hogg-Dubé (BHD) syndrome, which is caused by loss of function mutations in the folliculin (FLCN) gene. The pathogenic mechanisms underlying the cystic lung disease in BHD are poorly understood. METHODS: Mesenchymal Flcn was specifically deleted in mice or in cultured lung mesenchymal progenitor cells using a Cre/loxP approach. Dynamic changes in lung structure, cellular and molecular phenotypes and signalling were measured by histology, immunofluorescence staining and immunoblotting. RESULTS: Deletion of Flcn in mesoderm-derived mesenchymal cells results in significant reduction of postnatal alveolar growth and subsequent alveolar destruction, leading to cystic lesions. Cell proliferation and alveolar myofibroblast differentiation are inhibited in the Flcn knockout lungs, and expression of the extracellular matrix proteins Col3a1 and elastin are downregulated. Signalling pathways including mTORC1, AMP-activated protein kinase, ERK1/2 and Wnt-ß-catenin are differentially affected at different developmental stages. All the above changes have statistical significance (p<0.05). CONCLUSIONS: Mesenchymal Flcn is an essential regulator during alveolar development and maintenance, through multiple cellular and molecular mechanisms. The mesenchymal Flcn knockout mouse model provides the first in vivo disease model that may recapitulate the stages of cyst development in human BHD. These findings elucidate the developmental origins and mechanisms of lung disease in BHD.

New Developments in the Pathogenesis of Pulmonary Cysts in Birt-Hogg-Dubé Syndrome.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder caused by germline loss-of-function mutations in Folliculin gene (FLCN). BHD is characterized by lower lobe-predominant pulmonary cysts with risk of pneumothorax, benign skin tumors (fibrofolliculomas), and renal cell carcinoma, often of an unusual chromophobe/oncocytic hybrid histology. The FLCN protein functions in multiple signaling and metabolic pathways including positive regulation of mechanistic target of rapamycin complex 1 (mTORC1) activity via FLCN's GTPase (GAP) activity for Rag C, positive regulation of Wnt signaling (in mesenchymal cells), and negative regulation of TFE3 nuclear localization. Therefore, FLCN-deficient cells are predicted to have reduced mTORC1 and Wnt activity and enhanced TFE3 activity. Folliculin also has functions in autophagy, mitochondrial biogenesis, cell-cell adhesion, 5' AMP activated protein kinase activity, and other pathways. The specific contributions of these pathways to the lung manifestations of BHD are largely unknown. This review is focused on the pulmonary manifestations of BHD, highlighting selected recent advances in elucidating the cellular functions of FLCN and current hypotheses related to the pathogenesis of cystic lung disease in BHD, including the "stretch hypothesis." We also discuss important knowledge gaps in the field, including the genetic, cellular and physical mechanisms of cyst pathogenesis, and the timing of cyst initiation, which may occur during lung development.

Loss of FLCN inhibits canonical WNT signaling via TFE3.

Lower lobe predominant pulmonary cysts occur in up to 90% of patients with Birt-Hogg-Dubé (BHD) syndrome, but the key pathologic cell type and signaling events driving this distinct phenotype remain elusive. Through examination of the LungMAP database, we found that folliculin (FLCN) is highly expressed in neonatal lung mesenchymal cells. Using RNA-Seq, we found that inactivation of Flcn in mouse embryonic fibroblasts leads to changes in multiple Wnt ligands, including a 2.8-fold decrease in Wnt2. This was associated with decreased TCF/LEF activity, a readout of canonical WNT activity, after treatment with a GSK3-α/ß inhibitor. Similarly, FLCN deficiency in HEK293T cells decreased WNT pathway activity by 76% post-GSK3-α/ß inhibition. Inactivation of FLCN in human fetal lung fibroblasts (MRC-5) led to ~ 100-fold decrease in Wnt2 expression and a 33-fold decrease in Wnt7b expression-two ligands known to be necessary for lung development. Furthermore, canonical WNT activity was decreased by 60%. Classic WNT targets such as AXIN2 and BMP4, and WNT enhanceosome members including TCF4, LEF1 and BCL9 were also decreased after GSK3-α/ß inhibition. FLCN-deficient MRC-5 cells failed to upregulate LEF1 in response to GSK3-α/ß inhibition. Finally, we found that a constitutively active ß-catenin could only partially rescue the decreased WNT activity phenotype seen in FLCN-deficient cells, whereas silencing the transcription factor TFE3 completely reversed this phenotype. In summary, our data establish FLCN as a critical regulator of the WNT pathway via TFE3 and suggest that FLCN-dependent defects in WNT pathway developmental cues may contribute to lung cyst pathogenesis in BHD.

Mechanisms of pulmonary cyst pathogenesis in Birt-Hogg-Dube syndrome: The stretch hypothesis.

Loss-of-function mutations in the folliculin gene (FLCN) on chromosome 17p cause Birt-Hogg-Dube syndrome (BHD), which is associated with cystic lung disease. The risk of lung collapse (pneumothorax) in BHD patients is 50-fold higher than in the general population. The cystic lung disease in BHD is distinctive because the cysts tend to be basilar, subpleural and lentiform, differentiating BHD from most other cystic lung diseases. Recently, major advances in elucidating the primary functions of the folliculin protein have been made, including roles in mTOR and AMPK signaling via the interaction of FLCN with FNIP1/2, and cell-cell adhesion via the physical interaction of FLCN with plakophilin 4 (PKP4), an armadillo-repeat containing protein that interacts with E-cadherin and is a component of the adherens junctions. In addition, in just the last three years, the pulmonary impact of FLCN deficiency has been examined for the first time. In mouse models, evidence has emerged that AMPK signaling and cell-cell adhesion are involved in alveolar enlargement. In addition, the pathologic features of human BHD cysts have been recently comprehensively characterized. The "stretch hypothesis" proposes that cysts in BHD arise because of fundamental defects in cell-cell adhesion, leading to repeated respiration-induced physical stretch-induced stress and, over time, expansion of alveolar spaces particularly in regions of the lung with larger changes in alveolar volume and at weaker "anchor points" to the pleura. This hypothesis ties together many of the new data from cellular and mouse models of BHD and from the human pathologic studies. Critical questions remain. These include whether the consequences of stretch-induced cyst formation arise through a destructive/inflammatory program or a proliferative program (or both), whether cyst initiation involves a "second hit" genetic event inactivating the remaining wild-type copy of FLCN (as is known to occur in BHD-associated renal cell carcinomas), and whether cyst initiation involves exclusively the epithelial compartment versus an interaction between the epithelium and mesenchyme. Ultimately, understanding the mechanisms of cystic lung disease in BHD may help to elucidate the pathogenesis of primary spontaneous pneumothorax, with more than 20,000 cases reported annually in the United States alone.

Improved quantitative analysis of primary bone marrow megakaryocytes utilizing imaging flow cytometry.

Life-threatening thrombocytopenia can develop following bone marrow injury due to decreased platelet production from megakaryocytes (MKs). However, the study of primary MKs has been complicated by their low frequency in the bone marrow and by technical challenges presented by their unique maturation properties. More accurate and efficient methods for the analysis of in vivo MKs are needed to enhance our understanding of megakaryopoiesis and ultimately develop new therapeutic strategies for thrombocytopenia. Imaging flow cytometry (IFC) combines the morphometric capabilities of microscopy with the high-throughput analyses of flow cytometry (FC). Here, we investigate the application of IFC on the ImageStream(X) platform to the analysis of primary MKs isolated from murine bone marrow. Our data highlight and address technical challenges for conventional FC posed by the wide range of cellular size within the MK lineage as well as the shared surface phenotype with abundant platelet progeny. We further demonstrate that IFC can be used to reproducibly and efficiently quantify the frequency of primary murine MKs in the marrow, both at steady-state and in the setting of radiation-induced bone marrow injury, as well as assess their ploidy distribution. The ability to accurately analyze the full spectrum of maturing MKs in the bone marrow now allows for many possible applications of IFC to enhance our understanding of megakaryopoiesis and platelet production.

Chaperone-mediated autophagy is defective in mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is a lysosomal storage disorder caused by mutations in the MCOLN1 gene, a member of the transient receptor potential (TRP) cation channel gene family. The encoded protein, transient receptor potential mucolipin-1 (TRPML1), has been localized to lysosomes and late endosomes but the pathogenic mechanism by which loss of TRPML1 leads to abnormal cellular storage and neuronal cell death is still poorly understood. Yeast two-hybrid and co-immunoprecipitation (coIP) experiments identified interactions between TRPML1 and Hsc70 as well as TRPML1 and Hsp40. Hsc70 and Hsp40 are members of a molecular chaperone complex required for protein transport into the lysosome during chaperone-mediated autophagy (CMA). To determine the functional relevance of this interaction, we compared fibroblasts from MLIV patients to those from sex- and age-matched controls and show a defect in CMA in response to serum withdrawal. This defect in CMA was subsequently confirmed in purified lysosomes isolated from control and MLIV fibroblasts. We further show that the amount of lysosomal-associated membrane protein type 2A (LAMP-2A) is reduced in lysosomal membranes of MLIV fibroblasts. As a result of decreased CMA, MLIV fibroblasts have increased levels of oxidized proteins compared to control fibroblasts. We hypothesize that TRPML1 may act as a docking site for intralysosomal Hsc70 (ly-Hsc70) allowing it to more efficiently pull in substrates for CMA. It is also possible that TRPML1 channel activity may be required for CMA. Understanding the role of TRPML1 in CMA will undoubtedly help to characterize the pathogenesis of MLIV.

Mechanism of interferon-gamma-induced increase in T84 intestinal epithelial tight junction.

Interferon-gamma (IFN-gamma) is an important proinflammatory cytokine that plays a central role in the intestinal inflammatory process of inflammatory bowel disease. IFN-gamma induced disturbance of the intestinal epithelial tight junction (TJ) barrier has been postulated to be an important mechanism contributing to intestinal inflammation. The intracellular mechanisms that mediate the IFN-gamma induced increase in intestinal TJ permeability remain unclear. The aim of this study was to examine the role of the phosphatidylinositol 3-kinase (PI3-K) pathway in the regulation of the IFN-gamma induced increase in intestinal TJ permeability using the T84 intestinal epithelial cell line. IFN-gamma caused an increase in T84 intestinal epithelial TJ permeability and depletion of TJ protein, occludin. The IFN-gamma induced increase in TJ permeability and alteration in occludin protein was associated with rapid activation of PI3-K; and inhibition of PI3-K activation prevented the IFN-gamma induced effects. IFN-gamma also caused a delayed but more prolonged activation of nuclear factor-kappaB (NF-kappaB); inhibition of NF-kappaB also prevented the increase in T84 TJ permeability and alteration in occludin expression. The IFN-gamma induced activation of NF-kappaB was mediated by a cross-talk with PI3-K pathway. In conclusion, the IFN-gamma induced increase in T84 TJ permeability and alteration in occludin protein expression were mediated by the PI3-K pathway. These results show for the first time that the IFN-gamma modulation of TJ protein and TJ barrier function is regulated by a cross-talk between PI3-K and NF-kappaB pathways.

Cellular and molecular mechanisms of heat stress-induced up-regulation of occludin protein expression: regulatory role of heat shock factor-1.

The heat stress (HS)-induced increase in occludin protein expression has been postulated to be a protective response against HS-induced disruption of the intestinal epithelial tight junction barrier. The aim of this study was to elucidate the cellular and molecular processes that mediate the HS-induced up-regulation of occludin expression in Caco-2 cells. Exposure to HS (39 degrees C or 41 degrees C) resulted in increased expression of occludin protein; this was preceded by an increase in occludin mRNA transcription and promoter activity. HS-induced activation of heat shock factor-1 (HSF-1) resulted in cytoplasmic-to-nuclear translocation of HSF-1 and binding to its binding motif in the occludin promoter region. HSF-1 activation was associated with an increase in occludin promoter activity, mRNA transcription, and protein expression; which were abolished by the HSF-1 inhibitor quercetin. Targeted HSF-1 knock-down by siRNA transfection inhibited the HSF-1-induced increase in occulin expression and junctional localization of occulin protein. Site-directed mutagenesis of the HSF-1 binding motif in the occludin promoter region inhibited HS-induced binding of HSF-1 to the occludin promoter region and subsequent promoter activity. In conclusion, our data show for the first time that the HS-induced increase in occludin protein expression is mediated by HSF-1 activation and subsequent binding of HSF-1 to the occludin promoter, which initiates a series of molecular and cellular events culminating in increased junctional localization of occludin protein.

Mechanism of glucocorticoid regulation of the intestinal tight junction barrier.

A defective intestinal epithelial tight junction (TJ) barrier has been proposed as an important pathogenic factor contributing to the intestinal inflammation of Crohn's disease. Glucocorticoids are first-line therapeutic agents for the treatment of moderate to severe Crohn's disease. Glucocorticoid treatment has been shown to induce retightening of the intestinal TJ barrier defect in Crohn's disease patients. However, the mechanisms that mediate the glucocorticoid therapeutic action on intestinal TJ barrier function remain unknown. The aim of this study was to elucidate the mechanism of glucocorticoid modulation of the intestinal epithelial TJ barrier using an in vitro model system. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro model to examine the effects of glucocorticoids on basal intestinal epithelial TJ barrier function and on TNF-alpha-induced disruption of the TJ barrier. Glucocorticoids (prednisolone and dexamethasone) did not have a significant effect on baseline Caco-2 TJ barrier function but prevented the TNF-alpha-induced increase in Caco-2 TJ permeability. The glucocorticoid protective effect against the TNF-alpha-induced increase in Caco-2 TJ permeability required activation of the glucocorticoid receptor (GR) complex. The activation of the GR complex resulted in GR complex binding to the glucocorticoid response element (GRE) site on DNA and activation of a GR-responsive promoter. Glucocorticoids inhibited the TNF-alpha-induced increase in myosin light chain kinase (MLCK) protein expression, a key process mediating the TNF-alpha increase in intestinal TJ permeability. The glucocorticoid inhibition of the TNF-alpha-induced increase in MLCK protein expression was due to the binding of the GR complex to a GRE binding site on the MLCK promoter region suppressing the TNF-alpha-induced activation. Glucocorticoids inhibit the TNF-alpha-induced increase in Caco-2 TJ permeability. The prednisolone protective action was mediated by binding of activated GR complex to the GRE site on the MLCK promoter, suppressing the TNF-alpha-induced increase in MLCK gene activity, protein expression, and subsequent opening of the intestinal TJ barrier.

Etiology of decreased gastric nitric oxide in the critically ill.

BACKGROUND: Nitric oxide (NO) is present in the gas phase of the normal human stomach at a high concentration (1-10 ppm). The majority of this NO is produced from the reduction of dietary nitrate to nitrite and finally NO. Generation of this nonenzymatically produced gastric NO occurs only in an acidic environment. We examined NO concentrations in critically ill subjects and the mechanism for the observed perturbations. METHODS: Seven critically ill, intubated intensive care unit (ICU) patients (mean APACHE II score 16) and seven control patients were studied. Gastric NO concentrations were measured with a Sievers NO analyzer (GE, Boulder, CO). Nitrate and nitrite concentrations were determined by a modified Griess assay. Bacterial counts were determined by optical density at 600 nm. RESULTS: Gastric NO concentration was significantly lower in the critically ill group (102.7 ppb) compared with the control group (953.2 ppb), although this difference was abolished by treating the control group with omeprazole (54 ppb). Gastric nitrate and nitrite concentrations were similar in the control and ICU groups, suggesting that substrate deficiency was not a cause of the low intragastric NO. Gastric pH was significantly lower in the control subjects (3.0) compared with the ICU patients (6.3) and the control subjects after receiving omeprazole (6.5). ICU patients had a trend toward higher gastric bacterial load. CONCLUSION: In critically ill patients, markedly decreased NO concentrations are found in the gas of the stomach owing to a failure of gastric acidification.

Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans.

BACKGROUND: Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. Recently the MLIV gene, MCOLN1, has been identified as a new member of the transient receptor potential (TRP) cation channel superfamily. Here we report the cloning and characterization of the mouse homologue, Mcoln1, and report a novel splice variant that is not seen in humans. RESULTS: The human and mouse genes display a high degree of synteny. Mcoln1 shows 91% amino acid and 86% nucleotide identity to MCOLN1. Also, Mcoln1 maps to chromosome 8 and contains an open reading frame of 580 amino acids, with a transcript length of approximately 2 kb encoded by 14 exons, similar to its human counterpart. The transcript that results from murine specific alternative splicing encodes a 611 amino acid protein that differs at the c-terminus. CONCLUSIONS: Mcoln1 is highly similar to MCOLN1, especially in the transmembrane domains and ion pore region. Also, the late endosomal/lysosomal targeting signal is conserved, supporting the hypothesis that the protein is localized to these vesicle membranes. To date, there are very few reports describing species-specific splice variants. While identification of Mcoln1 is crucial to the development of mouse models for MLIV, the fact that there are two transcripts in mice suggests an additional or alternate function of the gene that may complicate phenotypic assessment.