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Patients with non-small cell lung cancer (NSCLC) harboring ROS proto-oncogene 1 (ROS1) gene rearrangements show dramatic response to the tyrosine kinase inhibitor (TKI) crizotinib. Current best practice guidelines recommend that all advanced stage non-squamous NSCLC patients be also tested for ROS1 gene rearrangements. Several studies have suggested that ROS1 immunohistochemistry (IHC) using the D4D6 antibody may be used to screen for ROS1 fusion positive lung cancers, with assays showing high sensitivity but moderate to high specificity. A break apart fluorescence in situ hybridization (FISH) test is then used to confirm the presence of ROS1 gene rearrangement. The goal of Canadian ROS1 (CROS) study was to harmonize ROS1 laboratory developed testing (LDT) by using IHC and FISH assays to detect ROS1 rearranged lung cancers across Canadian pathology laboratories. Cell lines expressing different levels of ROS1 (high, low, none) were used to calibrate IHC protocols after which participating laboratories ran the calibrated protocols on a reference set of 24 NSCLC cases (9 ROS1 rearranged tumors and 15 ROS1 non-rearranged tumors as determined by FISH). Results were compared using a centralized readout. The stained slides were evaluated for the cellular localization of staining, intensity of staining, the presence of staining in non-tumor cells, the presence of non-specific staining (e.g. necrosis, extracellular mater, other) and the percent positive cells. H-score was also determined for each tumor. Analytical sensitivity and specificity harmonization was achieved by using low limit of detection (LOD) as either any positivity in the U118 cell line or H-score of 200 with the HCC78 cell line. An overall diagnostic sensitivity and specificity of up to 100% and 99% respectively was achieved for ROS1 IHC testing (relative to FISH) using an adjusted H-score readout on the reference cases. This study confirms that LDT ROS1 IHC assays can be highly sensitive and specific for detection of ROS1 rearrangements in NSCLC. As NSCLC can demonstrate ROS1 IHC positivity in FISH-negative cases, the degree of the specificity of the IHC assay, especially in highly sensitive protocols, is mostly dependent on the readout cut-off threshold. As ROS1 IHC is a screening assay for a rare rearrangements in NSCLC, we recommend adjustment of the readout threshold in order to balance specificity, rather than decreasing the overall analytical and diagnostic sensitivity of the protocols.

Key points We have previously shown that carotid body stimulation by lysophosphatidic acid elicits a reflex stimulation of vagal efferent activity sufficient to cause bronchoconstriction in asthmatic rats. Here, we show that pathophysiological concentrations of asthma-associated prototypical Th2 cytokines also stimulate the carotid bodies. Stimulation of the carotid bodies by these asthmakines involves a PKCe-transient receptor potential vanilloid 1 (TRPV1) signalling mechanism likely dependent on TRPV1 S502 and T704 phosphorylation sites. As the carotid bodies' oxygen sensitivity is independent of PKCe-TRPV1 signalling, systemic blockade of PKCe may provide a novel therapeutic target to reduce allergen-induced asthmatic bronchoconstriction. Consistent with the therapeutic potential of blocking the PKCe-TRPV1 pathway, systemic delivery of a PKCe-blocking peptide suppresses asthmatic respiratory distress in response to allergen and reduces airway hyperresponsiveness to bradykinin. Abstract The autonomic nervous system orchestrates organ-specific, systemic and behavioural responses to inflammation. Recently, we demonstrated a vital role for lysophosphatidic acid in stimulating the primary autonomic oxygen chemoreceptors, the carotid bodies, in parasympathetic-mediated asthmatic airway hyperresponsiveness. However, the cacophony of stimulatory factors and cellular mechanisms of carotid body activation are unknown. Therefore, we set out to determine the intracellular signalling involved in carotid body-mediated sensing of asthmatic blood-borne inflammatory mediators. We employed a range of in vitro and rat in situ preparations, site-directed mutagenesis, patch-clamp, nerve recordings and pharmacological inhibition to assess cellular signalling. We show that the carotid bodies are also sensitive to asthma-associated prototypical Th2 cytokines which elicit sensory nerve excitation. This provides additional asthmatic ligands contributing to the previously established reflex arc resulting in efferent vagal activity and asthmatic bronchoconstriction. This novel sensing role for the carotid body is mediated by a PKCe-dependent stimulation of transient receptor potential vanilloid 1 (TRPV1), likely via TRPV1 phosphorylation at sites T704 and S502. Importantly, carotid body oxygen sensing was unaffected by blocking either PKCe or TRPV1. Further, we demonstrate that systemic PKCe blockade reduces asthmatic respiratory distress in response to allergen and airway hyperresponsiveness. These discoveries support an inflammation-dependent, oxygen-independent function for the carotid body and suggest that targeting PKCe provides a novel therapeutic option to abate allergic airway disease without altering life-saving autonomic hypoxic reflexes.

Waning immunity to COVID-19 vaccination is associated with increased risk of breakthrough infection, especially with highly transmissible variants of concern (VOC). Booster vaccination generates rapid immune recall in humans, which real-world observational studies suggest protects against VOC infection and associated disease, and modeling studies suggest could mitigate community spread. We directly tested the impact of booster vaccination on protection against Delta VOC infection, disease, and transmission to naïve cohorts in golden Syrian hamsters. Animals with waning immunity to bnt162b2 generated rapid immune recall and strong protection against upper- and lower-respiratory tract infection when boosted with bnt126b2, mRNA-1273 or AZD1222. Boosting with either mRNA vaccine generated moderate protection against lung inflammation and virus transmission to unvaccinated animals. Our data support booster vaccination as a tool to address emerging VOC in the COVID-19 pandemic.One-Sentence SummaryA booster vaccine delivered 9 months after primary bnt162b2 vaccination protects hamsters from Delta VOC infection, disease, and transmission.

The murine interleukin-4 treated macrophage (MIL4) exerts anti-inflammatory and pro-healing effects and has been shown to reduce the severity of chemical-induced colitis. Positing M(IL4) transfer as an anti-inflammatory therapy, the possibility of side-effects must be considered. Consequently, bone marrow-derived M(IL4)s were administered via intraperitoneal injection to mice concomitant with Citrobacter rodentium infection (infections colitis), azoxymethane/dextran sodium sulphate (AOM/DSS) treatment [a model of colorectal cancer (CRC)], or ovalbumin sensitization (airway inflammation). The impact of M(IL4) treatment on C. rodentium infectivity, colon histopathology, tumor number and size and tissue-specific inflammation was examined in these models. The anti-colitic effect of the M(IL4)s were confirmed in the di-nitrobenzene sulphonic acid model of colitis and the lumen-to-blood movement of 4kDa FITC-dextran and bacterial translocation to the spleen and liver was also improved by M(IL4) treatment. Analysis of the other models of disease, that represent comorbidities that can occur in human inflammatory bowel disease (IBD), revealed that M(IL4) treatment did not exaggerate the severity of any of the conditions. Rather, there was reduction in the size (but not number) of polyps in the colon of AOM/DSS-mice and reduced infectivity and inflammation in C. rodentium-infected mice in M(IL4)-treated mice. Thus, while any new therapy can have unforeseen side effects, our data confirm and extend the anti-colitic capacity of murine M(IL4)s and indicate that systemic delivery of one million M(IL4)s did not exaggerate disease in models of colonic or airways inflammation or colonic tumorigenesis.

Rationale Bronchial thermoplasty is a mechanical therapeutic intervention that has been advocated as an effective treatment option for severe asthma. The mechanism is promoted as being related to the attenuation of airway smooth muscle which has been shown to occur in the short-term. However, long-term studies of the effects of bronchial thermoplasty on airway remodeling are few with only limited assessment of airway remodeling indices. Objectives To evaluate the effect of bronchial thermoplasty on (a) airway epithelial and smooth muscle cells in culture, and (b), airway remodeling in patients with severe asthma who have been prescribed bronchial thermoplasty up to 12-months post-treatment. Methods The distribution of heat within the airway by bronchial thermoplasty was assessed in a porcine model. Culture of human airway smooth muscle cells and bronchial epithelial cells evaluated the impact of thermal injury. Histological evaluation and morphometric assessment were performed on bronchial biopsies obtained from severe asthma patients at baseline, 6-weeks, and 12-months following bronchial thermoplasty. Results Bronchial thermoplasty resulted in heterogenous heating of the airway wall. Airway smooth muscle cell cultures sustained thermal injury, whilst bronchial epithelial cells were relatively resistant to heat. Airway smooth muscle and neural bundles were significantly reduced at 6-weeks and 12-months post-treatment. At 6-weeks post treatment, submucosal collagen was reduced, and vessel density increased, with both indices returning to baseline at 12-months. Goblet cell numbers, submucosal gland area and subbasement membrane thickness, were not significantly altered at any timepoint examined. Conclusions Bronchial thermoplasty primarily affects airway smooth muscle and nerves with the effects still present at 12-months post-treatment.

Pulmonary immunity requires tight regulation, as interstitial inflammation can compromise gas exchange and lead to respiratory failure. Here we found a greater number of aged CD11bhiL-selectinloCXCR4+ polymorphonuclear leukocytes (PMNs) in lung vasculature than in the peripheral circulation. Using pulmonary intravital microscopy, we observed lung PMNs physically interacting with B cells via β2 integrins; this initiated neutrophil apoptosis, which led to macrophage-mediated clearance. Genetic deletion of B cells led to the accumulation of aged PMNs in the lungs without systemic inflammation, which caused pathological fibrotic interstitial lung disease that was attenuated by the adoptive transfer of B cells or depletion of PMNs. Thus, the lungs are an intermediary niche in the PMN lifecycle wherein aged PMNs are regulated by B cells, which restrains their potential to cause pulmonary pathology.

The evaluation of a patient with fibrotic interstitial lung disease (ILD) includes assessment of clinical, radiological, and often histopathological data. There are currently no specific recommenda...

Introduction The programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) assay is used to select patients for first or second-line pembrolizumab monotherapy in NSCLC. The PD-L1 IHC 22C3 pharmDx assay requires an Autostainer Link 48 instrument. Laboratories without this stainer have the option to develop a highly accurate 22C3 IHC laboratory-developed test (LDT) on other instruments. The Canadian 22C3 IHC LDT validation project was initiated to harmonize the quality of PD-L1 22C3 IHC LDT protocols across 20 Canadian pathology laboratories. Methods Centrally optimized 22C3 LDT protocols were distributed to participating laboratories. The LDT results were assessed against results using reference PD-L1 IHC 22C3 pharmDx. Analytical sensitivity and specificity were assessed using cell lines with varying PD-L1 expression levels (phase 1) and IHC critical assay performance controls (phase 2B). Diagnostic sensitivity and specificity were assessed using whole sections of 50 NSCLC cases (phase 2A) and tissue microarrays with an additional 50 NSCLC cases (phase 2C). Results In phase 1, 80% of participants reached acceptance criteria for analytical performance in the first attempt with disseminated protocols. However, in phase 2A, only 40% of participants reached the desired diagnostic accuracy for both 1% and 50% tumor proportion score cutoff. In phase 2B, further protocol modifications were conducted, which increased the number of successful laboratories to 75% in phase 2C. Conclusions It is possible to harmonize highly accurate 22C3 LDTs for both 1% and 50% tumor proportion score in NSCLC across many laboratories with different platforms. However, despite a centralized approach, diagnostic validation of predictive IHC LDTs can be challenging and not always successful.

The gut microbiome consists of a multi-kingdom microbial community. Whilst the role of bacteria as causal contributors governing host physiological development is well established, the role of fungi remains to be determined. Here, we use germ-free mice colonized with defined species of bacteria, fungi, or both to differentiate the causal role of fungi on microbiome assembly, immune development, susceptibility to colitis, and airway inflammation. Fungal colonization promotes major shifts in bacterial microbiome ecology, and has an independent effect on innate and adaptive immune development in young mice. While exclusive fungal colonization is insufficient to elicit overt dextran sulfate sodium-induced colitis, bacterial and fungal co-colonization increase colonic inflammation. Ovalbumin-induced airway inflammation reveals that bacterial, but not fungal colonization is necessary to decrease airway inflammation, yet fungi selectively promotes macrophage infiltration in the airway. Together, our findings demonstrate a causal role for fungi in microbial ecology and host immune functionality, and therefore prompt the inclusion of fungi in therapeutic approaches aimed at modulating early life microbiomes., The immunomodulatory role of commensal gut fungi and interactions with bacteria remain unclear. Here, using germ-free mice colonized with defined species of bacteria and fungi, the authors find that fungal colonization induces changes in bacterial microbiome ecology while having an independent effect on innate and adaptive immunity in mice.

Gut microbiomes make major contributions to the physiological and immunological development of the host, but the relative importance of their bacterial and fungal components, and how they interact, remain largely unknown. We applied carefully controlled experiments in gnotobiotic mice colonized with defined communities of bacteria, fungi, or both to differentiate the direct role of fungi on microbiome assembly, host development, and susceptibility to colitis and airway inflammation. Our results revealed that fungal colonization alone was insufficient to promote the intestinal anatomic and physiological changes seen in mice colonized by bacteria, and had limited impact on the fecal metabolome. However, fungal colonization promoted major shifts in bacterial microbiome ecology, and had an independent effect on the innate and adaptive immune development in young mice. Fungi further exacerbated some aspects of the inflammatory effects of the bacterial community during OVA-induced airway inflammation by promoting macrophage infiltration in the airway. Our results demonstrate a dominant ecological and physiological role of bacteria in gut microbiomes, but highlight fungi as an ecological factor shaping the assembly of the bacterial community and a direct capacity to impact immune system and modulate disease susceptibility. These findings demonstrate that studies focused on bacteria alone provide an incomplete portrayal on microbiome ecology and functionality, and prompt for the inclusion of fungi in human microbiome studies.

Summary During respiration, humans breathe in more than 10,000 liters of non-sterile air daily, allowing some pathogens access to alveoli. Interestingly, alveoli outnumber alveolar macrophages (AMs), which favors alveoli devoid of AMs. If AMs, like most tissue macrophages, are sessile, then this numerical advantage would be exploited by pathogens unless neutrophils from the blood stream intervened. However, this would translate to omnipresent persistent inflammation. Developing in vivo real-time intravital imaging of alveoli revealed AMs crawling in and between alveoli using the pores of Kohn. Importantly, these macrophages sensed, chemotaxed, and, with high efficiency, phagocytosed inhaled bacterial pathogens such as P. aeruginosa and S. aureus, cloaking the bacteria from neutrophils. Impairing AM chemotaxis toward bacteria induced superfluous neutrophil recruitment, leading to inappropriate inflammation and injury. In a disease context, influenza A virus infection impaired AM crawling via the type II interferon signaling pathway, and this greatly increased secondary bacterial co-infection.

Transgenic expression of a mutant hSTING cDNA under the control of the Vav1 gene promoter leads to a vasculopathy similar to that of the interferonopathy, STING-associated vasculopathy of infancy., STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disorder characterized by blood vessel occlusions, acral necrosis, myositis, rashes, and pulmonary inflammation that are the result of activating mutations in the STimulator of Interferon Genes (STING). We generated a transgenic line that recapitulates many of the phenotypic aspects of SAVI by targeting the expression of the human STING-N154S–mutant protein to the murine hematopoietic compartment. hSTING-N154S mice demonstrated failure to gain weight, lymphopenia, progressive paw swelling accompanied by inflammatory infiltrates, severe myositis, and ear and tail necrosis. However, no significant lung inflammation was observed. X-ray microscopy imaging revealed vasculopathy characterized by arteriole occlusions and venous thromboses. Type I interferons and proinflammatory mediators were elevated in hSTING-N154S sera. Importantly, the phenotype was prevented in hSTING-N154S mice lacking the type I interferon receptor gene (Ifnar1). This model, based on a mutant human STING protein, may shed light on the pathophysiological mechanisms operative in SAVI.

Airway remodeling is a characteristic feature of asthma and is thought to play an important role in the pathogenesis of airway hyperresponsiveness. Myofibroblasts are key structural cells involved in injury and repair, and there is evidence that dysregulation of their normal function contributes to airway remodeling. Despite the importance of myofibroblasts, a lack of specific cellular markers and inconsistent nomenclature have limited recognition of their key role in airway remodeling. Myofibroblasts are increased several-fold in the airways in asthma, in proportion to the severity of the disease. Myofibroblasts are postulated to be derived from both tissue-resident and bone marrow-derived cells, depending on the stage of injury and the tissue. A small number of studies have demonstrated attenuation of myofibroblast numbers and also reversal of established myofibroblast populations in asthma and other inflammatory processes. In this article, we review what is currently known about the biology of myofibroblasts in the airways in asthma and identify potential targets to reduce or reverse the remodeling process. However, further translational research is required to better understand the mechanistic role of the myofibroblast in asthma.

We report varicella-zoster virus (VZV) gastritis in a 70-year-old woman postchemotherapy for lymphoma, presenting with abdominal pain, vomiting, and delirium without rash. A gastric biopsy demonstrated viral inclusions but posed a diagnostic challenge as immunohistochemistry for cytomegalovirus and herpes simplex virus were negative, and VZV immunohistochemistry was not available. The patient developed a vesicular rash 7 days after her symptoms began. Molecular testing of the gastric biopsy and a skin swab both confirmed VZV infection. She also had probable involvement of her liver and pancreas based on imaging and serum chemistry, and possible central nervous system involvement. She recovered with appropriate antiviral therapy but later developed a postherpetic neuralgia, and chronic intrahepatic biliary strictures; liver biopsy demonstrated a cholangiopathy of uncertain etiology. A literature review of the pathogenesis, epidemiology and sequelae of VZV infection is included.

A 66-year-old man was referred to an urgent eye clinic after one week of experiencing painless proptosis and epiphora (abnormal overflow of tears down the cheek) in his right eye. The intraocular pressure was normal in both eyes. The patient had normal visual acuity but had right-gaze diplopia. No

Sepsis-associated encephalopathy manifesting as delirium is a common problem in critical care medicine. In this study, patients that had delirium due to sepsis had significant cognitive impairments at 12-18 months after hospital discharge when compared with controls and Cambridge Neuropsychological Automated Test Battery-standardized scores in spatial recognition memory, pattern recognition memory, and delayed-matching-to-sample tests but not other cognitive functions. A mouse model of S. pneumoniae pneumonia-induced sepsis, which modeled numerous aspects of the human sepsis-associated multiorgan dysfunction, including encephalopathy, also revealed similar deficits in spatial memory but not new task learning. Both humans and mice had large increases in chemokines for myeloid cell recruitment. Intravital imaging of the brains of septic mice revealed increased neutrophil and CCR2+ inflammatory monocyte recruitment (the latter being far more robust), accompanied by subtle microglial activation. Prevention of CCR2+ inflammatory monocyte recruitment, but not neutrophil recruitment, reduced microglial activation and other signs of neuroinflammation and prevented all signs of cognitive impairment after infection. Therefore, therapeutically targeting CCR2+ inflammatory monocytes at the time of sepsis may provide a novel neuroprotective clinical intervention to prevent the development of persistent cognitive impairments.

Purpose: Once lung cancer is detected due to clinical symptoms or by being visible on chest X-ray, it is usually high stage and non-operable. In order to improve mortality rates in lung cancer, low-dose CT (LDCT) screening of “high risk” individuals is gaining popularity. However, the rate of malignancy in LDCT detected sub-centimetre lung nodules is not clear. We aimed to analyze surgically resected specimens in this patient group to explore cost effectiveness and recommendations for clinical management of these nodules. Material & methods: Our hospital pathology database was searched for sub-centimeter lung nodules detected by LDCT screening which were resected. The patient demographics were collected and the radiologic and pathologic characteristics of those nodules were analyzed. Results: From the records, 44 patients with 46 resected subcentimetre nodules were identified. Patients were selected for surgery based on an irregular shape, growth in size during follow up, family history of lung cancer or personal history of cancer of other sites, previous lung disease, smoking and personal anxiety. Of the 44 patients, 33 were women and the ages ranged from 43 to 76 years (56.75 ± 8.44). All nodules were equal to, or less than 10 mm with a mean diameter of 7.81 ± 1.80 mm (SD). Out of 46 nodules, the pathological diagnoses were: invasive adenocarcinoma (ACa) in 4 (8.7%); adenocarcinoma in situ (AIS) or atypical adenomatous hyperplasia (AAH) in 29 (63%); benign fibrosis/fibrotic scar with inflammation or calcification in 12 (26.1%); an intrapulmonary benign lymph node in 1 (2.2%). Of the ACa, AIS and AAH groups (a total of 31 patients), 77% were women (24 vs. 7). The cancer or pre-cancer nodules (ACa, AIS and AAH) tended to be larger than benign fibrotic scars (P = 0.039). Amongst all characteristics, significant statistical differences were found when the following radiological features were considered: reconstructed nodule shape (P = 0.011), margin (P = 0.003) and ground glass pattern (P = 0.000). The patient’s age, the axial morphology of the lesion, relationship to major vessels or visceral pleura and location within the lung parenchyma were not predictive of the pathologic diagnosis. Only one of the 31 patients with a cancer or pre-cancer nodule was a smoker. Conclusion: ACa, AIS and AAH nodules detected on LDCT included more women (77%) than men in our cohort. Smoking as inclusive criteria for LDCT screening of lung cancer needs to be further evaluated in the Chinese population. The reconstructed nodule shape, density and margin may help radiologists to identify small cancer and pre-cancer nodules from benign conditions. Keywords: LDCT, Lung cancer screen, Lung nodule

BALF cellular analysis results are greatly impacted by processing technique #AdvancesinILD @ERSTalk http://ow.ly/u9js30i1dUP

Summary Candida albicans bloodstream infection causes fungal septicaemia and death in over half of afflicted patients. Polymorphonuclear leukocytes (PMN) mediate defense against invasive candidiasis, but their role in protection versus tissue injury and sepsis is unclear. We observe PMN intravascular swarming and subsequent clustering in response to C. albicans yeast in a lethal septic mouse and human pulmonary circulation model. Live C. albicans sequester to the endothelium and are immediately captured by complement-dependent PMN chemotaxis, which is required for host survival. However, complement activation also leads to Leukotriene B4 (LTB4)-mediated intravascular PMN clustering and occlusion, resulting in capillaritis with pulmonary hemorrhage and hypoxemia. This clustering is unique to fungi and triggered by fungal cell wall components. PMN clustering is absent in mice lacking LTB4-receptor, and capillaritis is attenuated upon pharmacological LTB4 blockade without affecting phagocytosis. Therefore, therapeutically disrupting infection-induced capillaritis may limit organ injury without impairing host defense during fungal sepsis.

BACKGROUND ThinPrep bronchial brush and wash accuracy in the diagnosis of pulmonary small cell carcinoma (pSCCa) and measured as sensitivity, specificity, positive and negative predictive values (PPV and NPV) is incompletely studied or unknown. METHODS Specimens collected over 5 years from 199 pSCCa and 938 negative (Neg) for pulmonary cancer individuals were selected by linking the laboratory file with the cancer registry. Results other than unsatisfactory were classified as true-positive and -negative, and false-positive and -negative tests so as to calculate accuracy estimates. Slides of all false-negative and -positive and randomly selected samples of true-positive and -negative tests were evaluated for 11 abnormal cell features typical of pSCCa in conventional preparations: distribution differences by disease status were tested for significance. RESULTS There were 129 brush and 170 wash in the pSCCa group and 365 brush and 1153 wash in the Neg group. Of all specimens, 1.2% were unsatisfactory. Brush sensitivity, specificity, PPV, and NPV were 61.9%, 99.4%, 97.5%, and 88%, respectively. Wash frequencies were 53.3%, 98.8%, 86.5%, and 93.5%, respectively. Abnormal cell features occurred in 29.9% of the selected pSCCa and 4.7% of the Neg specimens, and distribution differences were significant for each feature (P

We thank Moran-Mendoza et al. for their thoughtful comments regarding the recent Canadian Thoracic Society position statement on the evaluation of patients with fibrotic interstitial lung disease (...

Inflammatory myofibroblastic tumour (IMT) is a rare form of inflammatory pseudotumour of the lung. Complete surgical resection is the treatment of choice, with corticosteroid-based medical therapy reserved for cases where complete resection is not possible.1 To date, the majority of recurrent cases have occurred subsequent to incomplete resection or after discontinuation of steroids when used as primary therapy.1 2 There is a paucity of data guiding the follow-up of patients who undergo complete resection. In addition, the efficacy of oral steroids in the treatment of IMT remains poorly characterised and the optimal duration of therapy is unknown. A 46-year-old lifetime non-smoker presented in December 2015 with dyspnoea, fatigue, night sweats and weight loss. A chest X-ray demonstrated a new left lower lobe mass. His history was significant only for a right pneumonectomy in June 2005 for a large inflammatory pseudotumour. This pneumonectomy was performed for definitive surgical management, as the mass was adjacent to …

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

We assessed 6 cases acquired during routine surgical sign-out for IgG4-related disease (IRD) according to criteria from a recent consensus meeting. These cases fulfilled the morphologic criteria—that is, dense lymphoplasmacytic infiltrates, IgG4:IgG ratio greater than or equal to 0.4, and fibrosis (storiform in 4 cases—but were associated with malignancy or did not fulfill the criteria for a new site. These criteria include increased serum IgG4 (normal in the majority of IRD) and a response to glucocorticoids, which is not appropriate treatment for resectable lesions as in our cases. Until more is known about the natural history of the disease, we propose that the possibility of an early, localized, or forme fruste of IRD should be considered and that cases associated with malignancy should at least be documented. Although we acknowledge the value of the consensus criteria, their strict application may result in missed opportunities to study the disease.

Hepatitis induced by concanavalin A (Con A) in mice is well known to be a T-lymphocyte-mediated injury. It has been reported that T helper (Th)1 and Th2 lymphocytes use α4 integrin and vascular adhesion protein (VAP)−1, respectively, to adhere within the hepatic sinusoids. Therefore, we investigated whether inhibition of these molecules ameliorates or worsens the Con A-induced hepatic injury in vivo. Vehicle or antibody to α4 integrin or VAP-1 was intravenously administered 30 minutes before Con A administration. In control mice Con A markedly increased the serum alanine aminotransferase (ALT) level in a dose-dependent manner, and induced a massive infiltration of CD3, particularly interleukin (IL)−4 producing CD4 T cells and liver injury. Both parameters were reduced by anti-VAP-1 antibody despite antibody only blocking the adhesion, not the amine oxidase activity of VAP-1. Both activities of VAP-1 were eliminated in VAP-1-deficient mice and both Con A-induced liver injury and CD4 T-cell infiltration were eradicated. In contrast to anti-VAP-1, anti-α4 integrin antibody reduced interferon-gamma (IFN-γ)-producing CD3 T cells but this worsened Con A hepatitis, suggesting inhibition of a suppressor cell. Con A induced the recruitment of CD49d+ monocytic myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) into the liver. Anti-α4 integrin dramatically blocked the influx of MDSCs but not Tregs. Conclusion: Our findings show that VAP-1 and α4 integrin have opposing effects in Con A-induced hepatic injury, which is associated with blocking the recruitment of CD4 lymphocytes and monocytic MDSCs, respectively. Moreover, these data provide the rationale for a potential therapeutic approach to target adhesion molecules in autoimmune hepatitis. (Hepatology 2013;58:1413–1423)

Background Increased airway smooth muscle is observed in large and small airways in asthma. Semi-quantitative estimates suggest that cells containing alpha smooth muscle actin (α-SMA) are also increased in the lung parenchyma. This study quantified and characterized α-SMA positive cells (α-SMA+) in the lung parenchyma of non-asthmatic and asthmatic individuals. Methods Post-mortem sections of peripheral lung from cases of fatal asthma (FA), persons with asthma dying of non-respiratory causes (NFA) and non-asthma control subjects (NAC) were stained for α-SMA, quantified using point-counting and normalised to alveolar basement membrane length and interstitial area. Results α-SMA+ fractional area was increased in alveolar parenchyma in both FA (14.7 ± 2.8% of tissue area) and NFA (13.0 ± 1.2%), compared with NAC (7.4 ± 2.4%), p < 0.05 The difference was greater in upper lobes compared with lower lobes (p < 0.01) in both asthma groups. Similar changes were observed in alveolar ducts and alveolar walls. The electron microscopic features of the α-SMA+ cells were characteristic of myofibroblasts. Conclusions We conclude that in asthma there is a marked increase in α-SMA+ myofibroblasts in the lung parenchyma. The physiologic consequences of this increase are unknown.

Although inhaled glucocorticoids, or corticosteroids (ICS), are generally effective in asthma, understanding their anti‐inflammatory actions in vivo remains incomplete. To characterize glucocorticoid‐induced modulation of gene expression in the human airways, we performed a randomized placebo‐controlled crossover study in healthy male volunteers. Six hours after placebo or budesonide inhalation, whole blood, bronchial brushings, and endobronchial biopsies were collected. Microarray analysis of biopsy RNA, using stringent (≥2‐fold, 5% false discovery rate) or less stringent (≥1.25‐fold, P ≤ 0.05) criteria, identified 46 and 588 budesonide‐induced genes, respectively. Approximately two third of these genes are transcriptional regulators (KLF9, PER1, TSC22D3, ZBTB16), receptors (CD163, CNR1, CXCR4, LIFR, TLR2), or signaling genes (DUSP1, NFKBIA, RGS1, RGS2, ZFP36). Listed genes were qPCR verified. Expression of anti‐inflammatory and other potentially beneficial genes is therefore confirmed and consistent with gene ontology (GO) terms for negative regulation of transcription and gene expression. However, GO terms for transcription, signaling, metabolism, proliferation, inflammatory responses, and cell movement were also associated with the budesonide‐induced genes. The most enriched functional cluster indicates positive regulation of proliferation, locomotion, movement, and migration. Moreover, comparison with the budesonide‐induced expression profile in primary human airway epithelial cells shows considerable cell type specificity. In conclusion, increased expression of multiple genes, including the transcriptional repressor, ZBTB16, that reduce inflammatory signaling and gene expression, occurs in the airways and blood and may contribute to the therapeutic efficacy of ICS. This provides a previously lacking insight into the in vivo effects of ICS and should promote strategies to improve glucocorticoid efficacy in inflammatory diseases.

Pulmonary fibrosis, the end stage of a variety of fibroproliferative lung diseases, is usually induced after repetitive or chronic lung injury or inflammation. The mechanisms of fibroproliferation are poorly understood. Insulin-like growth factor-I (IGF-I) is significantly elevated in patients with pulmonary fibrosis and fibroproliferative acute respiratory distress syndrome. However, we showed that IGF-I overexpression alone in wild-type mouse lungs does not cause fibroproliferation. We therefore questioned whether IGF-I, acting together with active TGF-β1, a known profibrotic cytokine, enhances pulmonary fibroproliferation caused by active TGF-β1. A unique sequential adenoviral transgene mouse model was used expressing AdEmpty/AdTGF-β1 or AdhIGF-IB/AdTGF-β1 transgenes. IGF-IB plus active TGF-β1 transgene expression synergistically increased collagen deposition in the lung parenchyma compared with active TGF-β1 expression alone. The enhanced fibrosis was accompanied by an increased recruitment of macrophages and lymphocytes into the bronchoalveolar lavage fluid (BALF) and inflammatory cells in the lungs. α-Smooth muscle actin expression, a marker of myofibroblast proliferation and differentiation, was also increased. Finally, fibroblasts exposed ex vivo to BALF isolated from AdhIGF-IB/AdTGF-β1-transduced mice showed synergistic collagen induction compared with BALF from AdEmpty/AdTGF-β1-transduced mice. This study provides the first direct evidence that IGF-I is able to synergistically enhance pulmonary fibroproliferation in cooperation with TGF-β1.

BACKGROUND AND PURPOSE Inhaled corticosteroids (ICS) are the cornerstone of asthma pharmacotherapy and, acting via the glucocorticoid receptor (GR), reduce inflammatory gene expression. While this is often attributed to a direct inhibitory effect of the GR on inflammatory gene transcription, corticosteroids also induce the expression of anti-inflammatory genes in vitro. As there are no data to support this effect in asthmatic subjects taking ICS, we have assessed whether ICS induce anti-inflammatory gene expression in subjects with atopic asthma. EXPERIMENTAL APPROACH Bronchial biopsies from allergen-challenged atopic asthmatic subjects taking inhaled budesonide or placebo were subjected to gene expression analysis using real-time reverse transcriptase-PCR for the corticosteroid-inducible genes (official gene symbols with aliases in parentheses): TSC22D3 [glucocorticoid-induced leucine zipper (GILZ)], dual-specificity phosphatase-1 (MAPK phosphatase-1), both anti-inflammatory effectors, and FKBP5 [FK506-binding protein 51 (FKBP51)], a regulator of GR function. Cultured pulmonary epithelial and smooth muscle cells were also treated with corticosteroids before gene expression analysis. KEY RESULTS Compared with placebo, GILZ and FKBP51 mRNA expression was significantly elevated in budesonide-treated subjects. Budesonide also increased GILZ expression in human epithelial and smooth muscle cells in culture. Immunostaining of bronchial biopsies revealed GILZ expression in the airways epithelium and smooth muscle of asthmatic subjects. CONCLUSIONS AND IMPLICATIONS Expression of the corticosteroid-induced genes, GILZ and FKBP51, is up-regulated in the airways of allergen-challenged asthmatic subjects taking inhaled budesonide. Consequently, the biological effects of corticosteroid-induced genes should be considered when assessing the actions of ICS. Treatment modalities that increase or decrease GR-dependent transcription may correspondingly affect corticosteroid efficacy.

Background and objective: Both the efficacy and toxicity of sclerosing agents are likely to be dose-dependent. Clinical pleurodesis strategies typically involve single bolus dose administration of drugs. This study was designed to test whether repeated administration of low doses of silver nitrate (SN) could lead to effective pleurodesis. Methods: Intrapleural administration, to rabbits, of decreasing doses of SN or normal saline was undertaken daily over 1, 5 or 14 days. Assessment of the degree of pleurodesis was by visual inspection (score 1–8) and histological examination and scoring of inflammation and fibrosis (score 0–4). The untreated contralateral side was used as a control. A visual pleurodesis score of ≥5 was considered to be positive. Results: The lowest concentrations of SN leading to a visual pleurodesis score ≥5 were 0.425%, 0.085% and 0.05% for 1, 5 and 14 day administration protocols respectively (P

The endothelial response to LPS is critical in the recruitment of leukocytes, thereby allowing the host to survive Gram-negative infection. Herein, we investigated the roles of soluble CD14 (sCD14) and membrane CD14 (mCD14) in the endothelial response to low level LPS (0.1 ng/ml), intermediate level LPS (10 ng/ml), and high level LPS (1000 ng/ml). Removal of sCD14 from serum and sCD14-negative serum prevented low level LPS detection and subsequent response. Addition of recombinant sCD14 back into the endothelial system rescued the endothelial response. GPI-linked mCD14 removal from endothelium or endothelial treatment with a CD14 mAb prevented responses to low-level LPS even in the presence of sCD14. This demonstrates essential nonoverlapping roles for both mCD14 and sCD14 in the detection of low-level LPS. At intermediate levels of LPS, sCD14 was not required, but blocking mCD14 still prevented endothelial LPS detection and E-selectin expression, even in the presence of sCD14, suggesting that sCD14 cannot substitute for mCD14. At very high levels of LPS, the absence of mCD14 and sCD14 did not abrogate TLR4-dependent, E-selectin synthesis in response to LPS. The MyD88 independent pathway was detected in endothelium (presence of TRIF-related adaptor molecule TRAM). The MyD88-independent response (IFN-β) in endothelium required mCD14 even at the highest LPS dose tested. Our results demonstrate an essential role for endothelial mCD14 that cannot be replaced by sCD14. Furthermore, we have provided evidence for a TRAM pathway in endothelium that is dependent on mCD14 even when other responses are no longer mCD14 dependent.

Vaginal epithelium is regulated by female sex hormones and serves as the first line of innate immune defense against sexually transmitted infections (STIs). This occurs in part through recognition of pathogens via Toll-like receptors (TLRs); however, the expression and role of TLRs in reproductive tract immunity are poorly understood. Here, we have compared the effect of the estrous cycle and treatment with DepoProvera (Depo) on TLR mRNA expression in whole mouse vaginal tissue, vaginal epithelium isolated using laser capture microdissection (LCM) and in primary vaginal epithelial cells (ECs) grown in vitro. Distinct patterns of TLR expression were observed in LCM-isolated vaginal epithelium versus whole vaginal tissue. Absolute quantitative RT-PCR of LCM vaginal epithelium showed that expression of all TLRs, except TLR11, was significantly increased during the diestrus phase or following Depo-treatment. TLR2 mRNA showed an extraordinary increase in expression in both diestrus and following Depo-treatment (23-fold) over that in the estrus phase. Although TLR2 protein was expressed at similar levels over the estrous cycle in whole vaginal tissue, full-length TLR2 protein was only detected during diestrus or after Depo-treatment in LCM-isolated vaginal epithelium. Distinct TLR mRNA expression profiles were seen also in primary vaginal ECs in vitro and only expression of TLR2 was significantly decreased in ECs cultured in the presence of stromal cells. Thus, TLR expression in vaginal ECs is regulated by sex hormones and can be affected by stromal cells. These findings contribute to our understanding of innate immune defense against STIs and enhance the quality of woman's reproductive health.

Gamma-aminobutyric acid (GABA) is an important neurotransmitter that, through the subtype A GABA receptor (GABAAR), induces inhibition in the adult brain. Here we show that an excitatory, rather than inhibitory, GABAergic system exists in airway epithelial cells. Both GABAARs and the GABA synthetic enzyme glutamic acid decarboxylase (GAD) are expressed in pulmonary epithelial cells. Activation of GABAARs depolarized these cells. The expression of GAD in the cytosol and GABAARs in the apical membranes of airway epithelial cells increased markedly when mice were sensitized and then challenged with ovalbumin, an approach for inducing allergic asthmatic reactions. Similarly, GAD and GABAARs in airway epithelial cells of humans with asthma increased after allergen inhalation challenge. Intranasal application of selective GABAAR inhibitors suppressed the hyperplasia of goblet cells and the overproduction of mucus induced by ovalbumin or interleukin-13 in mice. These findings show that a previously unknown epithelial GABAergic system has an essential role in asthma.

Usual interstitial pneumonia (UIP) is characterized by extracellular matrix deposition and the development of pulmonary fibrosis. Fibroblastic foci found in the lung are believed to represent an early stage in the evolution of this disease.To compare gene expression profiles in different components of lung tissue (fibroblastic foci, adjacent epithelium, and areas of type 2 pneumocyte hyperplasia) from patients with UIP, and contrast these profiles to distal, uninvolved (control) alveolar tissue from patients undergoing lung resection for cancer.Lung resection tissue (UIP, n = 11; controls, n = 11) was snap-frozen for subsequent laser capture microdissection, followed by mRNA extraction, linear amplification, and quantitative real-time polymerase chain reaction.In patients with UIP, tissue inhibitor of matrix metalloprotease-1 and matrix metalloprotease (MMP)-2 gene expression was up-regulated within the fibroblastic foci compared with the overlying epithelium (p = 0.03, p = 0.02), and to control alveoli (p = 0.001, p = 0.04), respectively. MMP-9 and MMP-7, as well as osteopontin, were up-regulated in fibroblastic foci (p = 0.01, p = 0.08, p = 0.08), the adjacent epithelium (p = 0.001, p = 0.001, p = 0.03), and the hyperplastic type 2 pneumocytes (p = 0.02, p = 0.001, p = 0.08), respectively, compared with control alveoli.Altered gene expression of important profibrotic mediators in the different cellular lung compartments in patients with UIP likely plays an important role in pathogenesis of the deranged extracellular matrix deposition and subsequent fibrosis in this condition.

The beta-arrestins, a small family of G protein-coupled receptor (GPCR)-binding proteins involved in receptor desensitization, have been shown to bind extracellular signal-regulated kinases 1 and 2 (ERK1/2) and function as scaffolds for GPCR-stimulated ERK1/2 activation. To better understand the mechanism of beta-arrestin-mediated ERK1/2 activation, we compared ERK1/2 activation by the wild-type neurokinin 1 (NK1) receptor with a chimeric NK1 receptor having beta-arrestin1 fused to the receptor C terminus (NK1-betaArr1). The NK1 receptor couples to both G(s) and G(q/11), resides on the plasma membrane, and mediates rapid ERK1/2 activation and nuclear translocation in response to neurokinin A. In contrast, NK1-betaArr1 is a G protein-uncoupled "constitutively desensitized" receptor that resides almost entirely in an intracellular endosomal compartment. Despite its inability to respond to neurokinin A, we found that NK1-betaArr1 expression caused robust constitutive activation of cytosolic ERK1/2 and that endogenous Raf, MEK1/2, and ERK1/2 coprecipitated in a complex with NK1-betaArr1. While agonist-dependent ERK1/2 activation by the NK1 receptor was independent of protein kinase A (PKA) or PKC activity, NK1-betaArr1-mediated ERK1/2 activation was completely inhibited when basal PKA and PKC activity were blocked. In addition, the rate of ERK1/2 dephosphorylation was slowed in NK1-betaArr1-expressing cells, suggesting that beta-arrestin-bound ERK1/2 is protected from mitogen-activated protein kinase phosphatase activity. These data suggest that beta-arrestin binding to GPCRs nucleates the formation of a stable "signalsome" that functions as a passive scaffold for the ERK1/2 cascade while confining ERK1/2 activity to an extranuclear compartment.

Background Newer generations and formulations of inhaled corticosteroids have necessitated the development of a clinically relevant model to compare their clinical potency. Objective We evaluated whether sputum eosinophil counts could demonstrate a dose-response to inhaled corticosteroids, and compared the response with other inflammatory markers. Methods Fourteen steroid-naive patients with asthma with an initial sputum eosinophilia of ≥2.5% entered a 6-week sequential, placebo-controlled, patient-blinded, cumulative dose-response study. After 7 days of placebo, they received incremental doses of fluticasone propionate (FP), 50, 100, 200, and 400 μg/d, each for 7 days. Measurements were made of sputum and blood eosinophils, exhaled nitric oxide, spirometry, airway responsiveness to methacholine (methacholine PC 20 ), and symptom scores before and after each dose. Results Sputum eosinophils and exhaled nitric oxide were extremely sensitive to the effects of FP, and exhibited significant dose-dependent reductions of 99.4% and 99.8 parts per billion, respectively, where each variable was expressed per 100 μg/d FP. This compared with a 0.5 doubling dose increase of airway responsiveness to methacholine and a 0.3 decrease in symptom scores. Airway responsiveness to methacholine was the only variable that increased throughout the study. Conclusion These results suggest that the model of eosinophilic bronchitis could be used to compare the effect of cumulative doses of an inhaled corticosteroid delivered by different types of delivery systems or preparations using a relatively small number of patients. Clinical implications Future clinical studies based on this model will allow clinicians to make informed decisions regarding the relative potencies of different inhaled corticosteroids.

Airway remodeling, including subepithelial fibrosis, is a characteristic feature of asthma and likely contributes to the pathogenesis of airway hyperresponsiveness. We examined expression of genes related to airway wall fibrosis in a model of chronic allergen-induced airway dysfunction using laser capture microdissection and quantitative real-time PCR. BALB/c mice were sensitized and subjected to chronic ovalbumin exposure over a 12-wk period, after which they were rested and then harvested 2 and 8 wk after the last exposure. Chronic allergen-exposed mice had significantly increased indices of airway remodeling and airway hyperreactivity at all time points, although no difference in expression of fibrosis-related genes was found when mRNA extracted from whole lung was examined. In contrast, fibrosis-related gene expression was significantly upregulated in mRNA obtained from microdissected bronchial wall at 2 wk after chronic allergen exposure. In addition, when bronchial wall epithelium and smooth muscle were separately microdissected, gene expression of transforming growth factor-beta1 and plasminogen activating inhibitor-1 were significantly upregulated only in the airway epithelium. These data suggest that transforming growth factor-beta1 and other profibrotic mediators produced by airway wall, and specifically, airway epithelium, play an important role in the pathophysiology of airway remodeling.

Connective tissue growth factor (CTGF) is felt to be one of the key profibrotic factors and is a downstream effector molecule mediating the action of transforming growth factor (TGF)-beta1, a cytokine known to induce severe and progressive fibrosis. However, the in vivo fibrogenic effect of isolated CTGF expression is not well described. We used adenoviral gene transfer to transiently overexpress CTGF in rat lungs after intratracheal administration and compared it with transient overexpression of active TGF-beta1 delivered by a similar adenovirus vector. This high expression of CTGF over 6-10 days induced a moderate but reversible fibrosis. We observed an increase of fibronectin, procollagen 1a2, and endogenous CTGF gene expression at 14 days, which suggested an indirect activation by CTGF. Tissue inhibitor of metalloproteinase-1 was weakly and transiently upregulated after CTGF exposure. These same genes were robustly and persistently stimulated by TGF-beta1 from Day 3 to Day 21. This data suggested that CTGF may act as a TGF-beta1 cofactor rather than a direct fibrogenic factor. We demonstrate that CTGF overexpression can initiate fibrogenic activity but likely requires the presence of additional factors, such as tissue inhibitor of metalloproteinase-1, to maintain a nonfibrolytic environment and to cause progression of fibrosis.

BACKGROUND: Treatment with inhaled glucocorticosteroids attenuates allergen-induced airway inflammation but is less effective in people with asthma who have noneosinophilic airway inflammation.OBJECTIVE: Studies in which glucocorticosteroid treatment was used before allergen challenges were re-examined to determine whether the efficacy of steroid treatment could be predicted by baseline levels of sputum inflammatory cells.PATIENTS AND METHODS: Twenty-eight nonsmoking subjects with atopic asthma controlled by beta2-agonists participated in only one of three studies, each carried out with a double-blind, placebo controlled, randomized, crossover design. Subjects were treated with glucocorticosteroids or placebo for six to eight days and then underwent allergen inhalation challenge. Spirometry was measured for 7 h after allergen challenge, and then sputum inflammatory cells were measured. Sputum inflammatory cells were also measured before and after treatment, and 24 h after allergen challenge. The per cent inhibition of the allergen-induced airway responses by glucocorticosteroids was calculated.RESULTS: Inhaled gluticocorticosteroids significantly attenuated the early and late asthmatic responses, and the number of allergen-induced sputum eosinophils (PCONCLUSIONS: Baseline airway neutrophils, not eosinophils, can be used to predict the efficacy of inhaled steroids on allergen-induced sputum eosinophils.

IL-5 measurement in the fluid phase of induced sputum is considered to be important in the assessment of asthma, but the validity of these measurements is uncertain.We investigated the validity of sputum IL-5 measurements through a series of spiking experiments and examined the effect of dithiothreitol (DTT) on these measurements.Induced sputum from 26 asthmatic subjects was spiked with IL-5 and processed, and the percentage of recovery was measured by means of immunoassay. In 6 of the 26 samples the effect of adding albumin to the processing fluids was studied. In 3 separate samples radiolabeled IL-5 was added, and the recovery measured by means of gamma counting and immunoassay were compared. In addition, the effect of DTT on the immunoassay was examined.The mean +/- SD recovery of spiked IL-5 was 26.1% +/- 14.6% measured by means of immunoassay; adding albumin increased the recovery to 47.7% +/- 8.0% (P.001). The mean recovery measured by means of gamma counting was 84.8% +/- 5.7% (P.001); adding albumin had no effect on recovery. DTT had no significant effect on IL-5 measurement.The validity of IL-5 measurement by means of current methods is poor. The discrepancy in recovery as measured by gamma counting compared with immunoassay suggests that there is a problem with the recognition of IL-5 epitopes by immunoassay in induced sputum. This cannot be attributed to DTT but may be due to other interfering substances present in sputum, such as sputum proteases, soluble receptors, or autoantibodies.

Treatment with testosterone propionate (TP) and diethylstilbestrol (DES) or TP and estradiol (E2) for 8–9 months causes development of leiomyosarcomas in the vas deferens or uterus of Golden Syrian hamsters at a frequency of 100%. In males, treatment with estrogens alone results in renal tumors, fatal within 6 months. No leiomyosarcomas have been detected after treatment with estrogens alone, perhaps due to this high mortality rate. In tissue culture, treatment with the glucocorticoid (GC) triamcinolone acetonide (TA) results in an increased expression of a μ-class glutathione S-transferase (GST) (hGSTYBX). We have characterized this induction as a secondary response, i.e. requiring new protein synthesis. Here we describe homologies to known transcription factor-binding sites in the hGSTYBX promoter which may be involved in the induction event. hGSTYBX is a member of a superfamily of detoxification enzymes, induced by genotoxic compounds and reactive oxygen species (ROS). We also describe the effects of several known inducers of other GST family members on hGSTYBX. While implicated in many chemotherapeutic-resistant tumors, GST enzymes have not yet been characterized as a functional agent in hormonal carcinogenesis. This latter possibility is the focus of our investigations. To study the effects of hormone treatment on GST levels in vivo, we have developed a polyclonal antibody to hGSTYBX, and conducted immunohistochemistry on tissues from control and treated animals. Treatment with TP and E2 causes a loss of hGSTYBX expression in the epithelium of the vas deferens. We hypothesize that the loss of this protective enzyme leaves the cells vulnerable to the genotoxic effects of estrogen or estrogenic metabolites.