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In cystic lung diseases such as lymphangioleiomyomatosis (LAM), a CT-based cyst score that measures the percentage of the lung volume occupied by cysts is a common index of the cyst burden in the lungs. Although the current semi-automatic measurement of the cyst score is well established, it is susceptible to human operator variabilities. We recently developed a fully automatic method incorporating adaptive features in place of manual adjustments. In this clinical study, the automatic method is validated against the standard method in several aspects. These include the agreement between the cyst scores of the two methods, the agreement of each method with independent tests of pulmonary function, and the temporal consistency of the measurements in the consecutive visits of the same patients. We found that the automatic method agreed with the standard method as well as the agreement between two trained operators running the same standard method; both methods obtained the same level of correlation with laboratory pulmonary function tests; the automated method had better temporal consistency than the standard method (p < 0.0001). The study indicates that the automatic method could replace the standard method and provide better consistency in assessing the extent of cystic changes in the lungs of patients.

microRNAs (miRs) function in cancer progression as post-transcriptional regulators. We previously reported that endogenous circular RNAs (circRNAs) function as efficient miR sponges and could act as novel gene regulators in oral squamous cell carcinoma (OSCC). In this study, we carried out cellular and luciferase reporter assays to examine competitive inhibition of miR-1269a, which is upregulated expression in several cancers, by circRNA-1269a, a synthetic circRNA that contains miR-1269a binding sequences. We also used data-independent acquisition (DIA) proteomics and in silico analyses to determine how circRNA-1269a treatment affects molecules downstream of miR-1269a. First, we confirmed the circularization of the linear miR-1269a binding site sequence using RT-PCR with divergent/convergent primers and direct sequencing of the head-to-tail circRNA junction point. In luciferase reporter and cellular functional assays, circRNA-1269a significantly reduced miR-1269a function, leading to a significant decrease in cell proliferation and migration. DIA proteomics and gene set enrichment analysis of OSCC cells treated with circRNA-1269a indicated high differential expression for 284 proteins that were mainly enriched in apoptosis pathways. In particular, phospholipase C gamma 2 (PLCG2), which is related to OSCC clinical stage and overall survival, was affected by the circRNA-1269a/miR-1269a axis. Taken together, synthetic circRNA-1269a inhibits tumor progression via miR-1269a and its downstream targets, indicating that artificial circRNAs could represent an effective OSCC therapeutic.

Abstract Objectives Lymphangioleiomyomatosis (LAM) patients with severe lung disease may be considered for lung transplantation. Clinical, physiologic, and quality of life data are usually employed for referral. The aim of this study was to determine whether computed tomographic measurement of lung volume occupied by cysts (cyst score) complemented clinical and physiologic data in supporting referral for transplantation. Methods Forty-one patients were studied. Pre-referral clinical data, pulmonary function tests, exercise testing, and high-resolution computed tomography (HRCT) scans were obtained. From HRCT, a computer-aided diagnostic program was employed to calculate cyst scores. These data were compared to those of 41 age-matched LAM patients not referred for lung transplantation. Results Cyst score, and % predicted FEV1 and DLCO were respectively, 48.1 ± 9.4%, 36.5 ± 9.1%, and 35.0 ± 10.7%. For the control group, cyst score, FEV1, and DLCO were respectively, 14.8 ± 8.3%, 77.2 ± 20.3%, and 66.7 ± 19.3%. Cyst score values showed a normal distribution. However, the frequency distribution of FEV1 was skewed to the right while the distribution of DLCO was bimodal. Correlations between cyst score and FEV1 and DLCO for the study group were respectively, r = − 0.319 and r = − 0.421. Conclusions LAM patients referred for lung transplantation had nearly 50% of lungs occupied by cysts. Correlations between cyst score and FEV1 or DLCO were weak; as shown previously, DLCO was better related to cyst number while FEV1 had a better association with cyst size. Given its normal distribution, cyst score measurements may assist in evaluation of pre-transplant severity of lung disease before referral for transplantation.

(1) Background: Lymphangioleiomyomatosis is a genetic disease that affects mostly women of childbearing age. In the lungs, it manifests as the progressive formation of air-filled cysts and is associated with a decline in lung function. With a median survival of 29 years after the onset of symptoms, computed-tomographic monitoring of cystic changes in the lungs is a key part of the management of the disease. However, the current standard method to measure cyst burdens from CT is semi-automatic and requires manual adjustments from trained operators to obtain consistent results due to variabilities in CT technology and imaging conditions over the long course of the disease. This can be impractical for longitudinal studies involving large numbers of scans and is susceptible to subjective biases. (2) Methods: We developed an automated method of pulmonary cyst segmentation for chest CT images incorporating novel graphics processing algorithms. We assessed its performance against the gold-standard semi-automated method performed by experienced operators who were blinded to the results of the automated method. (3) Results: the automated method had the same consistency over time as the gold-standard method, but its cyst scores were more strongly correlated with concurrent pulmonary function results from the physiology laboratory than those of the gold-standard method. (4) Conclusions: The automated cyst segmentation is a competent replacement for the gold-standard semi-automated process. It is a solution for saving time and labor in clinical studies of lymphangioleiomyomatosis that may involve large numbers of chest CT scans from diverse scanner platforms and protocols.

Skin findings can be critical to determining whether a patient with lymphangioleiomyomatosis (LAM), a progressive pulmonary disease that predominantly affects adult women, has sporadic LAM or LAM in association with tuberous sclerosis complex (TSC). Three individuals with LAM underwent evaluation for TSC-associated mucocutaneous and internal findings. We used our previously published algorithm to confirm the clinical suspicion for mosaicism and guide the selection of tissue specimens and genetic workup. Next-generation sequencing of cutaneous findings was used to confirm clinical suspicion for mosaic TSC in individuals with LAM. Two individuals previously thought to have sporadic LAM were diagnosed with mosaic TSC-associated LAM upon next-generation sequencing of unilateral angiofibromas in one and an unusual cutaneous hamartoma in the other. A third individual, diagnosed with TSC in childhood, was found to have a mosaic pathogenic variant in TSC2 in cutaneous tissue from a digit with macrodactyly. Accurate diagnosis of mosaic TSC-associated LAM may require enhanced genetic testing and is important because of the implications regarding surveillance, prognosis, and risk of transmission to offspring.

Abstract Locus for Enterocyte Effacement (LEE)-positive Shiga-toxigenic Escherichia coli (STEC) contributes to many global foodborne diseases, with infection characterized by severe gastrointestinal symptoms, including bloody diarrhea. The incidence of LEE-negative STEC-mediated disease is also increasing globally. Subtilase cytotoxin (SubAB) is released by some LEE-negative STEC strains. It cleaves BiP, which is a chaperone protein located in the endoplasmic reticulum (ER), thereby causing apoptosis induced by ER stress. To date, the apoptotic signaling pathway mediated by SubAB has not been identified. In the current study, RNA-seq analysis showed that SubAB significantly induced the expression of Kelch domain containing 7B (KLHDC7B). We explored the role of KLHDC7B in the SubAB-induced apoptotic pathway. SubAB-induced KLHDC7B mRNA expression was increased after 12 h of incubation of toxin with HeLa cells. KLHDC7B expression was downregulated by knockdown of PKR-like endoplasmic reticulum kinase (PERK), CEBP homologous protein (CHOP), activating transcription factor 4 (ATF4), and CEBP β (CEBPB). KLHDC7B knockdown suppressed SubAB-stimulated CHOP expression, poly(ADP-ribose) polymerase (PARP) cleavage, and cytotoxicity. The over-expressed KLHDC7B was localized to the nucleus and cytosolic fractions. Next, we used RNA-seq to analyze the effect of KLHDC7B knockdown on apoptosis induced by SubAB, and found that the gene encoding for the pro-apoptotic Bcl-2 family protein, Harakiri (HRK), was upregulated in SubAB-treated control cells. However, this effect was not observed in SubAB-treated KLHDC7B-knockdown cells. Therefore, we identified the pathway through which SubAB-induced KLHDC7B regulates HRK expression, which is essential for apoptosis in toxin-mediated ER stress.

Abstract Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease with mortality driven primarily by respiratory failure. Patients with LAM frequently have respiratory infections, suggestive of a dysregulated microbiome. Here we demonstrate that end-stage LAM patients have a distinct microbiome signature compared to patients with end-stage chronic obstructive pulmonary disease.

Background Tuberous sclerosis complex (TSC) is a neurogenetic syndrome due to loss-of-function mutations in TSC2 or TSC1, characterized by tumors at multiple body sites, including facial angiofibroma (FAF). Here, an ultrasensitive assessment of the extent and range of UV-induced mutations in TSC facial skin was performed.Methods A multiplex high-sensitivity PCR assay (MHPA) was developed, enabling mutation detection at extremely low (

Mutations underlying disease in tuberous sclerosis complex (TSC) give rise to tumors with biallelic mutations in TSC1 or TSC2 and hyperactive mammalian target of rapamycin complex 1 (mTORC1). Benign tumors might exhibit de novo expression of immunogens, targetable by immunotherapy. As tumors may rely on ganglioside D3 (GD3) expression for mTORC1 activation and growth, we compared GD3 expression in tissues from patients with TSC and controls. GD3 was overexpressed in affected tissues from patients with TSC and also in aging Tsc2+/– mice. As GD3 overexpression was not accompanied by marked natural immune responses to the target molecule, we performed preclinical studies with GD3 chimeric antigen receptor (CAR) T cells. Polyfunctional CAR T cells were cytotoxic toward GD3-overexpressing targets. In mice challenged with Tsc2–/– tumor cells, CAR T cells substantially and durably reduced the tumor burden, correlating with increased T cell infiltration. We also treated aged Tsc2+/– heterozygous (>60 weeks) mice that carry spontaneous Tsc2–/– tumors with GD3 CAR or untransduced T cells and evaluated them at endpoint. Following CAR T cell treatment, the majority of mice were tumor free while all control animals carried tumors. The outcomes demonstrate a strong treatment effect and suggest that targeting GD3 can be successful in TSC.

The ARH family of ADP-ribose-acceptor hydrolases consists of three 39-kDa members (ARH1-3), with similarities in amino acid sequence. ARH1 was identified based on its ability to cleave ADP-ribosyl-arginine synthesized by cholera toxin. Mammalian ADP-ribosyltransferases (ARTCs) mimicked the toxin reaction, with ARTC1 catalyzing the synthesis of ADP-ribosyl-arginine. ADP-ribosylation of arginine was stereospecific, with β-NAD+ as substrate and, α-anomeric ADP-ribose-arginine the reaction product. ARH1 hydrolyzed α-ADP-ribose-arginine, in addition to α-NAD+ and O-acetyl-ADP-ribose. Thus, ADP-ribose attached to oxygen-containing or nitrogen-containing functional groups was a substrate. Arh1 heterozygous and knockout (KO) mice developed tumors. Arh1-KO mice showed decreased cardiac contractility and developed myocardial fibrosis. In addition to Arh1-KO mice showed increased ADP-ribosylation of tripartite motif-containing protein 72 (TRIM72), a membrane-repair protein. ARH3 cleaved ADP-ribose from ends of the poly(ADP-ribose) (PAR) chain and released the terminal ADP-ribose attached to (serine)protein. ARH3 also hydrolyzed α-NAD+ and O-acetyl-ADP-ribose. Incubation of Arh3-KO cells with H2O2 resulted in activation of poly-ADP-ribose polymerase (PARP)-1, followed by increased nuclear PAR, increased cytoplasmic PAR, leading to release of Apoptosis Inducing Factor (AIF) from mitochondria. AIF, following nuclear translocation, stimulated endonucleases, resulting in cell death by Parthanatos. Human ARH3-deficiency is autosomal recessive, rare, and characterized by neurodegeneration and early death. Arh3-KO mice developed increased brain infarction following ischemia-reperfusion injury, which was reduced by PARP inhibitors. Similarly, PARP inhibitors improved survival of Arh3-KO cells treated with H2O2. ARH2 protein did not show activity in the in vitro assays described above for ARH1 and ARH3. ARH2 has a restricted tissue distribution, with primary involvement of cardiac and skeletal muscle. Overall, the ARH family has unique functions in biological processes and different enzymatic activities.

Parthanatos is programmed cell death mediated by poly(ADP-ribose) polymerase 1 (PARP1) after DNA damage. PARP1 acts by catalyzing the transfer of poly(ADP-ribose) (PAR) polymers to various nuclear proteins. PAR is subsequently cleaved, generating protein-free PAR polymers, which are translocated to the cytoplasm where they associate with cytoplasmic and mitochondrial proteins, altering their functions and leading to cell death. Proteomic studies revealed that several proteins involved in endocytosis bind PAR after PARP1 activation, suggesting endocytosis may be affected by the parthanatos process. Endocytosis is a mechanism for cellular uptake of membrane-impermeant nutrients. Rab5, a small G-protein, is associated with the plasma membrane and early endosomes. Once activated by binding GTP, Rab5 recruits its effectors to early endosomes and regulates their fusion. Here, we report that after DNA damage, PARP1-generated PAR binds to Rab5, suppressing its activity. As a result, Rab5 is dissociated from endosomal vesicles, inhibiting the uptake of membrane-impermeant nutrients. This PARP1-dependent inhibition of nutrient uptake leads to cell starvation and death. It thus appears that this mechanism may represent a novel parthanatos pathway.

Melanoma is an aggressive cancer that utilizes multiple signaling pathways, including those that involve oncogenes, proto-oncogenes, and tumor suppressors. It has been suggested that melanoma formation requires cross-talk of the PI3K/Akt/mTOR and Ras-ERK pathways. This pathway cross-talk has been associated with aggressiveness, drug resistance, and metastasis; thus, simultaneous targeting of components of the different pathways involved in melanoma may aid in therapy. We have previously reported that bacterial cyclodipeptides (CDPs) are cytotoxic to HeLa cells and inhibit Akt phosphorylation. Here, we show that CDPs decreased melanoma size and tumor formation in a subcutaneous xenografted mouse melanoma model. In fact, CDPs accelerated death of B16-F0 murine melanoma cells. In mice, antitumor effect was improved by treatment with CDPs using cyclodextrins as drug vehicle. In tumors, CDPs caused nuclear fragmentation and changed the expression of the Bcl-2 and Ki67 apoptotic markers and promoted restoration of hyperactivation of the PI3K/Akt/mTOR pathway. Additionally, elements of several signaling pathways such as the Ras-ERK, PI3K/JNK/PKA, p27Kip1/CDK1/survivin, MAPK, HIF-1, epithelial–mesenchymal transition, and cancer stem cell pathways were also modified by treatment of xenografted melanoma mice with CDPs. The findings indicate that the multiple signaling pathways implicated in aggressiveness of the murine B16-F0 melanoma line are targeted by the bacterial CDPs. Molecular modeling of CDPs with protein kinases involved in neoplastic processes suggested that these compounds could indeed interact with the active site of the enzymes. The results suggest that CDPs may be considered as potential antineoplastic drugs, interfering with multiple pathways involved in tumor formation and progression.

Protein ADP-ribosylation has emerged as a key post translational modification that regulates several stress responses. Here the authors characterize ARH3 as a major serine-specific mono–ADP-­ribosylhydrolase and use a proteomics approach to identify the cellular targets of ARH3.

Helicobacter pylori, a major cause of gastroduodenal diseases, produces vacuolating cytotoxin (VacA) and cytotoxin-associated gene A (CagA), which seem to be involved in virulence. VacA exhibits pleiotropic actions in gastroduodenal disorders via its specific receptors. Recently, we found that VacA induced the phosphorylation of cellular Src kinase (Src) at Tyr418 in AZ-521 cells. Silencing of receptor protein tyrosine phosphatase (RPTP)α, a VacA receptor, reduced VacA-induced Src phosphorylation. Src is responsible for tyrosine phosphorylation of CagA at its Glu-Pro-Ile-Tyr-Ala (EPIYA) variant C (EPIYA-C) motif in Helicobacter pylori-infected gastric epithelial cells, resulting in binding of CagA to SHP-2 phosphatase. Challenging AZ-521 cells with wild-type H. pylori induced phosphorylation of CagA, but this did not occur when challenged with a vacA gene-disrupted mutant strain. CagA phosphorylation was observed in cells infected with a vacA gene-disrupted mutant strain after addition of purified VacA, suggesting that VacA is required for H. pylori-induced CagA phosphorylation. Following siRNA-mediated RPTPα knockdown in AZ-521 cells, infection with wild-type H. pylori and treatment with VacA did not induce CagA phosphorylation. Taken together, these results support our conclusion that VacA mediates CagA phosphorylation through RPTPα in AZ-521 cells. These data indicate the possibility that Src phosphorylation induced by VacA is mediated through RPTPα, resulting in activation of Src, leading to CagA phosphorylation at Tyr972 in AZ-521 cells.

Pathogenic microorganisms produce various virulence factors, e.g., enzymes, cytotoxins, effectors, which trigger development of pathologies in infectious diseases. Cholera toxin (CT) produced by O1 and O139 serotypes of Vibrio cholerae (V. cholerae) is a major cytotoxin causing severe diarrhea. Cholix cytotoxin (Cholix) was identified as a novel eukaryotic elongation factor 2 (eEF2) adenosine-diphosphate (ADP)-ribosyltransferase produced mainly in non-O1/non-O139 V. cholerae. The function and role of Cholix in infectious disease caused by V. cholerae remain unknown. The crystal structure of Cholix is similar to Pseudomonas exotoxin A (PEA) which is composed of an N-terminal receptor-recognition domain and a C-terminal ADP-ribosyltransferase domain. The endocytosed Cholix catalyzes ADP-ribosylation of eEF2 in host cells and inhibits protein synthesis, resulting in cell death. In a mouse model, Cholix caused lethality with severe liver damage. In this review, we describe the mechanism underlying Cholix-induced cytotoxicity. Cholix-induced apoptosis was regulated by mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways, which dramatically enhanced tumor necrosis factor-α (TNF-α) production in human liver, as well as the amount of epithelial-like HepG2 cancer cells. In contrast, Cholix induced apoptosis in hepatocytes through a mitochondrial-dependent pathway, which was not stimulated by TNF-α. These findings suggest that sensitivity to Cholix depends on the target cell. A substantial amount of information on PEA is provided in order to compare/contrast this well-characterized mono-ADP-ribosyltransferase (mART) with Cholix.

Arginine-specific mono-adenosine diphosphate (ADP)-ribosylation is a nicotinamide adenine dinucleotide (NAD)+-dependent, reversible post-translational modification involving the transfer of an ADP-ribose from NAD+ by bacterial toxins and eukaryotic ADP-ribosyltransferases (ARTs) to arginine on an acceptor protein or peptide. ADP-ribosylarginine hydrolase 1 (ARH1) catalyzes the cleavage of the ADP-ribose-arginine bond, regenerating (arginine)protein. Arginine-specific mono-ADP-ribosylation catalyzed by bacterial toxins was first identified as a mechanism of disease pathogenesis. Cholera toxin ADP-ribosylates and activates the α subunit of Gαs, a guanine nucleotide-binding protein that stimulates adenylyl cyclase activity, increasing cyclic adenosine monophosphate (cAMP), and resulting in fluid and electrolyte loss. Arginine-specific mono-ADP-ribosylation in mammalian cells has potential roles in membrane repair, immunity, and cancer. In mammalian tissues, ARH1 is a cytosolic protein that is ubiquitously expressed. ARH1 deficiency increased tumorigenesis in a gender-specific manner. In the myocardium, in response to cellular injury, an arginine-specific mono-ADP-ribosylation cycle, involving ART1 and ARH1, regulated the level and cellular distribution of ADP-ribosylated tripartite motif-containing protein 72 (TRIM72). Confirmed substrates of ARH1 in vivo are Gαs and TRIM72, however, more than a thousand proteins, ADP-ribosylated on arginine, have been identified by proteomic analysis. This review summarizes the current understanding of the properties of ARH1, e.g., bacterial toxin action, myocardial membrane repair following injury, and tumorigenesis.

Sarcoidosis is a devastating inflammatory disease affecting many organs, especially the lungs and lymph nodes. Bone marrow-derived mesenchymal stromal cells (MSCs) can “reprogram” various types of macrophages towards an anti-inflammatory phenotype. We wanted to determine whether alveolar macrophages from sarcoidosis subjects behave similarly by mounting an anti-inflammatory response when co-cultured with MSCs. Fifteen sarcoidosis and eight control subjects underwent bronchoscopy and bronchoalveolar lavage (BAL). Unselected BAL cells (70−94% macrophages) were isolated and cultured with and without MSCs from healthy adults. Following stimulation of the cultured cells with lipopolysaccharide, the medium was removed to measure interleukin 10 and tumor necrosis factor alpha (IL-10 and TNF-α). In two additional sarcoidosis subjects, flow cytometry was used to study intracellular cytokines and surface markers associated with alveolar macrophages to confirm the results. Unselected BAL cells from sarcoidosis subjects co-cultured with MSCs showed a reduction in TNF-α (pro-inflammatory M1) and an increase in IL-10 (anti-inflammatory M2) in 9 of 11 samples studied. Control subject samples showed few, if any, differences in cytokine production. Unselected BAL cells from two additional patients analyzed by flow cytometry confirmed a switch towards an anti-inflammatory state (i.e., M1 to M2) after co-culture with MSCs. These results suggest that, similarly to other macrophages, alveolar macrophages also respond to MSC contacts by changing towards an anti-inflammatory phenotype. Based on our results, we hypothesize that mesenchymal stromal cells applied to the airways might alleviate lung inflammation and decrease steroid need in patients with sarcoidosis.

Cholera toxin, an 84-kDa multimeric protein and a major virulence factor of Vibrio cholerae, uses the ADP-ribosyltransferase activity of its A subunit to intoxicate host cells. ADP-ribosylation is a posttranslational modification of proteins, in which the ADP-ribose moiety of NAD+ is transferred to an acceptor. In mammalian cells, ADP-ribosylation of acceptors appears to be reversible. ADP-ribosyltransferases (ARTs) catalyze the modification of acceptor proteins, and ADP-ribose-acceptor hydrolases (ARHs) cleave the ADP-ribose-acceptor bond. ARH1 specifically cleaves the ADP-ribose-arginine bond. We previously demonstrated a role for endogenous ARH1 in regulating the extent of cholera toxin-mediated fluid and electrolyte abnormalities in a mouse model of intoxication. Murine ARH1-knockout (KO) cells and ARH1-KO mice exhibited increased sensitivity to cholera toxin compared to their wild-type (WT) counterparts. In the current report, we examined the sensitivity to cholera toxin of male and female ARH1-KO and WT mice. Intestinal loops derived from female ARH1-KO mice when injected with cholera toxin showed increased fluid accumulation compared to male ARH1-KO mice. WT mice did not show gender differences in fluid accumulation, ADP-ribosylarginine content, and ADP-ribosyl Gαs levels. Injection of 8-Bromo-cAMP into the intestinal loops also increased fluid accumulation, however, there was no significant difference between female and male mice or in WT and KO mice. Female ARH1-KO mice showed greater amounts of ADP-ribosylated Gαs protein and increased ADP-ribosylarginine content both in whole intestine and in epithelial cells than did male ARH1-KO mice. These results demonstrate that female ARH1-KO mice are more sensitive to cholera toxin than male mice. Loss of ARH1 confers gender sensitivity to the effects of cholera toxin but not of cyclic AMP. These observations may in part explain the finding noted in some clinical reports of enhanced symptoms of cholera and/or diarrhea in women than men.

Increased mTORC1 signaling from TSC1/TSC2 inactivation is found in cancer and causes tuberous sclerosis complex (TSC). The role of mesenchymal-derived cells in TSC tumorigenesis was investigated through disruption of Tsc2 in craniofacial and limb bud mesenchymal progenitors. Tsc2cKOPrrx1-cre mice had shortened lifespans and extensive hamartomas containing abnormal tortuous, dilated vessels prominent in the forelimbs. Abnormalities were blocked by the mTORC1 inhibitor sirolimus. A Tsc2/mTORC1 expression signature identified in Tsc2-deficient fibroblasts was also increased in bladder cancers with TSC1/TSC2 mutations in the TCGA database. Signature component Lgals3 encoding galectin-3 was increased in Tsc2-deficient cells and serum of Tsc2cKOPrrx1-cre mice. Galectin-3 was increased in TSC-related skin tumors, angiomyolipomas, and lymphangioleiomyomatosis with serum levels in patients with lymphangioleiomyomatosis correlating with impaired lung function and angiomyolipoma presence. Our results demonstrate Tsc2-deficient mesenchymal progenitors cause aberrant morphogenic signals, and identify an expression signature including Lgals3 relevant for human disease of TSC1/TSC2 inactivation and mTORC1 hyperactivity.

Sex prevalence in lung disease suggests that sex-specific hormones may contribute to the pathogenesis and/or progression of at least some lung diseases, such as lung adenocarcinoma, lymphangioleiomyomatosis (LAM) and benign metastasising leiomyoma (BML). Oestrogen is an important hormone in normal lung development and in the pathogenesis of female predominant pulmonary diseases. In vivo and in vitro studies have facilitated our understanding of disease pathogenesis and discovery of potential therapeutic targets. Oestrogen promoted disease progression in cell and animal models of lung adenocarcinoma, LAM and BML. Specifically, oestrogen enhanced tumour growth and metastasis in animal models of these diseases. Furthermore, 17β-estradiol (E2), the most abundant form of oestrogen in humans, increased the size and proliferation of cultured cells of lung adenocarcinoma and LAM. Coupled with the known mechanisms of oestrogen metabolism and signalling, these model systems may provide insights into the diverse effects of oestrogen and other hormones on lung diseases. Anti-oestrogen treatments that target key events of oestrogen synthesis or signalling, such as aromatase activity, oestrogen receptors and signalling pathways, may offer additional opportunities for clinical trials.

The primary function of the lymphatic system is absorbing and transporting macromolecules and immune cells to the general circulation, thereby regulating fluid, nutrient absorption and immune cell trafficking. Lymphangiogenesis plays an important role in tissue inflammation and tumour cell dissemination. Lymphatic involvement is seen in lymphangioleiomyomatosis (LAM) and idiopathic pulmonary fibrosis (IPF). LAM, a disease primarily affecting females, involves the lung (cystic destruction), kidney (angiomyolipoma) and axial lymphatics (adenopathy and lymphangioleiomyoma). LAM occurs sporadically or in association with tuberous sclerosis complex (TSC). Cystic lung destruction results from proliferation of LAM cells, which are abnormal smooth muscle-like cells with mutations in the TSC1 or TSC2 gene. Lymphatic abnormalities arise from infiltration of LAM cells into the lymphatic wall, leading to damage or obstruction of lymphatic vessels. Benign appearing LAM cells possess metastatic properties and are found in the blood and other body fluids. IPF is a progressive lung disease resulting from fibroblast proliferation and collagen deposition. Lymphangiogenesis is associated with pulmonary destruction and disease severity. A macrophage subset isolated from IPF bronchoalveolar lavage fluid (BALF) express lymphatic endothelial cell markers in vitro, in contrast to the same macrophage subset from normal BALF. Herein, we review lymphatic involvement in LAM and IPF.

SUMMARY The mechanism of action of Salmonella enterotoxin (Stn) as a virulence factor in disease is controversial. Studies of Stn have indicated both positive and negative effects on Salmonella virulence. In this study, we attempted to evaluate Stn function and its effects on Salmonella virulence. To investigate Stn function, we first performed in vitro and in vivo analysis using mammalian cells and a murine ileal loop model. In these systems, we did not observe differences in virulence phenotypes between wild-type Salmonella and an stn gene-deleted mutant. We next characterized the phenotypes and molecular properties of the mutant strain under various in vitro conditions. The proteomic profiles of the total cell membrane protein fraction differed between wild type and mutant in that there was an absence of a protein in the mutant strain, which was identified as OmpA. By far-western blotting, OmpA was found to interact directly with Stn. To verify this result, the morphology of Salmonella was examined by transmission electron microscopy, with OmpA localization being analyzed by immunogold labeling. Compared with wild-type Salmonella, the mutant strain had a different pole structure and a thin periplasmic space; OmpA was not seen in the mutant. These results indicate that Stn, via regulation of OmpA membrane localization, functions in the maintenance of membrane composition and integrity.

Vacuolating cytotoxin A (VacA) is one of the important virulence factors produced by H. pylori. VacA induces apoptotic cell death, which is potentiated by ammonia. VacA also causes cell death by mitochondrial damage, via signaling pathways that are not fully defined. Our aim was to determine whether endoplasmic reticulum (ER) stress is associated with VacA-induced mitochondrial dysfunction and apoptosis. We found that C/EBP homologous protein (CHOP), a key signaling protein of ER stress-induced apoptosis, was transcriptionally up-regulated following incubation of gastric epithelial cells with VacA. The effect of VacA on CHOP induction was significantly enhanced by co-incubation with ammonium chloride. Phosphorylation of eukaryotic initiation factor 2 (eIF2)-alpha, which is known to occur downstream of the ER stress sensor PKR-like ER-localized eIF2-alpha kinase (PERK) and to regulate CHOP expression, was also observed following incubation with VacA in the presence of ammonium chloride. Knockdown of CHOP by siRNA resulted in inhibition of VacA-induced apoptosis. Further studies showed that silencing of the PERK gene with siRNA attenuated VacA-mediated phosphorylation of eIF2-alpha, CHOP induction, expression of BH3-only protein Bim and Bax activation, and cell death induced by VacA with ammonium chloride, indicating that ER stress may lead to mitochondrial dysfunction during VacA-induced toxicity. Activation of ER stress and up-regulation of BH3-only proteins were also observed in human H. pylori-infected gastric mucosa. Collectively, this study reveals a possible association between VacA-induced apoptosis in gastric epithelial cells, and activation of ER stress in H. pylori-positive gastric mucosa.

Helicobacter pylori (H. pylori), a major cause of gastroduodenal diseases, produces VacA, a vacuolating cytotoxin associated with gastric inflammation and ulceration. The C-terminal domain of VacA plays a crucial role in receptor recognition on target cells. We have previously identified three proteins (i.e., RPTPα, RPTPβ, and LRP1) that serve as VacA receptors. These receptors contribute to the internalization of VacA into epithelial cells, activate signal transduction pathways, and contribute to cell death and gastric ulceration. In addition, other factors (e.g., CD18, sphingomyelin) have also been identified as cell-surface, VacA-binding proteins. Since we believe that, following interactions with its host cell receptors, VacA participates in events leading to disease, a better understanding of the cellular function of VacA receptors may provide valuable information regarding the mechanisms underlying the pleiotropic actions of VacA and the pathogenesis of H. pylori-mediated disease. In this review, we focus on VacA receptors and their role in events leading to cell damage.

The mammalian target of rapamycin (mTOR) modulates immune responses and cellular proliferation. The objective of this study was to assess whether inhibition of mTOR with rapamycin modifies disease severity in two experimental murine models of house dust mite (HDM)-induced asthma. In an induction model, rapamycin was administered to BALB/c mice coincident with nasal HDM challenges for 3 weeks. In a treatment model, nasal HDM challenges were performed for 6 weeks and rapamycin treatment was administered during weeks 4 through 6. In the induction model, rapamycin significantly attenuated airway inflammation, airway hyperreactivity (AHR) and goblet cell hyperplasia. In contrast, treatment of established HDM-induced asthma with rapamycin exacerbated AHR and airway inflammation, whereas goblet cell hyperplasia was not modified. Phosphorylation of the S6 ribosomal protein, which is downstream of mTORC1, was increased after 3 weeks, but not 6 weeks of HDM-challenge. Rapamycin reduced S6 phosphorylation in HDM-challenged mice in both the induction and treatment models. Thus, the paradoxical effects of rapamycin on asthma severity paralleled the activation of mTOR signaling. Lastly, mediastinal lymph node re-stimulation experiments showed that treatment of rapamycin-naive T cells with ex vivo rapamycin decreased antigen-specific Th2 cytokine production, whereas prior exposure to in vivo rapamycin rendered T cells refractory to the suppressive effects of ex vivo rapamycin. We conclude that rapamycin had paradoxical effects on the pathogenesis of experimental HDM-induced asthma. Thus, consistent with the context-dependent effects of rapamycin on inflammation, the timing of mTOR inhibition may be an important determinant of efficacy and toxicity in HDM-induced asthma.