Development of a robotic cluster for automated and scalable cell therapy manufacturing.

BACKGROUND AIMS: The production of commercial autologous cell therapies such as chimeric antigen receptor T cells requires complex manual manufacturing processes. Skilled labor costs and challenges in manufacturing scale-out have contributed to high prices for these products. METHODS: We present a robotic system that uses industry-standard cell therapy manufacturing equipment to automate the steps involved in cell therapy manufacturing. The robotic cluster consists of a robotic arm and customized modules, allowing the robot to manipulate a variety of standard cell therapy instruments and materials such as incubators, bioreactors, and reagent bags. This system enables existing manual manufacturing processes to be rapidly adapted to robotic manufacturing, without having to adopt a completely new technology platform. Proof-of-concept for the robotic cluster's expansion module was demonstrated by expanding human CD8+ T cells. RESULTS: The robotic cultures showed comparable cell yields, viability, and identity to those manually performed. In addition, the robotic system was able to maintain culture sterility. CONCLUSIONS: Such modular robotic solutions may support scale-up and scale-out of cell therapies that are developed using classical manual methods in academic laboratories and biotechnology companies. This approach offers a pathway for overcoming manufacturing challenges associated with manual processes, ultimately contributing to the broader accessibility and affordability for personalized immunotherapies.

Filling the gap: the workforce of tomorrow for CGT manufacturing as the sector advances.

Workforce education and development are key cornerstones in advancing and maturing the Cell & Gene Therapy sector. A skilled worker shortage can significantly impact and delay progress as well as the quality of output for any developer, thereby negatively impacting a patient's access to life-saving treatments. Several roundtable discussions were held at the International Society for Cell & Gene Therapy (ISCT) 2023 Annual Meeting to dive deeper into the current state of workforce development and solutions to address this bottleneck. One roundtable discussion was co-hosted by the Alliance for Regenerative Medicine (ARM) and ISCT, which focused on the gap analysis provided for the United States Cell & Gene Therapy (CGT) sector, highlighting the lack of skilled workers in manufacturing and quality control. In this manuscript, the roundtable participants continue this conversation, review the roles and staffing requirements in both academic and industry as well as small and large company settings. The adoption of increased manufacturing automation is one promising solution to propel the sector forward. However, automation alone won't replace on-site staff, but will lower the bar to entry for a larger pool of people and require different training. This paper also addresses the workforce development and training paradigm shift as advanced manufacturing techniques are implemented, which will differ considerably based on the type of manufacturing efforts, thus emphasizing the need for a well-thought-out strategy to up-skill and re-skill the technical workforce to adapt to these advancements. Organizations such as ISCT and ARM have a role to play in propelling the field forward, providing awareness and education to stakeholders at all levels, as well as acting as a convener and participating as a key stakeholder in discussions and partnerships between academia and industry towards solutions for training the best personnel for CGT manufacturing. This scope includes novel digital tools and technologies to simplify training to increase access to new talent pools interested in careers in a rapidly advancing sector.

DNA methylation of insulin signaling pathways is associated with HOMA2-IR in primary myoblasts from older adults.

BACKGROUND: While ageing is associated with increased insulin resistance (IR), the molecular mechanisms underlying increased IR in the muscle, the primary organ for glucose clearance, have yet to be elucidated in older individuals. As epigenetic processes are suggested to contribute to the development of ageing-associated diseases, we investigated whether differential DNA methylation was associated with IR in human primary muscle stem cells (myoblasts) from community-dwelling older individuals. METHODS: We measured DNA methylation (Infinium HumanMethylationEPIC BeadChip) in myoblast cultures from vastus lateralis biopsies (119 males/females, mean age 78.24 years) from the Hertfordshire Sarcopenia Study extension (HSSe) and examined differentially methylated cytosine phosphate guanine (CpG) sites (dmCpG), regions (DMRs) and gene pathways associated with HOMA2-IR, an index for the assessment of insulin resistance, and levels of glycated hemoglobin HbA1c. RESULTS: Thirty-eight dmCpGs (false discovery rate (FDR) < 0.05) were associated with HOMA2-IR, with dmCpGs enriched in genes linked with JNK, AMPK and insulin signaling. The methylation signal associated with HOMA2-IR was attenuated after the addition of either BMI (6 dmCpGs), appendicular lean mass index (ALMi) (7 dmCpGs), grip strength (15 dmCpGs) or gait speed (23 dmCpGs) as covariates in the model. There were 8 DMRs (Stouffer < 0.05) associated with HOMA2-IR, including DMRs within T-box transcription factor (TBX1) and nuclear receptor subfamily-2 group F member-2 (NR2F2); the DMRs within TBX1 and NR2F2 remained associated with HOMA2-IR after adjustment for BMI, ALMi, grip strength or gait speed. Forty-nine dmCpGs and 21 DMRs were associated with HbA1c, with cg13451048, located within exoribonuclease family member 3 (ERI3) associated with both HOMA2-IR and HbA1c. HOMA2-IR and HbA1c were not associated with accelerated epigenetic ageing. CONCLUSIONS: These findings suggest that insulin resistance is associated with differential DNA methylation in human primary myoblasts with both muscle mass and body composition making a significant contribution to the methylation changes associated with IR.

Comparison of the Quality of Echocardiography Imaging Between the Left Lateral Decubitus and Supine Positions.

Introduction It is commonly taught that positioning the patient in the left lateral decubitus (LLD) position will improve transthoracic echocardiography (TTE) image quality. Despite this, no previous studies have been performed that study this practice. Our goal was to quantify the difference in image quality of TTE views between the supine and LLD positions.  Methods This was a prospective study in a single academic Emergency Department (ED) of a convenience sample of 30 patients. Three separate ED physicians performed TTE views in both the supine and LLD position on each patient. The order of position was randomized. Images were then reviewed on a previously validated TTE image quality scale by two blinded ED physicians with specialized training in ultrasound. The scale used a 0 to 5 (highest quality) metric for quality assessment. Interpretability of right ventricular and left ventricular function was also assessed. Results The mean image quality for the supine position was 2.85 (standard deviation {SD} 1.1) and 3.05 (SD 1.2) for the LLD position (p=0.044). In the subset of parasternal and apical windows, the mean quality for the supine position was 2.87 (SD 1.1) and 3.23 (SD 1.1) for the LLD position (p=0.003). The number of studies in which right ventricular function was interpretable was significantly higher in the LLD position (62% versus 42%, p=0.044). Conclusions There was a statistically significant increase in image quality when TTE was performed in the LLD position as compared to supine. This was especially pronounced in the apical four and parasternal windows.

Expanding Zirconocene Hydride Catalysis: In Situ Generation and Turnover of ZrH Catalysts Enabling Catalytic Carbonyl Reductions.

Despite the wide use and popularity of metal hydride catalysis, methods utilizing zirconium hydride catalysts remain underexplored. Here, we report the development of a mild method for the in situ preparation and use of zirconium hydride catalysts. This robust method requires only 2.5-5 mol % of zirconocene dichloride in combination with a hydrosilane as the stoichiometric reductant and does not require careful air- or moisture-free techniques. A key finding of this study concerns an amine-mediated ligand exchange en route to the active zirconocene hydride catalyst. A mechanistic investigation supports the intermediacy of an oxo-bridged dimer precatalyst. The application of this method to the reduction of a wide variety of carbonyl-containing substrates, including ketones, aldehydes, enones, ynones, and lactones, is demonstrated with up to 92% yield and exhibits broad functional group tolerability. These findings open up alternative avenues for the catalytic application of chlorozirconocenes, potentially serving as the foundation for broader applications of zirconium hydride catalysis.

Evaluation of Host Protein Biomarkers by ELISA From Whole Lysed Peripheral Blood for Development of Diagnostic Tests for Active Tuberculosis.

Tuberculosis (TB) remains a significant global health crisis and the number one cause of death for an infectious disease. The health consequences in high-burden countries are significant. Barriers to TB control and eradication are in part caused by difficulties in diagnosis. Improvements in diagnosis are required for organisations like the World Health Organisation (WHO) to meet their ambitious target of reducing the incidence of TB by 50% by the year 2025, which has become hard to reach due to the COVID-19 pandemic. Development of new tests for TB are key priorities of the WHO, as defined in their 2014 report for target product profiles (TPPs). Rapid triage and biomarker-based confirmatory tests would greatly enhance the diagnostic capability for identifying and diagnosing TB-infected individuals. Protein-based test methods e.g. lateral flow devices (LFDs) have a significant advantage over other technologies with regard to assay turnaround time (minutes as opposed to hours) field-ability, ease of use by relatively untrained staff and without the need for supporting laboratory infrastructure. Here we evaluate the diagnostic performance of nine biomarkers from our previously published biomarker qPCR validation study; CALCOCO2, CD274, CD52, GBP1, IFIT3, IFITM3, SAMD9L, SNX10 and TMEM49, as protein targets assayed by ELISA. This preliminary evaluation study was conducted to quantify the level of biomarker protein expression across latent, extra-pulmonary or pulmonary TB groups and negative controls, collected across the UK and India, in whole lysed blood samples (WLB). We also investigated associative correlations between the biomarkers and assessed their suitability for ongoing diagnostic test development, using receiver operating characteristic/area under the curve (ROC) analyses, singly and in panel combinations. The top performing single biomarkers for pulmonary TB versus controls were CALCOCO2, SAMD9L, GBP1, IFITM3, IFIT3 and SNX10. TMEM49 was also significantly differentially expressed but downregulated in TB groups. CD52 expression was not highly differentially expressed across most of the groups but may provide additional patient stratification information and some limited use for incipient latent TB infection. These show therefore great potential for diagnostic test development either in minimal configuration panels for rapid triage or more complex formulations to capture the diversity of disease presentations.

Human non-CpG methylation patterns display both tissue-specific and inter-individual differences suggestive of underlying function.

DNA methylation (DNAm) in mammals is mostly examined within the context of CpG dinucleotides. Non-CpG DNAm is also widespread across the human genome, but the functional relevance, tissue-specific disposition, and inter-individual variability has not been widely studied. Our aim was to examine non-CpG DNAm in the wider methylome across multiple tissues from the same individuals to better understand non-CpG DNAm distribution within different tissues and individuals and in relation to known genomic regulatory features.DNA methylation in umbilical cord and cord blood at birth, and peripheral venous blood at age 12-13 y from 20 individuals from the Southampton Women's Survey cohort was assessed by Agilent SureSelect methyl-seq. Hierarchical cluster analysis (HCA) was performed on CpG and non-CpG sites and stratified by specific cytosine environment. Analysis of tissue and inter-individual variation was then conducted in a second dataset of 12 samples: eight muscle tissues, and four aliquots of cord blood pooled from two individuals.HCA using methylated non-CpG sites showed different clustering patterns specific to the three base-pair triplicate (CNN) sequence. Analysis of CAC sites with non-zero methylation showed that samples clustered first by tissue type, then by individual (as observed for CpG methylation), while analysis using non-zero methylation at CAT sites showed samples grouped predominantly by individual. These clustering patterns were validated in an independent dataset using cord blood and muscle tissue.This research suggests that CAC methylation can have tissue-specific patterns, and that individual effects, either genetic or unmeasured environmental factors, can influence CAT methylation.

Developing an Agnostic Risk Prediction Model for Early AKI Detection in Cancer Patients.

Acute kidney injury (AKI) is a common complication among oncology patients associated with lower remission rates and higher mortality. To reduce the impact of this condition, we aimed to predict AKI earlier than existing tools, to allow clinical intervention before occurrence. We trained a random forest model on 597,403 routinely collected blood test results from 48,865 patients undergoing cancer treatment at The Christie NHS Foundation Trust between January 2017 and May 2020, to identify AKI events upcoming in the next 30 days. AKI risk levels were assigned to upcoming AKI events and tested through a prospective analysis between June and August 2020. The trained model gave an AUROC of 0.881 (95% CI 0.878-0.883), when assessing predictions per blood test for AKI occurrences within 30 days. Assigning risk levels and testing the model through prospective validation from the 1st June to the 31st August identified 73.8% of patients with an AKI event before at least one AKI occurrence, 61.2% of AKI occurrences. Our results suggest that around 60% of AKI occurrences experienced by patients undergoing cancer treatment could be identified using routinely collected blood results, allowing clinical remedial action to be taken and disruption to treatment by AKI to be minimised.

A long way from home: Biosecurity lessons learnt from the impact of La Niña on the transportation and establishment of tropical portunid species.

Marine ecosystems can be modified and shaped by irregular interannual variations in oceanic current patterns and temperatures, such as El Niño and La Niña. These large scale oceanic events have also been shown to influence environmental stressors such as invasive marine species (IMS). Our study indicates that there is a causative link between these climatic events, and atypical detections of native and IMS. Significant La Niña events between 1970 and 2014 were associated with sightings of tropical crab species in temperate waters following a lag period of 18-24 months from the onset of the event. We identified a total of 72 records of six tropical portunid crabs species (from both Charybdis and Scylla) in temperate waters of south-western Australia following these La Niña events, based on reports in published scientific literature, grey literature and museum records, as well as citizen science networks such as FishWatch and PestWatch apps. We suggest that La Niña conditions facilitated transportation and temporary establishment of crab larvae from their native tropical habitat to temperate regions. As the strength of La Niña events is likely to increase into the future due to the escalating effects of climate change, it is likely that there will be a growth in associated atypical establishment events of IMS. Consequently, biosecurity managers will need to reprioritise resources in order to accommodate the potential impacts of these large scale oceanic events as part of their surveillance programmes.

The application of graph theory and percolation analysis for assessing change in the spatial configuration of pond networks.

Pond networks support high levels of biodiversity when compared to other freshwater ecosystems such as rivers, lakes and streams. The persistence of species in these small, sometimes ephemeral, aquatic habitats depends on the dispersal of individuals among ponds in the landscape. However, the number of ponds across the landscape is at a historical low as urbanisation and intensified agricultural practices have led to a substantial loss of ponds (nodes in the pond network) over more than a century. Here, we examine the extent and drivers of pond loss in a heavily urbanised landscape (Birmingham, UK) over 105 years and determine how pond loss influences key structural properties of the pond network using graph theoretic approaches. Specifically, we calculated minimum spanning trees (MST) and performed percolation analyses to determine changes in both the spatial configuration and resilience of the pond network through time. Pond numbers declined by 82% between ca1904 and 2009, such that pond density decreased from 7.1 km-2 to 1.3 km-2. The MST analyses revealed increased distance between ponds in the network (i.e. edge length increased) by up to 49% over the 105-year period, indicating that ponds in the modern landscape (2009) were considerably more isolated, with fewer neighbours. This study demonstrates that graph theory has an excellent potential to inform the management of pond networks in order to support ecological communities that are less vulnerable to environmental change.

Pharmacology of Bradykinin-Evoked Coughing in Guinea Pigs.

Bradykinin has been implicated as a mediator of the acute pathophysiological and inflammatory consequences of respiratory tract infections and in exacerbations of chronic diseases such as asthma. Bradykinin may also be a trigger for the coughing associated with these and other conditions. We have thus set out to evaluate the pharmacology of bradykinin-evoked coughing in guinea pigs. When inhaled, bradykinin induced paroxysmal coughing that was abolished by the bradykinin B2 receptor antagonist HOE 140. These cough responses rapidly desensitized, consistent with reports of B2 receptor desensitization. Bradykinin-evoked cough was potentiated by inhibition of both neutral endopeptidase and angiotensin-converting enzyme (with thiorphan and captopril, respectively), but was largely unaffected by muscarinic or thromboxane receptor blockade (atropine and ICI 192605), cyclooxygenase, or nitric oxide synthase inhibition (meclofenamic acid and N(G)-nitro-L-arginine). Calcium influx studies in bronchopulmonary vagal afferent neurons dissociated from vagal sensory ganglia indicated that the tachykinin-containing C-fibers arising from the jugular ganglia mediate bradykinin-evoked coughing. Also implicating the jugular C-fibers was the observation that simultaneous blockade of neurokinin2 (NK2; SR48968) and NK3 (SR142801 or SB223412) receptors nearly abolished the bradykinin-evoked cough responses. The data suggest that bradykinin induces coughing in guinea pigs by activating B2 receptors on bronchopulmonary C-fibers. We speculate that therapeutics targeting the actions of bradykinin may prove useful in the treatment of cough.

Aerosolization of respirable droplets from a domestic spa pool and the use of MS-2 coliphage and Pseudomonas aeruginosa as markers for Legionella pneumophila.

Legionnaires' disease can result when droplets or aerosols containing legionella bacteria are inhaled and deposited in the lungs. A number of outbreaks have been associated with the use of a spa pool where aeration, a high water temperature, and a large and variable organic load make disinfectant levels difficult to maintain. Spa pool ownership is increasing, and the aim of this study, using two surrogate organisms (MS-2 coliphage and Pseudomonas aeruginosa [a natural contaminant]), was to assess the potential risk to domestic users when disinfection fails. A representative "entry level" domestic spa pool was installed in an outdoor courtyard. The manufacturer's instructions for spa pool maintenance were not followed. A cyclone sampler was used to sample the aerosols released from the spa pool with and without activation of the air injection system. Samples were taken at increasing heights and distances from the pool. An aerodynamic particle sizer was used to measure the water droplet size distribution at each sample point. When the air injection system was inactivated, neither surrogate organism was recovered from the air. On activation of the air injection system, the mean mass of droplets within the respirable range (10 cm above the water line) was 36.8 µg cm(-3). This corresponded to a mean air concentration of P. aeruginosa of 350 CFU m(-3). From extrapolation from animal data, the estimated risk of infection from aerosols contaminated with similar concentrations of Legionella pneumophila was 0.76 (males) and 0.65 (females). At 1 m above and/or beyond the pool, the mean aerosol mass decreased to 0.04 µg cm(-3) and corresponded to a 100-fold reduction in mean microbial air concentration. The estimated risk of infection at this distance was negligible.

Contrasting influences of Drosophila white/mini-white on ethanol sensitivity in two different behavioral assays.

BACKGROUND: The fruit fly Drosophila melanogaster has been used extensively to investigate genetic mechanisms of ethanol (EtOH)-related behaviors. Many past studies in flies, including studies from our laboratory, have manipulated gene expression using transposons carrying the genetic-phenotypic marker mini-white(mini-w), a derivative of the endogenous gene white(w). Whether the mini-w transgenic marker or the endogenous w gene influences behavioral responses to acute EtOH exposure in flies has not been systematically investigated. METHODS: We manipulated mini-w and w expression via (i) transposons marked with mini-w, (ii) RNAi against mini-w and w, and (iii) a null allele of w. We assessed EtOH sensitivity and tolerance using a previously described eRING assay (based on climbing in the presence of EtOH) and an assay based on EtOH-induced sedation. RESULTS: In eRING assays, EtOH-induced impairment of climbing correlated inversely with expression of the mini-w marker from a series of transposon insertions. Additionally, flies harboring a null allele of w or flies with RNAi-mediated knockdown of mini-w were significantly more sensitive to EtOH in eRING assays than controls expressing endogenous w or the mini-w marker. In contrast, EtOH sensitivity and rapid tolerance measured in the EtOH sedation assay were not affected by decreased expression of mini-w or endogenous w in flies. CONCLUSIONS: EtOH sensitivity measured in the eRING assay is noticeably influenced by w and mini-w, making eRING problematic for studies on EtOH-related behavior in Drosophila using transgenes marked with mini-w. In contrast, the EtOH sensitivity assay described here is a suitable behavioral paradigm for studies on EtOH sensitivity and rapid tolerance in Drosophila including those that use widely available transgenes marked with mini-w.

Neoadjuvant treatment of endometrial cancer using anastrozole: a randomised pilot study.

OBJECTIVE: Excessive oestrogenic stimulation is a well-known risk factor for the development and progression of endometrial cancer. Aromatase is the key enzyme which catalyses the conversion of androgens to oestrogens in postmenopausal women. Inhibition of aromatase may therefore be a useful strategy in the management of endometrial cancer. A pilot study was designed to assess the feasibility of a neoadjuvant model and understand the biological effects of anastrozole, an aromatase inhibitor, in the treatment of endometrial cancer. METHODS: Patients with endometrial cancer who consented to participate in the study were randomised to receive anastrozole or placebo for a minimum of 14 days prior to definitive surgery. Endometrial samples were obtained before and after treatment. Immunohistochemistry was performed to ascertain the expression of oestrogen receptor alpha (ERα), progesterone receptor (PR), androgen receptor (AR), ki-67 and Bcl2 before and after treatment in glands and stroma of the endometrium. RESULTS: A total of 16 patients were randomised to the anastrozole arm and 8 to the placebo arm (2:1 randomisation). A significant decrease in the glandular expression of ERα and AR was observed in the anastrozole arm. There was no significant change in the expression of PR or Bcl2. Expression of ki-67, a proliferation marker, also decreased significantly following treatment with anastrozole. CONCLUSIONS: Treatment with anastrozole caused a significant decrease in proliferation as demonstrated by decreased ki-67 expression. A large randomised controlled trial is warranted to fully assess the role of anastrozole in the neoadjuvant treatment of endometrial cancer.

Respiratory stimulant drugs in the post-operative setting.

Drug-induced respiratory depression (DIRD) is a common problem encountered post-operatively and can persist for days after surgery. It is not always possible to predict the timing or severity of DIRD due to the number of contributing factors. A safe and effective respiratory stimulant could improve patient care by avoiding the use of reversal agents (e.g., naloxone, which reverses analgesia as well as respiratory depression) thereby permitting better pain management by enabling the use of higher doses of analgesics, facilitate weaning from prolonged ventilation, and ameliorate sleep-disordered breathing peri-operatively. The purpose of this review is to discuss the current pharmaceutical armamentarium of drugs (doxapram and almitrine) that are licensed for use in humans as respiratory stimulants and that could be used to reverse drug-induced respiratory depression in the post-operative period. We also discuss new chemical entities (AMPAkines and GAL-021) that have been recently evaluated in Phase 1 clinical trials and where the initial regulatory registration would be as a respiratory stimulant.

Coughing precipitated by Bordetella pertussis infection.

Infections with the gram-negative bacteria Bordetella pertussis (B. pertussis) have long been recognized as a significant threat to children and are increasingly recognized as a cause of cough in adolescents and adults. Antibiotic therapy, when administered during the virulent stages of the disease, can reduce the duration and severity of symptoms. Unfortunately, there are no effective treatments for the persistent coughing that accompanies and follows the infection. The pathogenesis of B. pertussis infection is briefly reviewed. Also discussed is the evidence supporting the hypothesis that the inflammatory peptide bradykinin may be responsible for the persistent, paroxysmal coughing associated with B. pertussis-initiated illness.

Requisite role for the dectin-1 beta-glucan receptor in pulmonary defense against Aspergillus fumigatus.

Immune suppression increases the incidence of invasive fungal infections, particularly those caused by the opportunistic mold Aspergillus fumigatus. Previous investigations revealed that members of the TLR family are not absolutely required for host defense against A. fumigatus in nonimmunosuppressed hosts, suggesting that other pattern recognition receptors are involved. We show in this study that naive mice (i.e., not pharmacologically immunosuppressed) lacking the beta-glucan receptor Dectin-1 (Dectin-1(-/-)) are more sensitive to intratracheal challenge with A. fumigatus than control mice, exhibiting >80% mortality within 5 days, ultimately attributed to a compromise in respiratory mechanics. In response to A. fumigatus challenge, Dectin-1(-/-) mice demonstrated impaired IL-1alpha, IL-1beta, TNF-alpha, CCL3/MIP-1alpha, CCL4/MIP-1beta, and CXCL1/KC production, which resulted in insufficient lung neutrophil recruitment and uncontrolled A. fumigatus lung growth. Alveolar macrophages from Dectin-1(-/-) mice failed to produce proinflammatory mediators in response to A. fumigatus, whereas neutrophils from Dectin-1(-/-) mice had impaired reactive oxygen species production and impaired killing of A. fumigatus. We further show that IL-17 production in the lung after A. fumigatus challenge was Dectin-1 dependent, and that neutralization of IL-17 significantly impaired A. fumigatus clearance. Collectively, these results support a requisite role for Dectin-1 in in vivo defense against A. fumigatus.

Loss of Thy-1 inhibits alveolar development in the newborn mouse lung.

Transforming growth factor (TGF)-beta mediates hypoxia-induced inhibition of alveolar development in the newborn lung. TGF-beta is regulated primarily at the level of activation of latent TGF-beta. Fibroblasts expressing Thy-1 (CD90) inhibit TGF-beta activation. We hypothesized that loss of Thy-1 due to hypoxia may be a mechanism by which hypoxia increases TGF-beta activation and that animals deficient in Thy-1 will simulate the effects of hypoxia on lung development. To determine if loss of Thy-1 occurred during hypoxia, non-transgenic (C57BL/6) wild-type (WT) mice exposed to hypoxia were evaluated for Thy-1 mRNA and protein. To determine if Thy-1 deficiency simulated hypoxia, WT and Thy-1 null (Thy-1(-/-)) mice were exposed to air or hypoxia from birth to 2 wk, the critical period of lung development, and lung histology, function, parameters related to TGF-beta signaling, and extracellular matrix protein content were measured. To test if the phenotype in Thy-1(-/-) mice was due to excessive TGF-beta signaling, measurements were also performed in Thy-1(-/-) mice administered TGF-beta neutralizing antibody (1D11). We observed that hypoxia reduced Thy-1 mRNA and Thy-1 staining in WT mice. Thy-1(-/-) mice had impaired alveolarization, increased TGF-beta signaling, reduced lung epithelial and endothelial cell proliferation but increased fibroblast proliferation, and increased collagen and elastin. Lung compliance was lower, and tissue but not airway resistance was higher in Thy-1(-/-) mice at 2 wk. Thy-1(-/-) mice given 1D11 had improved alveolar development and lung function. These data support the hypothesis that hypoxia, by reducing Thy-1, increases TGF-beta activation, and thereby inhibits normal alveolar development.

Potential teratogenic effects of benomyl in rat embryos cultured in vitro.

A possible association between environmental exposure to benomyl and anophthalmia has been suggested. The aim of the present study was to investigate potential teratogenic effects of benomyl using the 9.5 day rat embryo culture method using rat and human serum. Explanted rat embryos were cultured in rat serum (n=121) or human serum (n=90) with differing concentrations of benomyl [170 nM to 13.6 microM], dissolved in ethanol (0.136%), at least five embryos per concentration being cultured. In addition, 18 embryos were cultured in both human and rat serum with the equivalent concentration of ethanol to act as a vehicle control. The cultured embryos were then measured and scored for growth and differentiation by two blinded observers. Embryotoxic effects were considered to be demonstrated by a decrease in parameters of growth such as crown rump length, yolk sac diameter and protein content, whereas embryopathic effects were considered to be those causing a decease in parameters of differentiation such as morphological score, somite number and optic development. Benomyl [> or =5 microM] produced a significant concentration dependent deterioration in morphological score, somite number and optic development. Gross toxic effects were noticed at concentrations of >12 microM in rat serum and >10microM in human serum as indicated by a significant effect on parameters measuring size (crown rump length; yolk sac diameter and protein content). This study provides evidence that benomyl is a potential developmental toxicant, affecting many parameters of differentiation, including optic development at levels below those that could be considered embryotoxic.