MicroRNA-mediated Krüppel-like factor 4 upregulation induces alternatively activated macrophage-associated marker and chemokine transcription in 4,4'-methylene diphenyl diisocyanate exposed macrophages.

1. Occupational exposure to 4,4'-methylene diphenyl diisocyanate (MDI) is associated with occupational asthma (OA) development. Alveolar macrophage-induced recruitment of immune cells to the lung microenvironment plays an important role during asthma pathogenesis. Previous studies identified that MDI/MDI-glutathione (GSH)-exposure downregulates endogenous hsa-miR-206-3p/hsa-miR-381-3p. Our prior report shows that alternatively activated (M2) macrophage-associated markers/chemokines are induced by MDI/MDI-GSH-mediated Krüppel-Like Factor 4 (KLF4) upregulation in macrophages and stimulates immune cell chemotaxis. However, the underlying molecular mechanism(s) by which MDI/MDI-GSH upregulates KLF4 remain unclear.2. Following MDI-GSH exposure, microRNA(miR)-inhibitors/mimics or plasmid transfection, endogenous hsa-miR-206-3p/hsa-miR-381-3p, KLF4, or M2 macrophage-associated markers (CD206, TGM2), and chemokines (CCL17, CCL22, CCL24) were measured by either RT-qPCR, western blot, or luciferase assay.3. MDI-GSH exposure downregulates hsa-miR-206-3p/hsa-miR-381-3p by 1.46- to 9.75-fold whereas upregulates KLF4 by 1.68- to 1.99-fold, respectively. In silico analysis predicts binding between hsa-miR-206-3p/hsa-miR-381-3p and KLF4. Gain- and loss-of-function, luciferase reporter assays and RNA-induced silencing complex-immunoprecipitation (RISC-IP) studies confirm the posttranscriptional regulatory roles of hsa-miR-206-3p/hsa-miR-381-3p and KLF4 in macrophages. Furthermore, hsa-miR-206-3p/hsa-miR-381-3p regulate the expression of M2 macrophage-associated markers and chemokines via KLF4.4. In conclusion, hsa-miR-206-3p/hsa-miR-381-3p play a major role in regulation of MDI/MDI-GSH-induced M2 macrophage-associated markers and chemokines by targeting the KLF4 transcript, and KLF4-mediated regulation in macrophages.

Ruxolitinib for Refractory PL-12 Antisynthetase Syndrome-Associated Angioedema-Like Panniculitis With Clonal T-Cell Receptor Gene Rearrangement.

This case report describes a woman in her 30s who presented with a 3-year history of anti­PL-12 antisynthetase syndrome characterized by interstitial lung disease, arthritis, and myositis and was diagnosed with antisynthetase syndrome­associated panniculitis.

4,4'-Methylene diphenyl diisocyanate exposure induces expression of alternatively activated macrophage-associated markers and chemokines partially through Krüppel-like factor 4 mediated signaling in macrophages.

Occupational exposure to the most widely used monomeric diisocyanate (dNCO), 4,4'-methylene diphenyl diisocyanate (MDI), may lead to the development of occupational asthma (OA). Alveolar macrophages with alternatively activated (M2) phenotype have been implicated in allergic airway responses and the pathogenesis of asthma. Recent in vivo studies demonstrate that M2 macrophage-associated markers and chemokines are induced by MDI-exposure, however, the underlying molecular mechanism(s) by which this proceeds is unclear.Following MDI exposure (in vivo and in vitro) M2 macrophage-associated transcription factors (TFs), markers, and chemokines were determined by RT-qPCR, western blots, and ELISA.Expression of M2 macrophage-associated TFs and markers including Klf4/KLF4, Cd206/CD206, Tgm2/TGM2, Ccl17/CCL17, Ccl22/CCL22, and CCL24 were induced by MDI/MDI-GSH exposure in bronchoalveolar lavage cells (BALCs)/THP-1 macrophages. The expression of CD206, TGM2, CCL17, CCL22, and CCL24 are upregulated by 3.83-, 7.69-, 6.22-, 6.08-, and 1.90-fold in KLF4-overexpressed macrophages, respectively. Endogenous CD206 and TGM2 were downregulated by 1.65-5.17-fold, and 1.15-1.78-fold, whereas CCL17, CCL22, and CCL24 remain unchanged in KLF4-knockdown macrophages. Finally, MDI-glutathione (GSH) conjugate-treated macrophages show increased chemotactic ability to T-cells and eosinophils, which may be attenuated by KLF4 knockdown.Our data suggest that MDI exposure may induce M2 macrophage-associated markers partially through induction of KLF4.

Characterization of inhalation exposures at a wildfire incident during the Wildland Firefighter Exposure and Health Effects (WFFEHE) Study.

Wildland firefighters (WFFs) are exposed to many inhalation hazards working in the wildland fire environment. To assess occupational exposures and acute and subacute health effects among WFFs, the wildland firefighter exposure and health effects study collected data for a 2-year repeated measures study. This manuscript describes the exposure assessment from one Interagency Hotshot Crew (N = 19) conducted at a wildfire incident. Exposures to benzene, toluene, ethylbenzene, xylene isomers, formaldehyde, acetaldehyde, and naphthalene were measured through personal air sampling each work shift. Biological monitoring was done for creatinine-adjusted levoglucosan in urine pre- and post-shift. For 3 days sampling at the wildfire incident, benzene, toluene, ethylbenzene, xylene isomers (m and p, and o) exposure was highest on day 1 (geometric mean [GM] = 0.015, 0.042, 0.10, 0.42, and 0.15 ppm, respectively) when WFFs were not exposed to smoke but used chainsaws to remove vegetation and prepare fire suppression breaks. Exposure to formaldehyde and acetaldehyde was highest on day 2 (GM = 0.03 and 0.036 ppm, respectively) when the WFFs conducted a firing operation and were directly exposed to wildfire smoke. The greatest difference of pre- and post-shift levoglucosan concentrations were observed on day 3 (pre-shift: 9.7 and post-shift: 47 µg/mg creatinine) after WFFs conducted mop up (returned to partially burned area to extinguish any smoldering vegetation). Overall, 65% of paired samples (across all sample days) showed a post-shift increase in urinary levoglucosan and 5 firefighters were exposed to benzene at concentrations at or above the National Institute for Occupational Safety and Health (NIOSH) recommended exposure limit. Our findings further demonstrate that exposure to inhalation hazards is one of many risks that wildland firefighters experience while suppressing wildfires.

Automated crude oil vapor inhalation exposure system.

Objective: Inhalation exposure systems are tools for delivering compounds (particles, vapors, and gases) under well-controlled conditions for toxicological testing. The objective of this project was to develop an automated computer-controlled system to expose small laboratory animals to precise concentrations of crude oil vapor (COV).Materials and Methods: Vapor from heated Deepwater Horizon surrogate oil was atomized into a fine mist then diluted with filtered air, then the air/droplet mixture was routed into an evaporation column with an high efficiency particulate air (HEPA) filter on its exit port. The HEPA filter was used to remove oil particles, thus ensuring only vapor would pass. The vapor was then introduced into a custom-built exposure chamber housing rats. A calibrated flame ionization detector was used to read the total volatile organic compounds (TVOC) in real time, and custom software was developed to automatically adjust the amount of oil entering the atomizer with a syringe pump. The software also controlled relative humidity and pressure inside the exposure chamber. Other exposure chamber environmental parameters, e.g. temperature and CO2 levels, were monitored. Four specific components within the COV were monitored during each exposure: benzene, toluene, ethylbenzene, and xylenes.Results: The TVOC vapor concentration control algorithm maintained median concentrations to within ±2 ppm of the target concentration (300 ppm) of TVOC during exposures lasting 6 h. The system could reach 90% of the desired target in less than 15 min, and repeat exposures were consistent and reproducible.Conclusion: This exposure system provided a highly automated tool for conducting COV inhalation toxicology studies.

MicroRNA-mediated calcineurin signaling activation induces CCL2, CCL3, CCL5, IL8, and chemotactic activities in 4,4'-methylene diphenyl diisocyanate exposed macrophages.

Occupational exposure to 4,4'-methylene diphenyl diisocyanate (MDI), the most widely used monomeric diisocyanate, is one of the leading causes of occupational asthma (OA). Previously, we identified microRNA (miR)-206-3p/miR-381-3p-mediated PPP3CA/calcineurin signalling regulated iNOS transcription in macrophages and bronchoalveolar lavage cells (BALCs) after acute MDI exposure; however, whether PPP3CA/calcineurin signalling participates in regulation of other asthma-associated mediators secreted by macrophages/BALCs after MDI exposure is unknown.Several asthma-associated, macrophage-secreted mediator mRNAs from MDI exposed murine BALCs and MDI-glutathione (GSH) conjugate treated differentiated THP-1 macrophages were analysed using RT-qPCR.Endogenous IL1B, TNF, CCL2, CCL3, CCL5, and TGFB1 were upregulated in MDI or MDI-GSH conjugate exposed BALCs and macrophages, respectively. Calcineurin inhibitor tacrolimus (FK506) attenuated the MDI-GSH conjugate-mediated induction of CCL2, CCL3, CCL5, and CXCL8/IL8 but not others. Transfection of either miR-inhibitor-206-3p or miR-inhibitor-381-3p in macrophages induced chemokine CCL2, CCL3, CCL5, and CXCL8 transcription, whereas FK506 attenuated the miR-inhibitor-206-3p or miR-inhibitor-381-3p-mediated effects. Finally, MDI-GSH conjugate treated macrophages showed increased chemotactic ability to various immune cells, which may be attenuated by FK506.In conclusion, these results indicate that MDI exposure to macrophages/BALCs may recruit immune cells into the airway via induction of chemokines by miR-206-3p and miR-381-3p-mediated calcineurin signalling activation.

A comparison of performance metrics for cloth face masks as source control devices for simulated cough and exhalation aerosols.

Universal mask wearing is recommended by the Centers for Disease Control and Prevention to help control the spread of COVID-19. Masks reduce the expulsion of respiratory aerosols (called source control) and offer some protection to the wearer. However, masks vary greatly in their designs and construction materials, and it is not clear which are most effective. Our study tested 15 reusable cloth masks (which included face masks, neck gaiters, and bandanas), two medical masks, and two N95 filtering facepiece respirators as source control devices for aerosols ≤ 7 µm produced during simulated coughing and exhalation. These measurements were compared with the mask filtration efficiencies, airflow resistances, and fit factors. The source control collection efficiencies for the cloth masks ranged from 17% to 71% for coughing and 35% to 66% for exhalation. The filtration efficiencies of the cloth masks ranged from 1.4% to 98%, while the fit factors were 1.3 to 7.4 on an elastomeric manikin headform and 1.0 to 4.0 on human test subjects. The correlation coefficients between the source control efficacies and the other performance metrics ranged from 0.31 to 0.66 and were significant in all but one case. However, none of the alternative metrics were strong predictors of the source control performance of cloth masks. Our results suggest that a better understanding of the relationships between source control performance and metrics like filtration efficiency, airflow resistance, and fit factor are needed to develop simple methods to estimate the effectiveness of masks as source control devices for respiratory aerosols.

A comparison of performance metrics for cloth masks as source control devices for simulated cough and exhalation aerosols.

Universal mask wearing is recommended to help control the spread of COVID-19. Masks reduce the expulsion of aerosols of respiratory fluids into the environment (called source control) and offer some protection to the wearer. Masks are often characterized using filtration efficiency, airflow resistance, and manikin or human fit factors, which are standard metrics used for personal protective devices. However, none of these metrics are direct measurements of how effectively a mask blocks coughed and exhaled aerosols. We studied the source control performance of 15 cloth masks (face masks, neck gaiters, and bandanas), two medical masks, and two N95 filtering facepiece respirators by measuring their ability to block aerosols ≤ 7 µm expelled during simulated coughing and exhalation (called source control collection efficiency). These measurements were compared with filtration efficiencies, airflow resistances, and fit factors measured on manikin headforms and humans. Collection efficiencies for the cloth masks ranged from 17% to 71% for coughing and 35% to 66% for exhalation. Filtration efficiencies for the cloth masks ranged from 1.4% to 98%, while the fit factors were 1.3 to 7.4 on headforms and 1.0 to 4.0 on human subjects. The Spearman's rank correlation coefficients between the source control collection efficiencies and the standard metrics ranged from 0.03 to 0.68 and were significant in all but two cases. However, none of the standard metrics were strongly correlated with source control performance. A better understanding of the relationships between source control collection efficiency, filtration efficiency, airflow resistance, and fit factor is needed.

Efficacy of face masks, neck gaiters and face shields for reducing the expulsion of simulated cough-generated aerosols.

Face masks are recommended to reduce community transmission of SARS-CoV-2. One of the primary benefits of face masks and other coverings is as source control devices to reduce the expulsion of respiratory aerosols during coughing, breathing, and speaking. Face shields and neck gaiters have been proposed as an alternative to face masks, but information about face shields and neck gaiters as source control devices is limited. We used a cough aerosol simulator with a pliable skin headform to propel small aerosol particles (0 to 7 µm) into different face coverings. An N95 respirator blocked 99% (standard deviation (SD) 0.3%) of the cough aerosol, a medical grade procedure mask blocked 59% (SD 6.9%), a 3-ply cotton cloth face mask blocked 51% (SD 7.7%), and a polyester neck gaiter blocked 47% (SD 7.5%) as a single layer and 60% (SD 7.2%) when folded into a double layer. In contrast, the face shield blocked 2% (SD 15.3%) of the cough aerosol. Our results suggest that face masks and neck gaiters are preferable to face shields as source control devices for cough aerosols.

Urushiol Compounds Detected in Toxicodendron-Labeled Consumer Products Using Mass Spectrometry.

BACKGROUND: Urushiol, the culprit allergen in Toxicodendron plants such as poison ivy, is an oily mixture of 15 and 17 carbon side chain alk-(en)-yl catechols. Recently, consumer products have been identified that contain Toxicodendron as an ingredient on their label; however, no studies have assessed whether urushiol is indeed present within these products. OBJECTIVE: The aim of the study was to determine whether urushiol compounds are present in consumer products labeled as containing Toxicodendron species. METHODS: Gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry were performed on 9 consumer products labeled as containing Toxicodendron species, including topical homeopathic remedies. Single ion monitoring gas chromatography-mass spectrometry was programmed in selective ion mode to detect 3-methylcatechol characteristic fragment ions of alk-(en)-yl catechols after silanization. Similarly, single ion monitoring liquid chromatography-tandem mass spectrometry was programmed to detect 4 urushiol pentadecylcatechols and 5 urushiol heptadecylcatechols using previously reported mass-to-charge ratios. RESULTS: Gas chromatography-mass spectrometry detected alk-(en)-yl catechols in 67% (6/9) of the products tested. Liquid chromatography-tandem mass spectrometry detected multiple urushiol pentadecylcatechols and heptadecylcatechols in 44% (4/9) of the products tested. CONCLUSIONS: Alk-(en)-yl catechols and multiple urushiols were detected in consumer products listing Toxicodendron species as an ingredient. Clinicians should be aware of these known allergenic ingredients in consumer products.

Chemical Identification and Confirmation of Contact Allergens.

Identification of the etiological chemical agent(s) associated with a case(s) of allergic contact dermatitis (ACD) is important for both patient management and public health surveillance. Traditional patch testing can identify chemical allergens to which the patient is allergic. Confirmation of allergen presence in the causative ACD-associated material is presently dependent on labeling information, which may not list the allergenic chemical on the product label or safety data sheet. Dermatologists have expressed concern over the lack of laboratory support for chemical allergen identification and possibly quantification from patients' ACD-associated products. The aim of this review was to provide the clinician a primer to better understand the analytical chemistry of contact allergen confirmation and unknown identification, including types of analyses, required instrumentation, identification levels of confidence decision tree, limitations, and costs.

Acute 4,4'-Methylene Diphenyl Diisocyanate Exposure-Mediated Downregulation of miR-206-3p and miR-381-3p Activates Inducible Nitric Oxide Synthase Transcription by Targeting Calcineurin/NFAT Signaling in Macrophages.

Exposure to 4,4'-methylene diphenyl diisocyanate (MDI) in the occupational setting may lead to development of occupational asthma (OA), and the underlying molecular mechanisms of MDI-induced disease pathogenesis remain an active area of research. Using a nose-only mouse inhalation model, we find that circulating microRNA (miR)-206-3p and miR-381-3p are downregulated after MDI exposure; however, cellular miR-206-3p and miR-381-3p responses after MDI aerosol exposure and their pathophysiological roles in MDI-OA are unknown. We hypothesize that miR-206-3p and miR-381-3p-regulated mechanisms cause increased expression of the inducible nitric oxide synthase (iNOS) after MDI aerosol exposure. We examined cellular miR-206-3p and miR-381-3p, calcineurins, nuclear factors of activated T cells (NFATs), and iNOS levels from both nose-only exposed murine bronchoalveolar lavage cells (BALCs) and differentiated THP-1 macrophages treated with MDI-glutathione (GSH) conjugates. Both in vivo murine MDI aerosol exposure and in vitro MDI-GSH exposures in THP-1 macrophages result in downregulation of endogenous miR-206-3p and miR-381-3p and upregulation of PPP3CA and iNOS expression. Transfection of THP-1 macrophages with miR-inhibitor-206-3p and miR-inhibitor-381-3p resulted in the upregulation of PPP3CA and iNOS. Using RNA-induced silencing complex immunoprecipitation and translational reporter assays, we verified that PPP3CA, but not iNOS, is directly targeted by both miR-206-3p and miR-381-3p. Downregulation of miR-206-3p and miR-381-3p following by MDI exposure induces calcineurin/NFAT signaling-mediated iNOS transcription in macrophages and BALCs.

A rare case of rectal prolapse after Deloyers procedure in a patient with Hirschsprung's disease: A case report.

INTRODUCTION: Deloyers procedure has been reported in the literature as a viable alternative to the more commonly performed Swenson, Soave and Duhamel methods. As of yet, the long term sequelae of this procedure for patients with Hirschsprung's disease have not been studied in depth. PRESENTATION OF CASE: We report the first case in the literatures of a 27-year-old man presenting with rectal prolapse due to colorectal anastomotic intussusception after Deloyers procedure for Hirschsprung's disease. DISCUSSION: Few studies with low case volume have been performed investigating the long term sequelae of Deloyers procedure as a mainstay in patients undergoing operative treatment for Hirschsprung's disease. This procedure allows for preservation of a longer segment of colon, in turn potentially improving absorption and continence compared to other methods. Studies are limited and as of yet the viability of Deloyers as a mainstay of treatment for Hirschsprung's disease is inconclusive. CONCLUSION: We report the first adult case of prolapsed colorectal anastomotic intussusception after Deloyers procedure for Hirschsprung's disease. Further study is required to delineate long-term complications and viability of this method in these patients.

Circulating miRs-183-5p, -206-3p and -381-3p may serve as novel biomarkers for 4,4'-methylene diphenyl diisocyanate exposure.

BACKGROUND: Occupational exposure to the most widely used diisocyanate, 4,4'-methylene diphenyl diisocyanate (MDI), is a cause of occupational asthma (OA). Early recognition of MDI exposure and sensitization is essential for the prevention of MDI-OA. OBJECTIVE: Identify circulating microRNAs (miRs) as novel biomarkers for early detection of MDI exposure and prevention of MDI-OA. MATERIALS AND METHODS: Female BALB/c mice were exposed to one of three exposure regimens: dermal exposure to 1% MDI in acetone; nose-only exposure to 4580 ± 1497 µg/m3 MDI-aerosol for 60 minutes; or MDI dermal exposure/sensitization followed by MDI-aerosol inhalation challenge. Blood was collected and miRCURY™ miRs qPCR Profiling Service was used to profile circulate miRs from dermally exposed mice. Candidate miRs were identified and verified from mice exposed to three MDI-exposure regimens by TaqMan® miR assays. RESULTS: Up/down-regulation patterns of circulating mmu-miRs-183-5p, -206-3p and -381-3p were identified and verified. Circulating mmu-miR-183-5p was upregulated whereas mmu-miRs-206-3p and -381-3p were downregulated in mice exposed via all three MDI exposure regimens. DISCUSSION AND CONCLUSION: Upregulation of circulating miR-183-5p along with downregulation of circulating miRs-206-3p and -381-3p may serve as putative biomarkers of MDI exposure and may be considered as potential candidates for validation in exposed human worker populations.

Mass spectrometry-based analysis of murine bronchoalveolar lavage fluid following respiratory exposure to 4,4'-methylene diphenyl diisocyanate aerosol.

1. Diisocyanates are highly reactive electrophiles utilized in the manufacture of a wide range of polyurethane products and have been identified as causative agents of occupational allergic respiratory disease. However, in spite of the significant occupational health burden associated with diisocyanate-induced asthma, the mechanism of disease pathogenesis remains largely unknown. 2. To better understand the fate of inhaled diisocyanates, a nose-only aerosol exposure system was constructed and utilized to expose a BALB/c mouse model to an aerosol generated from 4,4'-methylene diphenyl diisocyanate (MDI). Tissue and bronchoalveolar lavage samples were evaluated 4 and 24 h post-exposure for evidence of diisocyanate-protein haptenation, and a label-free quantitative proteomics strategy was employed to evaluate relative changes to the protein content of the cellular fraction of the lavage fluid. 3. Following MDI aerosol exposure, expression of the number of proteins with immunological or xenobiotic metabolism relevance is increased, including endoplasmin, cytochrome P450 and argininosuccinate synthase. Western blot analysis indicated MDI-conjugated protein in the lavage fluid, which was identified as serum albumin. 4. Tandem mass spectrometry analysis of MDI-albumin revealed MDI conjugation occurs at a dilysine motif at Lys525, as well as at a glutamine-lysine motif at Lys414, in good agreement with previously published in vitro data on diisocyanate-conjugated serum albumin.

The influence of diisocyanate antigen preparation methodology on monoclonal and serum antibody recognition.

Exposure to diisocyanates (dNCOs), such as methylene diphenyl diisocyanate (MDI) can cause occupational asthma (OA). Currently, lab tests for dNCO specific IgE are specific, but not sensitive, which limits their utility in diagnosing dNCO asthma. This may be due to variable preparation and poor characterization of the standard antigens utilized in these assays. The aim of this study was to produce and characterize a panel of antigens prepared using three different commonly employed methods and one novel method. The conjugates were examined for recognition by anti-MDI monoclonal antibodies (mAbs) in varying enzyme linked immunosorbant assay (ELISA) formats, extent of crosslinking, total amount of MDI, the sites of MDI conjugation, relative shape/charge, and reactivity with human serum with antibodies from sensitized, exposed workers. Results indicate that while there are minimal differences in the total amount of MDI conjugated, the extent of crosslinking, and the conjugation sites, there are significant differences in the recognition of differently prepared conjugates by mAbs. Native and denaturing polyacrylamide gel electrophoresis demonstrate differences in the mobility of different conjugates, indicative of structural changes that are likely important for antigenicity. While mAbs exhibited differential binding to different conjugates, polyclonal serum antibodies from MDI exposed workers exhibited equivalent binding to different conjugates by ELISA. While differences in the recognition of the different conjugates exist by mAb detection, differences in antigenicity could not be detected using human serum from MDI-sensitized individuals. Thus, although dNCO conjugate preparation can, depending on the immunoassay platform, influence binding of specific antibody clones, serologic detection of the dNCO-exposure-induced polyclonal antibody response may be less sensitive to these differences.

Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin.

Diisocyanates (dNCOs) are low molecular weight chemical sensitizers that react with autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCO-specific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25% of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may be modified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb-TDI antigenicity was approximately 30% that of HSA-TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs between the two diisocyanate species studied, which may influence their relative immunogenicity and/or antigenicity.

Parent stem cells can serve as niches for their daughter cells.

Stem cells integrate inputs from multiple sources. Stem cell niches provide signals that promote stem cell maintenance, while differentiated daughter cells are known to provide feedback signals to regulate stem cell replication and differentiation. Recently, stem cells have been shown to regulate themselves using an autocrine mechanism. The existence of a 'stem cell niche' was first postulated by Schofield in 1978 to define local environments necessary for the maintenance of haematopoietic stem cells. Since then, an increasing body of work has focused on defining stem cell niches. Yet little is known about how progenitor cell and differentiated cell numbers and proportions are maintained. In the airway epithelium, basal cells function as stem/progenitor cells that can both self-renew and produce differentiated secretory cells and ciliated cells. Secretory cells also act as transit-amplifying cells that eventually differentiate into post-mitotic ciliated cells . Here we describe a mode of cell regulation in which adult mammalian stem/progenitor cells relay a forward signal to their own progeny. Surprisingly, this forward signal is shown to be necessary for daughter cell maintenance. Using a combination of cell ablation, lineage tracing and signalling pathway modulation, we show that airway basal stem/progenitor cells continuously supply a Notch ligand to their daughter secretory cells. Without these forward signals, the secretory progenitor cell pool fails to be maintained and secretory cells execute a terminal differentiation program and convert into ciliated cells. Thus, a parent stem/progenitor cell can serve as a functional daughter cell niche.

Injury induces direct lineage segregation of functionally distinct airway basal stem/progenitor cell subpopulations.

Following injury, stem cells restore normal tissue architecture by producing the proper number and proportions of differentiated cells. Current models of airway epithelial regeneration propose that distinct cytokeratin 8-expressing progenitor cells, arising from p63(+) basal stem cells, subsequently differentiate into secretory and ciliated cell lineages. We now show that immediately following injury, discrete subpopulations of p63(+) airway basal stem/progenitor cells themselves express Notch pathway components associated with either secretory or ciliated cell fate commitment. One basal cell population displays intracellular Notch2 activation and directly generates secretory cells; the other expresses c-myb and directly yields ciliated cells. Furthermore, disrupting Notch ligand activity within the basal cell population at large disrupts the normal pattern of lineage segregation. These non-cell-autonomous effects demonstrate that effective airway epithelial regeneration requires intercellular communication within the broader basal stem/progenitor cell population. These findings have broad implications for understanding epithelial regeneration and stem cell heterogeneity.

Arterial stiffness, oxidative stress, and smoke exposure in wildland firefighters.

OBJECTIVES: To assess the association between exposure, oxidative stress, symptoms, and cardiorespiratory function in wildland firefighters. METHODS: We studied two Interagency Hotshot Crews with questionnaires, pulse wave analysis for arterial stiffness, spirometry, urinary 8-iso-prostaglandin F2α (8-isoprostane) and 8-hydroxy-2'-deoxyguanosine (8-OHdG), and the smoke exposure marker (urinary levoglucosan). Arterial stiffness was assessed by examining levels of the aortic augmentation index, expressed as a percentage. An oxidative stress score comprising the average of z-scores created for 8-OHdG and 8-isoprostane was calculated. RESULTS: Mean augmentation index % was higher for participants with higher oxidative stress scores after adjusting for smoking status. Specifically for every one unit increase in oxidative stress score the augmentation index % increased 10.5% (95% CI: 2.5, 18.5%). Higher mean lower respiratory symptom score was associated with lower percent predicted forced expiratory volume in one second/forced vital capacity. CONCLUSIONS: Biomarkers of oxidative stress may serve as indicators of arterial stiffness in wildland firefighters.