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Glycosaminoglycans (GAGs) such as heparan sulfate and chondroitin sulfate decorate all mammalian cell surfaces. These mucopolysaccharides act as coreceptors for extracellular ligands, regulating cell signaling, growth, proliferation, and adhesion. In glioblastoma, the most common type of primary malignant brain tumor, dysregulated GAG biosynthesis results in altered chain length, sulfation patterns, and the ratio of contributing monosaccharides. These events contribute to the loss of normal cellular function, initiating and sustaining malignant growth. Disruption of the aberrant cell surface GAGs with small molecule inhibitors of GAG biosynthetic enzymes is a potential therapeutic approach to blocking the rogue signaling and proliferation in glioma, including glioblastoma. Previously, 4-azido-xylose-α-UDP sugar inhibited both xylosyltransferase (XYLT-1) and β−1,4-galactosyltransferase-7 (β-GALT-7)—the first and second enzymes of GAG biosynthesis—when microinjected into a cell. In another study, 4-deoxy-4-fluoro-β-xylosides inhibited β-GALT-7 at 1 mM concentration in vitro. In this work, we seek to solve the enduring problem of drug delivery to human glioma cells at low concentrations. We developed a library of hydrophobic, presumed prodrugs 4-deoxy-4-fluoro-2,3-dibenzoyl-(α- or β-) xylosides and their corresponding hydrophilic inhibitors of XYLT-1 and β-GALT-7 enzymes. The prodrugs were designed to be activatable by carboxylesterase enzymes overexpressed in glioblastoma. Using a colorimetric MTT assay in human glioblastoma cell lines, we identified a prodrug–drug pair (4-nitrophenyl-α-xylosides) as lead drug candidates. The candidates arrest U251 cell growth at an IC(50) = 380 nM (prodrug), 122 μM (drug), and U87 cells at IC(50) = 10.57 μM (prodrug). Molecular docking studies were consistent with preferred binding of the α- versus β-nitro xyloside conformer to XYLT-1 and β-GALT-7 enzymes.

Non-invasive methods to study glucocorticoid receptor (GR) signaling are urgently needed to reveal the complexity of GR signaling in normal physiology and human disorders, as well as to identify selective GR modulators to treat diseases. Here, we report evidence supporting translational studies with (±)-[11C]-5-(4-fluorobenzyl)-10-methoxy-2,2,4-trimethyl-2,5-dihydro-1H-chromeno[3,4-f ]-quinoline (named as (±)-[11C]YJH08), a radioligand for positron emission tomography (PET) that engages the ligand binding domain on GR. Methods: (±)-[11C]YJH08 was synthesized by reacting the phenol precursor with [11C]methyl iodide. The biodistribution was studied in vivo with PET/CT and autoradiography. A library of analogues were synthesized and studied in vitro and in vivo to understand the (±)-[11C]YJH08 structure activity relationship. Rodent dosimetry studies were performed to estimate the human equivalent doses of (±)-[11C]YJH08. Results: (±)-[11C]YJH08 was synthesized by reaction of the phenolic precursor with [11C]methyl iodide giving a radiochemical yield of 51.7 ± 4.7% (decay corrected to starting [11C]methyl iodide). An analysis of the (±)-YJH08 structure-activity relationship with molecular dynamics simulations showed that (R)- and (S)-enantiomers occupy the ligand binding domain on GR with different binding modes and have indistinguishable patterns of biodistribution in vivo. Moreover, a focused chemical screen of analogues revealed that the aryl fluoride motif on YJH08 is essential for high affinity GR binding in vitro, high tissue uptake in vivo, and passage across the blood brain barrier. Lastly, we performed dosimetry studies in rodents, from which we showed estimated human equivalent doses of (±)-[11C]YJH08 to be commensurate with widely used carbon-11 and fluorine-18 tracers. Conclusion: In summary, these studies reveal the molecular determinants of a high affinity and selectivity ligand-receptor interaction and support the use of (±)-[11C]YJH08 PET to make the first measurements of GR expression in human subjects.

Aberrantly high mTORC1 signaling is a known driver of many cancers and human disorders, yet pharmacological inhibition of mTORC1 rarely confers durable clinical responses. To explore alternative therapeutic strategies, herein we conducted a proteomics survey to identify cell surface proteins upregulated by mTORC1. A comparison of the surfaceome from Tsc1(−/−) versus Tsc1(+/+) mouse embryonic fibroblasts revealed 59 proteins predicted to be significantly overexpressed in Tsc1(−/−) cells. Further validation of the data in multiple mouse and human cell lines showed that mTORC1 signaling most dramatically induced the expression of the proteases neprilysin (NEP/CD10) and aminopeptidase N (APN/CD13). Functional studies showed that constitutive mTORC1 signaling sensitized cells to genetic ablation of NEP and APN, as well as the biochemical inhibition of APN. In summary, these data show that mTORC1 signaling plays a significant role in the constitution of the surfaceome, which in turn may present novel therapeutic strategies.

Boron neutron capture therapy (BNCT) is an encouraging therapeutic modality for cancer treatment. Prostate-specific membrane antigen (PSMA) is a cell membrane protein that is abundantly overexpressed in prostate cancer and can be targeted with radioligand therapies to stimulate clinical responses in patients. In principle, a spatially targeted neutron beam together with specifically targeted PSMA ligands could enable prostate cancer-targeted BNCT. Thus, we developed and tested PSMA-targeted poly(lactide

PurposeRadioligand therapy (RLT) is relatively unexplored in metastatic castration resistant prostate cancer (mCRPC), with much of the focus having been on bone seeking radionuclides and PSMA-directed RLT. Herein, we evaluated if CUB domain containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, particularly for subsets like small cell neuroendocrine prostate cancer (SCNC) that would not be expected to respond to current options.Experimental DesignCDCP1 mRNA levels were evaluated in the RNA-seq data from 119 recent mCRPC biopsies. Protein expression was assessed in twelve SCNC and adenocarcinoma patient derived xenografts. Saturation binding assays were performed with 4A06, a recombinant human antibody that targets the CDCP1 ectodomain. The feasibility of imaging and treating mCRPC in vivo was tested with 89Zr-4A06 and 177Lu-4A06.ResultsCDCP1 mRNA expression was observed in over 90% of mCRPC biopsies, including SCNC and in adenocarcinoma with low FOLH1 (PSMA) levels. A modest anticorrelation was observed between CDCP1 and PTEN. Overall survival was not significantly different based on CDCP1 mRNA levels, regardless of PTEN status. Full length and/or cleaved CDCP1 was expressed in ten of twelve PDX samples. Bmax values of ~22,000 and ~6,200 fmol/mg were calculated for two human prostate cancer cell lines. Five prostate cancer models were readily detected in vivo with 89Zr-4A06. 177Lu-4A06 significantly suppressed the growth of DU145 tumors compared to control.ConclusionsThe antitumor data and the overexpression of CDCP1 reported herein provide the first evidence promoting CDCP1 directed RLT as a treatment strategy for mCRPC.Statement of Translational RelevanceNew targets for RLT are needed to address the subset of mCRPC that cannot be treated with bone seeking radionuclides or PSMA directed RLT. We report herein the first data credentialing CDCP1 as a target for mCRPC, in both adenocarcinoma and neuroendocrine subtypes. Combined with low expression in normal human tissues, these data provide a compelling scientific rationale for testing CDCP1 directed RLT clinically in mCRPC patients alone or in combination with other systemic therapies.

Purpose: We recently identified CD46 as a novel therapeutic target in prostate cancer. In this study, we developed a CD46-targeted PET radiopharmaceutical, [89Zr]DFO-YS5, and evaluated its performance for immunoPET imaging in murine prostate cancer models. Experimental Design: [89Zr]DFO-YS5 was prepared and its in vitro binding affinity for CD46 was measured. ImmunoPET imaging was conducted in male athymic nu/nu mice bearing DU145 [AR−, CD46+, prostate-specific membrane antigen–negative (PSMA−)] or 22Rv1 (AR+, CD46+, PSMA+) tumors, and in NOD/SCID gamma mice bearing patient-derived adenocarcinoma xenograft, LTL-331, and neuroendocrine prostate cancers, LTL-331R and LTL-545. Results: [89Zr]DFO-YS5 binds specifically to the CD46-positive human prostate cancer DU145 and 22Rv1 xenografts. In biodistribution studies, the tumor uptake of [89Zr]DFO-YS5 was 13.3 ± 3.9 and 11.2 ± 2.5 %ID/g, respectively, in DU145 and 22Rv1 xenografts, 4 days postinjection. Notably, [89Zr]DFO-YS5 demonstrated specific uptake in the PSMA- and AR-negative DU145 model. [89Zr]DFO-YS5 also showed uptake in the patient-derived LTL-331 and -331R models, with particularly high uptake in the LTL-545 neuroendocrine prostate cancer tumors (18.8 ± 5.3, 12.5 ± 1.8, and 32 ± 5.3 %ID/g in LTL-331, LTL-331R, and LTL-545, respectively, at 4 days postinjection). Conclusions: [89Zr]DFO-YS5 is an excellent PET imaging agent across a panel of prostate cancer models, including in both adenocarcinoma and neuroendocrine prostate cancer, both cell line- and patient-derived xenografts, and both PSMA-positive and -negative tumors. It demonstrates potential for clinical translation as an imaging agent, theranostic platform, and companion biomarker in prostate cancer.

Background: Immune checkpoint inhibitors (CPIs) are being used to effectively treat a growing number of cancers but can cause immune related adverse events (irAE). Thyroid dysfunction is the most common endocrine irAE. A meta-analysis of clinical trials estimated that following CPI exposure, 6.6% will become hypothyroid and 2.9% will have hyperthyroidism1. It is unclear if this reflects the real-world incidence of these irAEs. We used electronic health record (EHR) data to identify patients who developed thyroid dysfunction after CPI to estimate the real-world incidence of these irAEs. Methods: Data were derived from the EHR of a large U.S. academic center. We identified subjects treated with CPIs between 2012 and 2018 and excluded those with thyroid cancer or pre-existing thyroid disease. Thyroid dysfunction was identified as either a TSH > 10, an abnormal free T4 or a prescription for thyroid hormone replacement or anti-thyroid medication. Those with thyroid dysfunction were then categorized as having pre-existing disease or a new-onset thyroid irAE based on the timing of CPI initiation. Logistic regression was used to evaluate the association of thyroid irAE with age, gender, CPI and type of cancer. Results: In total, 1146 individuals without pre-existing thyroid disease that received CPIs were assessed. Pembrolizumab was the most common treatment (45%), followed by nivolumab (20%). Less than 10% of subjects received atezolizumab, durvalumab, ipilimumab monotherapy, combined ipilimumab/nivolumab, or other combinations of CPIs. Melanoma was the most common cancer treated (32%), followed by non-small cell lung cancer (13%). The prevalence of any other cancer was < 10% each. Overall, 19% developed thyroid irAEs. After adjustment for gender and age, the type of cancer was significantly associated with new onset thyroid dysfunction (p=0.01). The rates of thyroid irAEs ranged from 10% in glioblastoma to 40% in renal cell cancer. Although there was no significant association between irAEs and specific CPIs in the overall analysis, thyroid irAEs were more common in subjects who received combined ipilimumab/ nivolumab (31%) compared to pembrolizumab (18%, p=0.03), nivolumab (18%, p Conclusion: Thyroid irAEs are much more common in real world practice than in clinical trials and there is emerging evidence that certain cancer types incur a higher risk of thyroid irAEs even after adjustment for CPI exposure. Clinicians and patients should be educated about these risks. Future work should focus on exploring the reasons underlying the differing rates of thyroid irAEs among different cancers including effect on cancer outcomes. 1Barroso-Sousa et al. Incidence of Endocrine Dysfunction Following the Use of Different Immune Checkpoint Inhibitor Regimens. JAMA Oncol. 2017; 02215: 1–10.

Boron neutron capture therapy (BNCT) is a therapeutic modality which has been used for the treatment of cancers, including brain and head and neck tumors. For effective treatment via BNCT, efficient and selective delivery of a high boron dose to cancer cells is needed. Prostate specific membrane antigen (PSMA) is a target for prostate cancer imaging and drug delivery. In this study, we conjugated boronic acid or carborane functional groups to a well-established PSMA inhibitor scaffold to deliver boron to prostate cancer cells and prostate tumor xenograft models. Eight boron-containing PSMA inhibitors were synthesized. All of these compounds showed strong binding affinity to PSMA in a competition radioligand binding assay (IC(50) from 555.7 nM-20.3 nM). Three selected compounds 1a, 1d and 1f were administered to mice, and their in vivo blocking of (68)Ga-PSMA-11 uptake was demonstrated through a positron emission tomography (PET) imaging and biodistribution experiment. Biodistribution analysis demonstrated boron uptake of 4–7 μg/gram in 22Rv1 prostate xenograft tumors and similar tumor/muscle ratios compared to the ratio for the most commonly used BNCT compound, 4-borono-L-phenylalanine (BPA). Taken together, this data suggest a potential role for PSMA targeted BNCT agents in prostate cancer therapy following suitable optimization.

Noninvasive tools that target tumor cells could improve the management of glioma. Cancer generally has a high demand for Fe(III), an essential nutrient for a variety of biochemical processes. We tested whether 68Ga-citrate, an Fe(III) biomimetic that binds to apo-transferrin in blood, detects glioma in preclinical models and patients using hybrid PET/MRI. Mouse PET/CT studies showed that 68Ga-citrate accumulates in subcutaneous U87MG xenografts in a transferrin receptor-dependent fashion within 4 hours after injection. Seventeen patients with WHO grade III or IV glioma received 3.7-10.2 mCi 68Ga-citrate and were imaged with PET/MR 123-307 minutes after injection to establish that the radiotracer can localize to human tumors. Multiple contrast-enhancing lesions were PET avid, and tumor to adjacent normal white matter ratios were consistently greater than 10:1. Several contrast-enhancing lesions were not PET avid. One minimally enhancing lesion and another tumor with significantly reduced enhancement following bevacizumab therapy were PET avid. Advanced MR imaging analysis of one patient with contrast-enhancing glioblastoma showed that metabolic hallmarks of viable tumor spatially overlaid with 68Ga-citrate accumulation. These early data underscore that high-grade glioma may be detectable with a radiotracer that targets Fe(III) transport.

While there have been tremendous efforts to target oncogenic RAS signaling from inside the cell, little effort has focused on the cell-surface. Here, we used quantitative surface proteomics to reveal a signature of proteins that are upregulated on cells transformed with KRASG12V, and driven by MAPK pathway signaling. We next generated a toolkit of recombinant antibodies to seven of these RAS-induced proteins. We found that five of these proteins are broadly distributed on cancer cell lines harboring RAS mutations. In parallel, a cell-surface CRISPRi screen identified integrin and Wnt signaling proteins as critical to RAS-transformed cells. We show that antibodies targeting CDCP1, a protein common to our proteomics and CRISPRi datasets, can be leveraged to deliver cytotoxic and immunotherapeutic payloads to RAS-transformed cancer cells and report for RAS signaling status in vivo. Taken together, this work presents a technological platform for attacking RAS from outside the cell.

PURPOSE: Since the introduction of PSMA PET/CT with 68Ga-PSMA-11, this modality for imaging prostate cancer (PC) has spread worldwide. Preclinical studies have demonstrated that short-term androgen deprivation therapy (ADT) can significantly increase PSMA expression on PC cells. Additionally, retrospective clinical data in large patient cohorts suggest a positive association between ongoing ADT and a pathological PSMA PET/CT scan. The present evaluation was conducted to further analyse the influence of long-term ADT on PSMA PET/CT findings. METHODS: A retrospective analysis was performed of all 1,704 patients who underwent a 68Ga-PSMA-11 PET/CT scan at our institution from 2011 to 2017 to detect PC. Of 306 patients scanned at least twice, 10 had started and continued ADT with a continuous clinical response between the two PSMA PET/CT scans. These ten patients were included in the current analysis which compared the tracer uptake intensity and volume of PC lesions on PSMA PET/CT before and during ongoing ADT. RESULTS: Overall, 31 PC lesions were visible in all ten patients before initiation of ADT. However, during ongoing ADT (duration 42-369 days, median 230 days), only 14 lesions were visible in eight of the ten patients. The average tracer uptake values decreased in 71% and increased in 12.9% of the PC lesions. Of all lesions, 33.3% were still visible in six patients with a complete PSA response (≤0.1 ng/ml). CONCLUSION: Continuous long-term ADT significantly reduces the visibility of castration-sensitive PC on PSMA PET/CT. If the objective is visualization of the maximum possible extent of disease, we recommend referring patients for PSMA PET/CT before starting ADT. KEYWORDS: 68Ga-PSMA-11; Androgen deprivation therapy; PET/CT; PSMA; Prostate cancer; Prostate-specific membrane antigen

PurposeThe management of advanced or recurrent prostate cancer is limited in part by the lack of effective imaging agents. Metabolic changes in prostate cancer have previously been exploited for imaging, culminating in the recent US FDA approval of [11C]choline for the detection of subclinical recurrent disease after definitive local therapy. Despite this milestone, production of [11C]choline requires an on-site cyclotron, limiting the scope of medical centers at which this scan can be offered. In this pilot study, we tested whether prostate cancer could be imaged with positron emission tomography (PET) using [68Ga]citrate, a radiotracer that targets iron metabolism but is produced without a cyclotron.ProceduresEight patients with castrate-resistant prostate cancer were enrolled in this single-center feasibility study. All patients had evidence of metastatic disease by standard of care imaging [X-ray computed tomography (CT), bone scan, or magnetic resonance imaging (MRI)] prior to PET with [68Ga]citrate. Patients were intravenously injected with increasing doses of [68Ga]citrate (136.9 to a maximum of 259MBq). Uptake time was steadily increased from 1h to approximately 3.5h for the final 4 patients, and all patients were imaged with a PET/MRI. Qualitative and semi-quantitative (maximum standardized uptake value (SUVmax)) assessment of the metastatic lesions was performed and compared to the standard of care imaging.ResultsAt 1- and 2-h imaging times post injection, there were no detectable lesions with [68Ga]citrate PET. At 3- to 4-h uptake time, there were a total of 71 [68Ga]citrate-positive lesions (67 osseous, 1 liver, and 3 lymph node). Of these, 65 lesions were visible on the standard of care imaging (CT and/or bone scan). One PET-avid osseous vertebral body metastasis was not apparent on either CT or bone scan. Twenty-five lesions were not PET-avid but seen on CT and bone scan (17 bone, 6 lymph node, 1 pleural, and 1 liver). The average of the maximum SUVs for bone or soft tissue metastases for patients treated at higher doses and uptake time was statistically higher than the corresponding parameter in normal liver, muscle, and bone. Visually obvious blood pool activity was observed even 3-4h post injection, suggesting that further optimization of the [68Ga]citrate imaging protocol is required to maximize signal-to-background ratios.ConclusionsOur preliminary results support that PET with [68Ga]citrate may be a novel tool for imaging prostate cancer. Future studies are needed to determine the optimal imaging protocol, the clinical significance of [68Ga]citrate uptake, and its role in therapeutic decisions.

Early life is a dynamic period of growth for the lung and immune system. We hypothesized that ambient ozone exposure during postnatal development can affect the innate immune response to other environmental challenges in a persistent fashion. To test this hypothesis, we exposed infant rhesus macaque monkeys to a regimen of 11 ozone cycles between 30 days and 6 mo of age; each cycle consisted of ozone for 5 days (0.5 parts per million at 8 h/day) followed by 9 days of filtered air. Animals were subsequently housed in filtered air conditions and challenged with a single dose of inhaled LPS at 1 yr of age. After completion of the ozone exposure regimen at 6 mo of age, total peripheral blood leukocyte and polymorphonuclear leukocyte (PMN) numbers were reduced, whereas eosinophil counts increased. In lavage, total cell numbers at 6 mo were not affected by ozone, however, there was a significant reduction in lymphocytes and increased eosinophils. Following an additional 6 mo of filtered air housing, only monocytes were increased in blood and lavage in previously exposed animals. In response to LPS challenge, animals with a prior history of ozone showed an attenuated peripheral blood and lavage PMN response compared with controls. In vitro stimulation of peripheral blood mononuclear cells with LPS resulted in reduced secretion of IL-6 and IL-8 protein in association with prior ozone exposure. Collectively, our findings suggest that ozone exposure during infancy can result in a persistent effect on both pulmonary and systemic innate immune responses later in life.

The recent, dramatic increase in the incidence of childhood asthma suggests a role for environmental contaminants in the promotion of interactions between allergens and the respiratory system of young children. To establish whether exposure to an environmental stressor, ozone (O3), and an allergen, house dust mite (HDMA), during early childhood promotes remodeling of the epithelial-mesenchymal trophic unit (EMTU) of the tracheobronchial airway wall by altering postnatal development, infant rhesus monkeys were exposed to cyclic episodes of filtered air (FA), HDMA, O3, or HDMA plus O3. The following alterations in the EMTU were found after exposure to HDMA, O3, or HDMA plus O3: (1) reduced airway number; (2) hyperplasia of bronchial epithelium; (3) increased mucous cells; (4) shifts in distal airway smooth muscle bundle orientation and abundance to favor hyperreactivity; (5) interrupted postnatal basement membrane zone differentiation; (6) modified epithelial nerve fiber distribution; and (7) reorganization of the airway vascular and immune system. Conclusions: cyclic challenge of infants to toxic stress during postnatal lung development modifies the EMTU. This exacerbates the allergen response to favor development of intermittent airway obstruction associated with wheeze. And, exposure of infants during early postnatal lung development initiates compromises in airway growth and development that persist or worsen as growth continues, even with cessation of exposure.

Inner city children exposed to high levels of ozone suffer from an increased prevalence of respiratory diseases. Lung development in children is a long-term process, and there is a significant period of time during development when children growing up in urban areas are exposed to oxidant air pollution. This study was designed to test whether repeating cycles of injury and repair caused by episodes of ozone exposure lead to chronic airway disease and decreased lung function by altering normal lung maturation. We evaluated postnatal lung morphogenesis and function of infant monkeys after 5 mo of episodic exposure of 0.5 parts per million ozone beginning at 1 mo of age. Nonhuman primates were chosen because their airway structure and postnatal lung development is similar to those of humans. Airway morphology and structure were evaluated at the end of the 5-mo exposure period. Compared with control infants, ozone-exposed animals had four fewer nonalveolarized airway generations, hyperplastic bronchiolar epithelium, and altered smooth muscle bundle orientation in terminal and respiratory bronchioles. These results suggest that episodic exposure to environmental ozone compromises postnatal morphogenesis of tracheobronchial airways.

Development of smooth muscle in conducting airways begins early in fetal life. Whereas the pattern and regulation of smooth muscle differentiation are well-defined, the impact of airway growth on the process is not. To evaluate the transformations in organization during postnatal growth, smooth muscle bundle organization (size, abundance, and orientation) was mapped in five generations of distal airways of infant rhesus monkeys (5 days and 1, 2, 3, and 6 mo old). On the basis of direct measurement of the bronchiole proximal to the terminal bronchiole, length increased by 2-fold, diameter by 1.35-fold, and surface area by 2.8-fold between 5 days and 6 mo of age. Smooth muscle bundle size was greater in proximal bronchioles than in respiratory bronchioles and did not change with age. However, relative bundle size decreased in proportion to airway size as the airways grew. Relative bundle abundance was constant regardless of airway generation or age. The distribution of smooth muscle bundle orientation changed with age in each airway generation, and there were significant changes in the terminal and respiratory bronchioles. We conclude that smooth muscle undergoes marked organizational changes as airways grow during postnatal development.

The effect of sidestream tobacco smoke combined with other pollutants is largely unknown. Previously, we found that distal airway epithelial repair was inhibited in mice exposed to sidestream tobacco smoke (TS) for 5 days followed by single exposure to naphthalene (NA), a common polycyclic aromatic hydrocarbon found in cigarette smoke, diesel exhaust, and pesticide formulations. The main injury target of NA is the nonciliated (Clara) bronchiolar cell. NA injury normally resolves in two weeks. Repair in mice exposed to TS and NA was unresolved in the distal bronchioles 14 days post-NA injury. We hypothesized that repair inhibition persisted as a first step towards long-term airway remodeling and expanded the previous study by evaluating repair 21 days after acute NA injury. Repair was evaluated using high resolution histopathology, TEM, and quantitative morphometry. In animals exposed to TS and NA, repair was still impaired; re-differentiation of Clara cells at the bronchoalveolar duct junction was incomplete, indicating repair was continuing. Compared to 14 days post-NA-injury, repair at 21 days post-NA treatment was more extensive. Animals exposed only to TS had epithelium similar to controls. While TS exposure impairs bronchiolar epithelial repair after NA exposure, this effect appears to be slowly resolving over time.

Summary Background In previous studies, we showed that repeated exposure to (1) house dust mite allergen (HDMA) (Dermatophagoides farinae) caused thickening of the basement membrane zone (BMZ) and (2) HDMA+ozone (O3) caused depletion of BMZ perlecan and atypical development of BMZ collagen (irregular thin areas

Development of the basement membrane zone (BMZ) occurs postnatally in the rhesus monkey. The purpose of this study was to determine whether house dust mite allergen (HDMA) plus ozone altered this process. Rhesus monkeys were exposed to a regimen of HDMA and/or ozone or filtered air for 6 mo. To detect structural changes in the BMZ, we measured immunoreactivity of collagen I. To detect functional changes in the BMZ, we measured perlecan and fibroblast growth factor-2 (FGF-2). We also measured components of the FGF-2 ternary signaling complex [fibroblast growth factor receptor-1 (FGFR-1) and syndecan-4]. The width of the BMZ was irregular in the ozone groups, suggesting atypical development of the BMZ. Perlecan was also absent from the BMZ. In the absence of perlecan, FGF-2 was not bound to the BMZ. However, FGF-2 immunoreactivity was present in basal cells, the lateral intercellular space (LIS), and attenuated fibroblasts. FGFR-1 immunoreactivity was downregulated, and syndecan-4 immunoreactivity was upregulated in the basal cells. This suggests that FGF-2 in basal cells and LIS may be bound to the syndecan-4. We conclude that ozone and HDMA plus ozone effected incorporation of perlecan into the BMZ, resulting in atypical development of the BMZ. These changes are associated with specific alterations in the regulation of FGF-2, FGFR-1, and syndecan-4 in the airway epithelial-mesenchymal trophic unit, which may be associated with the developmental problems of lungs associated with exposure to ozone.

Twenty-four infant rhesus monkeys (30 days old) were exposed to 11 episodes of filtered air (FA), house dust mite allergen aerosol (HDMA), ozone (O 3 ), or HDMA + O 3 (5 days each followed by 9 days of FA). Ozone was delivered for 8 h/day at 0.5 ppm. Twelve of the monkeys were sensitized to house dust mite allergen ( Dermatophagoides farinae ) at ages 14 and 28 days by subcutaneous inoculation (SQ) of HDMA in alum and intraperitoneal injection of heat-killed Bordetella pertussis cells. Sensitized monkeys were exposed to HDMA aerosol for 2 h/day on days 3–5 of either FA ( n = 6) or O 3 ( n = 6) exposure. Nonsensitized monkeys were exposed to either FA ( n = 6) or O 3 ( n = 6). During the exposure regimen, parameters of allergy (i.e., serum IgE, histamine, and eosinophilia), airways resistance, reactivity, and structural remodeling were evaluated. Eleven repeated 5-day cycles of inhaling 0.5 ppm ozone over a 6-month period had only mild effects on the airways of nonsensitized infant rhesus monkeys. Similarly, the repeated inhalation of HDMA by HDMA-sensitized infant monkeys resulted in only mild airway effects, with the exception of a marked increase in proximal airway and terminal bronchiole content of eosinophils. In contrast, the combined cyclic inhalation of ozone and HDMA by HDMA sensitized infants monkeys resulted in a marked increase in serum IgE, serum histamine, and airways eosinophilia. Furthermore, combined cyclic inhalation of ozone and HDMA resulted in even greater alterations in airway structure and content that were associated with a significant elevation in baseline airways resistance and reactivity. These results suggest that ozone can amplify the allergic and structural remodeling effects of HDMA sensitization and inhalation.

Remodeling of the epithelial basement membrane zone (BMZ) involves increased deposition of collagen, resulting in thickening of the BMZ. The current study focuses on fibroblast growth factor-2 (FGF-2) in the tracheal BMZ in house dust mite allergen (HDMA)-sensitized infant rhesus monkeys, challenged with HDMA at a time when the BMZ is undergoing active postnatal development. To detect structural changes in the BMZ, we measured collagens I, III, and V. To detect changes in the function of the BMZ, we measured immunoreactivity of the heparan sulfate proteoglycan, perlecan, and FGF-2. We found significant thickening of the tracheal BMZ (p < 0.05) with each of these parameters. We also found that all HDMA tracheal samples expressed thin focal areas of the BMZ associated with leukocyte trafficking. These areas were depleted of perlecan and FGF-2; however, increased FGF-2 immunoreactivity was present in the adjacent basal cells. We conclude that basal cells and FGF-2 are involved with significant remodeling of the BMZ in the developing trachea of infant rhesus monkeys exposed to HDMA.

UnlabelledAntibodies and antibody-drug conjugates targeting the cell surface protein 6 transmembrane epithelial antigen of prostate 1 (STEAP1) are in early clinical development for the treatment of castration-resistant prostate cancer (PCa). In general, antigen expression directly affects the bioactivity of therapeutic antibodies, and the biologic regulation of STEAP1 is unusually complicated in PCa. Paradoxically, STEAP1 can be induced or repressed by the androgen receptor (AR) in different human PCa models, while also expressed in AR-null PCa. Consequently, there is an urgent need to translate diagnostic strategies to establish which regulatory mechanism predominates in patients to situate the appropriate therapy within standard of care therapies inhibiting AR.MethodsTo this end, we prepared and evaluated (89)Zr-labeled MSTP2109A ((89)Zr-2109A), a radiotracer for PET derived from a fully humanized monoclonal antibody to STEAP1 in preclinical PCa models.Results(89)Zr-2109A specifically localized to the STEAP1-positive human PCa models CWR22Pc, 22Rv1, and PC3. Moreover, (89)Zr-2109A sensitively measured treatment-induced changes (∼66% decline) in STEAP1 expression in CWR22PC in vitro and in vivo, a model we showed to express STEAP1 in an AR-dependent manner.ConclusionThese findings highlight the ability of immuno-PET with (89)Zr-2109A to detect acute changes in STEAP1 expression and argue for an expansion of ongoing efforts to image PCa patients with (89)Zr-2109A to maximize the clinical benefit associated with antibodies or antibody-drug conjugates to STEAP1.

Exposure to ozone has been associated with increased incidence of respiratory morbidity in humans; however the mechanism(s) behind the enhancement of susceptibility are unclear. We have previously reported that exposure to episodic ozone during postnatal development results in an attenuated peripheral blood cytokine response to lipopolysaccharide (LPS) that persists with maturity. As the lung is closely interfaced with the external environment, we hypothesized that the conducting airway epithelium of neonates may also be a target of immunomodulation by ozone. To test this hypothesis, we evaluated primary airway epithelial cell cultures derived from juvenile rhesus macaque monkeys with a prior history of episodic postnatal ozone exposure. Innate immune function was measured by expression of the proinflammatory cytokines IL-6 and IL-8 in primary cultures established following in vivo LPS challenge or, in response to in vitro LPS treatment. Postnatal ozone exposure resulted in significantly attenuated IL-6 mRNA and protein expression in primary cultures from juvenile animals; IL-8 mRNA was also significantly reduced. The effect of antecedent ozone exposure was modulated by in vivo LPS challenge, as primary cultures exhibited enhanced cytokine expression upon secondary in vitro LPS treatment. Assessment of potential IL-6-targeting microRNAs miR-149, miR-202, and miR-410 showed differential expression in primary cultures based upon animal exposure history. Functional assays revealed that miR-149 is capable of binding to the IL-6 39 UTR and decreasing IL-6 protein synthesis in airway epithelial cell lines. Cumulatively, our findings suggest that episodic ozone during early life contributes to the molecular programming of airway epithelium, such that memory from prior exposures is retained in the form of a dysregulated IL-6 and IL-8 response to LPS; differentially expressed microRNAs such as miR-149 may play a role in the persistent modulation of the epithelial innate immune response towards microbes in the mature lung. ©Clay et al.

Basal cells exist as a separate layer of cells covering most of the airway basal lamina. In this central position, they can interact with columnar epithelium, neurons, basement membrane, and the underlying mesenchymal cells. In addition, they interact with inflammatory cells, lymphocytes and dendritic cells. These interactions take place in the lateral intercellular space between basal cells. In this central position basal cells become a very important part of the epithelial-mesenchymal trophic unit of larger airways. In this review it is shown that basal cells may function as progenitor cells of airway epithelium and have a role in attachment of columnar epithelium with the basement membrane. They also have the potential to function in regulation of neurogenic inflammation, the inflammatory response, transepithelial water movement, oxidant defense of the tissue and formation of the lateral intercellular space. Other characteristics of basal cells were not clearly associated with a particular function. The functions for basal cells listed attempt to explain the presence of recently identified molecules in basal cells of airway epithelium. It should be pointed out that specific studies have not been carried out which test the relationship between the molecular functions we describe in this review and the basal cell in airway epithelium.

A noninvasive technology that quantitatively measures the activity of oncogenic signaling pathways could have a broad impact on cancer diagnosis and treatment with targeted therapies. Here we describe the development of (89)Zr-desferrioxamine-labeled transferrin ((89)Zr-transferrin), a new positron emission tomography (PET) radiotracer that binds the transferrin receptor 1 (TFRC, CD71) with high avidity. The use of (89)Zr-transferrin produces high-contrast PET images that quantitatively reflect treatment-induced changes in MYC-regulated TFRC expression in a MYC-driven prostate cancer xenograft model. Moreover, (89)Zr-transferrin imaging can detect the in situ development of prostate cancer in a transgenic MYC prostate cancer model, as well as in prostatic intraepithelial neoplasia (PIN) before histological or anatomic evidence of invasive cancer. These preclinical data establish (89)Zr-transferrin as a sensitive tool for noninvasive measurement of oncogene-driven TFRC expression in prostate and potentially other cancers, with prospective near-term clinical application.

The risks for infants and young children receiving inhaled corticosteroid (ICS) therapy are largely unknown. Recent clinical studies indicate that ICS therapy in pre-school children with symptoms of asthma result in decreased symptoms without influencing the clinical disease course, but potentially affect postnatal growth and development. The current study employs a primate experimental model to identify the risks posed by ICS therapy.To (1) establish whether ICS therapy in developing primate lungs reverses pulmonary pathobiology associated with allergic airway disease (AAD) and (2) define the impact of ICS on postnatal lung growth and development in primates.Infant rhesus monkeys were exposed, from 1 through 6 months, to filtered air (FA) with house dust mite allergen and ozone using a protocol that produces AAD (AAD monkeys), or to FA alone (Control monkeys). From three through 6 months, the monkeys were treated daily with ICS (budesonide) or saline.Several AAD manifestations (airflow restrictions, lavage eosinophilia, basement membrane zone thickening, epithelial mucin composition) were reduced with ICS treatment, without adverse effects on body growth or adrenal function; however, airway branching abnormalities and intraepithelial innervation were not reduced. In addition, several indicators of postnatal lung growth and differentiation: vital capacity, inspiratory capacity, compliance, non-parenchymal lung volume and alveolarization, were increased in both AAD and Control monkeys that received ICS treatment.Incomplete prevention of pathobiological changes in the airways and disruption of postnatal growth and differentiation of airways and lung parenchyma in response to ICS pose risks for developing primate lungs. These responses also represent two mechanisms that could compromise ICS therapy's ability to alter clinical disease course in young children.

The primary function of basal cells is to attach columnar epithelium to the basal lamina. They may also have limited stem cell potential, but very little is known of other biological functions. Basal cells lie on the basal lamina beneath the ciliated and secretory cells and do not reach the surface of the epithelium. The position of the cell beneath the ciliated and secretory cell epithelium makes their in situ study difficult. In order to further aid in the study of basal cells, we have developed an in situ preparation technique in which ciliated and secretory cells are removed. Treatment of rat tracheas with a 20 mM Na 2 EDTA solution, pH 7.4, results in partial removal of columnar epithelium from the basal lamina. The percent of denuded columnar epithelial cells per mm of basal lamina is 43.9 ± 7.8% at 60 min, 47.6 ± 8.4% at 90 min, and 52.6 ± 2.7% at 120 min. The viability of the exposed basal cells was the same at both 60 and 90 min of treatment (79.4 ± 7.8 and 78.0 ± 8.5, respectively). Morphologically, the exposed basal cells are attached to the basal lamina by hemidesmosomes and are similar to those in the intact animal.

The basal cell in airway epithelium plays a major role in attachment of ciliated and nonciliated columnar cells to the basal lamina. As the airway grows in diameter and the columnar epithelium in height, the number of basal cells and the amount of tonofilaments (cytokeratin filaments) and anchoring junctions increase. In this way they maintain a constant attachment strength between the increased volume of the epithelium and the basal lamina. The purpose of this study was to determine which cytokeratins (CKs) are expressed in growing basal cells of the rat and demonstrate where they are localized in the cytoskeleton. Sprague Dawley rats 10, 30 and 90 days of age were used in this study. For light microscopy, tracheal samples were fixed in 95% alcohol or 4% formalin for 2 hr and then embedded in paraffin. For electron microscopy, the tracheal samples were placed in 20 mM EDTA in HBSS media minus Ca ++ and Mg ++ at pH 7.4 for 60 min to permeabilize the cells and expose the intracellular structures. Antibodies to cytokeratins 7, 8, 10, 13 and 18 did not react to basal cells at any age studied. Antibodies to CKs 5 + 8, 14, 16 + 13, and 19 gave a positive reaction with basal cells at each age studied. Immunogold particles representing antibodies to CK 14 were heavily distributed over intermediate filaments making up the cytoskeleton. Both CK 16 + 13 and 19 were also over intermediate filaments but at a much lower density. Expression of CKs 16 and 19 may occur in basal cells that are actively proliferating and growing. Expression of CK 14 is thought to play a role in cells involved with adhesion. Expression patterns of CKs in these experiments are in agreement with the role of the airway basal cell in attachment of ciliated and nonciliated columnar cells to the basal lamina.

In the connective tissue underlying the epithelium of the airways, there are thin attenuated cells that appear to be fibroblasts. These cells are close to the basal lamina and delineate an area equivalent to the basement membrane zone (BMZ). Using the mean data from transversely and longitudinally sectioned attenuated fibroblasts, we estimate that the diameter of the attenuated fibroblast is approximately 28.0 microns and its thickness 0.55 microns. Serial sections indicate there are pores of various sizes in the attenuated fibroblast. Also, the presence of cell segments less than 5 microns in length suggests there are processes from the edge of the cell which would give it a stellate appearance. These large flat cells form a sheath covering 66 to 70% of the BMZ at an average distance of 1.9 microns from the basal lamina. In addition, they make contact with the basal lamina approximately 7,000 times per mm2 of basal lamina. The majority of the contacts with the basal lamina are beneath basal cells. Cells of the attenuated fibroblast sheath are probably associated with lung morphogenesis, wound healing, and development of the BMZ. Their existence as a sheath demonstrates an anatomical unit associated with fibroblast-epithelial cell interactions in airway epithelium.

Collagen VII anchoring fibrils in the basement membrane zone (BMZ) are part of a supracellular anchoring network that attaches the epithelium to the BMZ. Sloughing of airway epithelium in asthmatics (creola bodies) is a pathology associated with the supracellular anchoring network. In a rhesus monkey model of house dust mite (HDM)-induced allergic asthma, we found increased deposition of collagen I in the BMZ. In this study, we determine whether HDM also affected deposition of collagen VII in the BMZ. In the developing airway of rhesus monkeys, the width of collagen VII anchoring fibrils in the BMZ was 0.02 +/- 0.04 microm at 1 mo of age. At 6 mo the width had increased to 1.28 +/- 0.34 microm and at 12 mo 2.15 +/- 0.13 microm. In animals treated with HDM, we found a 42.2% reduction in the width of collagen VII layer in the BMZ at 6 mo (0.74 +/- 0.15 microm; P0.05). During recovery, the rate of collagen VII deposition returned to normal. However, the amount of collagen VII lost was not recovered after 6 mo. We concluded that normal development of the collagen VII attachment between the epithelium and BMZ occurs in coordination with development of the BMZ. However, in HDM-treated animals, the collagen VII attachment with the epithelium was significantly reduced. Such a reduction in collagen VII may weaken the supracellular anchoring network and be associated with sloughing of the epithelium and formation of creola bodies in asthmatics.

The impact of topical antimicrobial agents on improving the survival of patients with major thermal injuries is significant. However, the effects of these agents on cells responsible for wound healing has only recently received attention. Fresh human basal keratinocytes were grown in serum-free modified MCDB 153 medium under standard tissue culture conditions. Cells were subsequently exposed to concentrations of silver sulfadiazine and mafenide acetate as low as 1/100 of that used clinically over a period of 5-7 days. Cellular responses documented with hemocytometer cells counts, cellular protein assays, phase-contrast microscopy, and transmission electron microscopy show only severe toxicity to mafenide acetate. Such data imply that inhibition of wound epithelialization is greater with the use of mafenide acetate than with the use of silver sulfadiazine.

We tested the hypothesis that enhanced cell division accounted for the augmented numbers of monocytic phagocytes with characteristics attributed to alveolar macrophages (AM) found in the lungs of habitual tobacco (T) and marijuana (M) smokers. The monocytic phagocytes, that is, alveolar macrophages, were obtained by bronchoalveolar lavage (BAL) from 12 nonsmoking subjects; 10 subjects who smoked T only (TS); 13 subjects who smoked M only (MS); and 6 smokers of both T and M (MTS). The replication of these cells was determined by measuring the incorporation of [3H]thymidine into the DNA of dividing cells and visually counting 2,000 cells on autoradiographically prepared cytocentrifuge cell preparations. This study demonstrated that the number of [3H]thymidine-labeled monocytic phagocytes with characteristics of alveolar macrophages from either TS or MS have a higher proliferative index compared to cells (macrophages) from nonsmokers, p less than 0.05 by one-way ANOVA. The total number of BAL macrophages that are in mitosis in TS (17.90 +/- 4.50 labeled AM x 10(3)/ml) or MTS (10.50 +/- 4.20 labeled AM x 10(3)/ml) are 18- and 10-fold greater, respectively, than the number obtained from nonsmokers (1.01 +/- 0.18 labeled AM x 10(3)/ml). Interestingly, the number of [3H]thymidine-labeled macrophages from MS (2.90 +/- 0.66 labeled AM x 10(3)/ml) are also greater than the number obtained from nonsmokers, although this is not statistically significant. The stimulus augmenting alveolar macrophage replication is as yet unknown but may likely be found in the T or M smoke.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated rat lung (IPL) studies have suggested that the pulmonary uptake of inhaled nitrogen dioxide (NO2) is governed via a chemical reaction-dependent process which results in NO2-derived reaction products diffusing into the vascular space. Experimental results indicated that substantial proportions of this reactive absorption occur in distal sites. However, gas phase deposition in proximal locations cannot be ruled out due to the lack of information on bronchial perfusion in rat IPL preparations. Consequently, we evaluated the presence of pulmonary-to-bronchial anastomotic perfusate flow in control and NO2-exposed (10.3 ppm) rat IPL. Monastral blue (MB) was used as a vascular marker and was infused into the pulmonary artery catheter either for recirculation at time zero or as an end-experiment (60 min) bolus. In addition, MB was infused into control in situ preparations to observe intact bronchial circulations. Lungs were prepared for routine evaluation by light microscopy. In situ MB was observed in all pulmonary and bronchial vessels. In IPL, MB was observed only in far terminal airway-associated vessels. No differences were observed in MB distribution between bolus (end-experiment) and recirculated (time zero) applications. NO2 exposure produced no effect on MB distribution. We conclude that in rat IPL: (1) negligible anastomotic flow occurs from the pulmonary into the bronchial circulation, (2) nonedemagenic NO2 exposures do not alter existing perfusate distribution, and (3) the perfusate appearance of inhalation-derived species results from gas phase deposition only in distal sites which have ready accessibility to the pulmonary circulation.

The etiology of fibrous capsular contractures in patients with silicone prostheses is unclear. However, cellular responses to the silicone polymers of the prostheses have not been examined. The exposure of human dermal fibroblasts to the components of the silicone gel prosthesis results in a significant change in cellular configuration and a progressive reduction in cell proliferation as determined by total matrix protein assays and hemocytometer cell counts. Transmission electron microscopy, however, documents a twofold increase in the rough endoplasmic reticulum when cells are exposed to the silicone gel. These findings suggest significant alterations in the behavior of human fibroblast subpopulations in response to silicone polymers.

The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.

Chemical genomics aims to discover small molecules that affect biological processes through the perturbation of protein function. However, determining the protein targets of bioactive compounds remains a formidable challenge. We address this problem here through the creation of a natural product-inspired small-molecule library bearing protein-reactive elements. Cell-based screening identified a compound, MJE3, that inhibits breast cancer cell proliferation. In situ proteome reactivity profiling revealed that MJE3, but not other library members, covalently labeled the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), resulting in enzyme inhibition. Interestingly, MJE3 labeling and inhibition of PGAM1 were observed exclusively in intact cells. These results support the hypothesis that cancer cells depend on glycolysis for viability and promote PGAM1 as a potential therapeutic target. More generally, the incorporation of protein-reactive compounds into chemical genomics screens offers a means to discover targets of bioactive small molecules in living systems, thereby enabling downstream mechanistic investigations.

Lung development is both a pre- and postnatal process. Although many lung diseases have their origins in early childhood, few quantitative data are available on the normal growth and differentiation of both the conducting airways and the airway epithelium during the postnatal period. We examined rhesus monkey lungs from five postnatal ages: 4–6 days and 1, 2, 3, and 6 mo. Airways increase significantly in both length and circumference as monkeys increase significantly in body weight from 5 days to 6 mo. In this study we asked: as basement membrane surface area increases, does the epithelial cell organization change? To answer this question, we quantified total epithelial cell mass using high-resolution light micrographs and morphometric techniques on sections from defined airway regions: trachea, proximal intrapulmonary bronchus (generations 1 or 2), and distal intrapulmonary bronchus (generations 6–8). Epithelial thickness decreased in the smaller, more distal, airways compared with trachea but did not change with age in the trachea and proximal bronchus. The volume fraction of all cell types measured did not change significantly. Ciliated cells in the distal bronchus and goblet cells in the trachea both decreased in abundance with increasing age. Overall, the epithelial cell populations changed little in terms of mass or relative abundance to each other during this period of active postnatal lung growth. Regarding the proximal conducting airway epithelium, we conclude that 1) the steady-state abundance is tightly regulated to keep the proportion of cell types constant, and 2) establishment of these cell types occurs before 4–6 days postnatal age. We conclude that growth of the proximal airways occurs primarily in length and lags behind that of the lung parenchyma.

Nerves and neuroendocrine cells located within the airway epithelium are ideally situated to sample a changing airway environment, to transmit that information to the central nervous system, and to promote trophic interactions between epithelial and mesenchymal cellular and acellular components. We tested the hypothesis that the environmental stresses of ozone (O(3)) and house dust mite allergen (HDMA) in atopic infant rhesus monkeys alter the distribution of airway nerves. Midlevel bronchi and bronchioles from 6-month-old infant monkeys that inhaled filtered air (FA), house dust mite allergen HDMA, O(3), or HDMA + O(3) for 11 episodes (5 days each, 0.5 ppm O(3), 8 h/day followed by 9 days recovery) were examined using immunohistochemistry for the presence of Protein gene product 9.5 (PGP 9.5), a nonspecific neural indicator, and calcitonin gene-related peptide (CGRP). Along the axial path between the sixth and the seventh intrapulmonary airway generations, there were small significant (P < 0.05) decrements in the density of epithelial nerves in monkeys exposed to HDMA or O(3), while in monkeys exposed to HDMA + O(3) there was a greater significant (P < 0.05) reduction in epithelial innervation. In animals exposed to O(3) or HDMA + O(3) there was a significant increase in the number of PGP 9.5 positive/CGRP negative cells that were anchored to the basal lamina and emitted projections in primarily the lateral plain and often intertwined with projections and cell bodies of other similar cells. We conclude that repeated cycles of acute injury and repair associated with the episodic pattern of ozone and allergen exposure alter the normal development of neural innervation of the epithelial compartment and the appearance of a new population of undefined PGP 9.5 positive cells within the epithelium.

Thickening of the basement membrane zone (BMZ) is a characteristic of several airway diseases; however, very little is known about how this process occurs. The purpose of this study was to define development of the BMZ in the trachea of growing rhesus monkeys at 1, 2, 3, and 6 mo of age. We measured immunoreactivity of collagen types I, III, and V to detect structural changes in the developing BMZ. To detect more dynamic, functional components of the epithelial-mesenchymal trophic unit, we evaluated the distribution of perlecan, fibroblast growth factor-2 (FGF-2), and fibroblast growth factor receptor-1 (FGFR-1). One-month-old monkeys had a mean collagen BMZ width of 1.5 ± 0.7 μm that increased to 4.4 ± 0.4 μm in 6-mo-old monkeys. Perlecan was localized in the BMZ of the epithelium at all ages. FGF-2 was strongly expressed in basal cells at 1–3 mo. At 6 mo, FGF-2 was expressed throughout the BMZ and weakly in basal cells. FGFR-1 immunoreactivity was expressed by basal cells and cilia and weakly in the nuclei of columnar cells at all time points. These data indicate that development of the BMZ is a postnatal event in the rhesus monkey that involves FGF-2.

The bronchiolar injury/repair response to naphthalene (NA) in mice includes acute distal airway epithelial injury that is followed by epithelial proliferation and redifferentiation, which result in repair of the epithelium within 14 days. To test whether prior exposure to aged and diluted sidestream cigarette smoke (TS) would alter the injury/repair response of the airway epithelium, adult mice were exposed to either filtered air (FA) or smoke for 5 days before injection with either corn oil carrier (CO) or naphthalene. Mice were killed 1 and 14 days after naphthalene injury. Lung and lobar bronchus were examined and measured using high-resolution epoxyresin sections. The control group (FACOFA) that was exposed to filtered air/corn oil/filtered air contained airway epithelium similar to untreated controls at all airway levels. The group exposed to tobacco smoke/corn oil/filtered air (TSCOFA) contained some rounded cells in the small airways and some expansion of the lateral intercellular space in the larger airways. Necrotic or vacuolated cells were not observed. As expected, the epithelium in the group exposed to filtered air/naphthalene/filtered air (FANAFA) contained many light-staining vacuolated Clara cells and squamated ciliated cells within distal bronchioles during the acute injury phase. Repair (including redifferentiation of epithelial cells and restoration of epithelial thickness) was nearly complete 14 days after injury. The extent of Clara cell injury, as assessed in lobar bronchi, was not different between the four groups. Although the FANAFA group contained greater initial injury in the distal airways at 1 day, the group exposed to tobacco smoke/naphthalene/filtered air (TSNAFA) had the least amount of epithelial repair at 14 days after naphthalene treatment; many terminal bronchioles contained abundant squamated undifferentiated epithelium. We conclude that tobacco smoke exposure prior to injury (1) does not change the target site or target cell type of naphthalene injury, since Clara cells in terminal bronchioles are still selectively injured; (2) results in slightly diminished acute injury from naphthalene in distal bronchioles; and (3) delays bronchiolar epithelial repair.

The airway basement membrane zone is a region specialized for the attachment of the epithelium with the matrix. The epithelium is attached to the lamina densa, which, in turn, is connected to types I and III collagen of the lamina reticularis with anchoring fibrils. The purpose of this study was to define the three-dimensional organization of the structural proteins of the lamina reticularis in the rat trachea. We approached this problem by using whole mounts to look down on the flat surface of the basement-membrane zone rather than a cross section of its thin profile. Fluorescent microscopy with long working distance water immersion objectives and scanning electron microscopy revealed that the structural proteins are arranged as a mat of large fibers oriented along the longitudinal axis of the airway. Smaller fibers are crosslinked with the larger fibers to complete this structure. Other small fibers are oriented around the large fibers and an amorphous material covers individual fibers. The large fibers oriented along the longitudinal axis of the airway are consistent with prior descriptions of fibers composed of collagen III with some collagen I and V; small fibers encircling the large fibers may be collagen VI. The crosslinking fibers are made up of elastin and probably elastin-associated microfibrils. The amorphous proteins covering the fibrous framework may contain proteoglycans and other nonstructural proteins reported to be in the lamina reticularis. The present studies demonstrate that the structural proteins of the lamina reticularis in the rat trachea are arranged as fibers in a highly organized manner.

Interactions between epithelial, mesenchymal, and neural tissue and also the extracellular matrix are necessary to initiate numerous cellular functions of the lung (1). The most common of these functions include differentiation during lung growth, repair of damaged tissue, and regulation of the inflammatory response. Each of these processes requires a localized response to a specific stimulus. Fibroblasts, especially those in close proximity to the airway epithelium, are likely regulators of local responses. In a recent commentary, Smith and colleagues discussed the possibility that resident fibroblasts may act as sentinel cells for these responses (2). In addition to their role as connective tissue cells, fibroblasts also produce cytokines and chemokines in response to various stimuli. Their fixed position in the tissue suggests that they can respond in a local manner to bacterial products, tissue injury, or other environmental factors. The relationship between cytokines and inflammatory cells in asthmatic airways also indicates a similar role for fibroblasts. In the asthmatic lung, the fibroblast plays a key role as a resident mesenchymal cell beneath the epithelium, receiving and sending information to epithelial and inflammatory cells (3, 4). Additionally, these fibroblasts are thought to be responsible for the subepithelial fibrosis associated with asthma. The significance of resident fibroblasts in the airway during inflamation has been described; however, the concept of an anatomically distinct group of fibroblasts associated with airway epithelium has not been explored. In 1990, Brewster and associates described a layer of subepithelial fibroblasts in the bronchi of normal and asthmatic human subjects that were positioned to allow close interaction with the epithelium, neural tissues, and extracellular matrix (5). The population of cells was shown to comprise fibroblasts and myofibroblasts, and individual cells were reported to be as large as 100 m m in diameter. A detailed description of the subepithelial layer of resident fibroblasts in the rat trachea was reported by Evans and coworkers (6). In tissue sections, the cells appear as a layer of attenuated cell processes closely opposed to the lamina reticularis of the basement membrane zone, about 1.9 m m beneath the epithelial basal lamina. The cells are intermeshed with each other, about 40 m m in diameter with the attenuated portions about 0.55 m m in thickness. The cell is thicker near the nucleus and contains abundant rough endoplasmic reticulum near the nucleus. There were no apparent bundles of microfilaments in the thin or thick portions of the cell as there are in myofibroblasts. The cells were determined to be stellate in shape. They exist as a layer of large, flat cells covering about 70% of the interstitial surface of the lamina reticularis, and make numerous contacts with the lamina densa of the basement membrane zone (approximately 7,000 times per mm 2 ). This layer of thin mesenchymal cells was named the attenuated fibroblast sheath (6), with properties of a layer of similar cells in the gut identified as the pericryptal fibroblast sheath (7). On the basis of the data from this previous paper (6), we constructed a three-dimensional model of the attenuated fibroblast sheath (Figure 1). The total attenuated fibroblast sheath, with its large surface area and close proximity to the epithelial/environmental interface, defines an anatomic unit of resident fibroblasts that could respond in a local manner to various stimuli. In this role, the attenuated fibroblast sheath represents the mesenchymal component of interactions with the epithelium, extracellular matrix, neural tissues, and migratory cells of the inflammatory response. The anatomic and functional relationship between the attenuated fibroblast sheath, epithelial and neural tissue, and also the extracellular matrix appears to serve as an epithelial–mesenchymal trophic unit. The epithelial–mesenchymal trophic unit would allow local exchange of information between the different tissue elements in response to various stimuli. This concept is similar to that of other investigators, with the exception that it recognizes that the subepithelial fibroblasts exist as a specific layer of resident fibroblasts beneath the epithelium instead of being randomly distributed in the lamina propria (2–4). Of the tissues in the epithelial–mesenchymal trophic unit, the least is known about the attenuated fibroblast sheath. Examples of the attenuated fibroblast sheath can be seen in most published electron microscope studies of the upper respiratory tract as a thin layer of attenuated cell processes immediately beneath the epithelium. Present in all animal species examined to date, the attenuated fibroblast sheath extends from the proximal to the distal regions of the conducting airways (5, 6, 8). Conceptually, it continues into the gas exchange region as interstitial fibroblasts in ( Received in original form May 14, 1999 and in revised form July 9, 1999 )

Auditory display designers are making increasingly effective and creative use of our ability to localise sound; to particular auditory events as occurring at particular locations. Many applications in which spatial audio has been applied could also benefit from exploiting another important ability of the auditory system; the detection and identification of sound source motion. The display of moving sources could improve usability, provide additional variables in sonification, make virtual environments more perceptually realistic and provide new creative possibilities for designers. Transaural cancellation allows the creation of spatial audio with just two loudspeakers. These techniques are now extended to create the illusion of a sound source moving along an arbitrary trajectory at an arbitrary rate. This paper discusses the application of synthesised sound source movement to a number of practical applications in auditory display. We seek to extend the use of Head-Related Transfer Functions (HRTFs) in stationary sound spatialisation to encompass movement synthesis. The detection of moving sources is not time-invariant so we propose and demonstrate the use of time-frequency spectrograms as a mechanism for characterising source movement. There are an infinite number of such trajectory-related spectrograms and we address the need for a continuous directional model to accommodate this.

One of the most important initial events of colonization and infection of epithelial tissues is the adherence of bacteria to mucosal surfaces. Bacterial adhesion to the epithelial cell may be mediated by a variety of adhesins, including exoproducts. One of these exoproducts, exotoxin A (EA) is a three-domain bacterial toxin that kills mammalian cells by gaining entry to the cytosol and inactivating protein synthesis. In the present study, HTE cultures, 2-4 weeks in vitro (containing both ciliated and non-ciliated cells), were treated for 1 hr with two different non-mucoid strains of Pseudomonas aeruginosa (1 x 10(8) organisms/ml) in the presence of anti-EA. 50 randomly selected fields were evaluated via SEM at x2500 magnification and the number of bacterial clusters/field quantitated. The results of this study indicate, first, that both piliated (ATCC15692) and non-piliated (PAKp) P. aeruginosa will bind to the HTE cells and, second, that treatment of HTE cells with either strain of P. aeruginosa in the presence of anti-EA will reduce bacterial binding by 25% to 50%. Thus, EA may participate in the adhesion of P. aeruginosa to respiratory tract epithelia.

Neutrophils are the primary vectors that produce lung parenchymal injury after smoke inhalation as a result of their transmigration through endothelial and epithelial gap junctions toward the airway surfaces. LFA-1 (CDlla/CD18) on the neutrophil surface is essential for this transmigration in many tissues. We hypothesized that anti-CD18 antibodies would block such movement and prevent lung damage. Ten sheep received nebulized cotton smoke in our standardized model of inhalation injury. The sham group ( η = 2) was insufflated with air only, the second group (η = 4) was insufflated with smoke but received no antibody, and the third group (η = 4) was given monoclonal antibody R15.7 before smoke injury. A similar degree of parenchymal injury and an alveolar epithelial reparative response were measured in both groups receiving injury. Neutrophil counts in the interstitium of both injured groups were also similar. However, in the R15.7 pretreated group, neutrophils remained inside the capillary in far larger numbers (ρ < 0.05). The CD11/18 adhesion complex may not be necessary for the pulmonary microvascular changes associated with inhalation injury. (J Burn Care Rehabil 1993;14:503-ll)

Summary Hamster tracheal epithelial (HTE) cell cultures when grown on collagen coated Millicell-HA filters for 28 d in vitro have an incomplete basal lamina-like structure at the basal plasmalemma/collagen interface. EM evaluation of 42 d HTE cell cultures also revealed the presence of hemidesmosome-like structures on the basal plasmalemma. In order to better characterize this discontinuous basal lamina-like material, HTE cultures at 7, 14, 21, and 28 d were evaluated for the presence of type IV collagen, a basal lamina component. Immunocytochemical treatment of HTE cultures at these time points resulted in the presence of reaction product at the base of the cell layer. When immunocytochemical procedures for the localization of type IV collagen were carried out with normal mouse trachea, the results were also positive. Immunoblotting evaluation of HTE cell supernatants and conditioned media also indicated the presence of type IV collagen. Taken altogether, the presence of what appears to be basal lamina, hemidesmosome-like structures and immunocytochemical and immunoblot data showing the presence of type IV collagen in HTE cultures suggest that HTE cells may be producing basal lamina components but cannot organize them into a complete basal lamina.

The cellular repair process of injured tracheal epithelium is described for sheep after exposure to toxic smoke containing high concentrations of acrolein. Fourteen fasted 3-4-year-old ewes had a portion of their cervical trachea exposed to cotton smoke for 20 min and then were sacrificed at various time intervals ranging from 1 to 22 days after exposure. Within 1 day of injury, columnar epithelium sloughed intact from the trachea with a concomitant reduction of nearly 35% in the basal cell population. At 2 days of recovery, the cellularity of the epithelium had increased and mitotic figures were observed in some tracheal epithelial and gland cells. By 8 days, undifferentiated hyperplastic cells increased to 30/100 microns, differentiated nonciliated columnar cells first appeared, and the basal cell population returned to a normal count of 13 cells/100 microns. Thirteen days after exposure, the undifferentiated hyperplastic cell population had declined to 7 cells/100 microns, nonciliated columnar cells were at control values, and some ciliated cells were identified. At 18 and 22 days, epithelium was normal in appearance and the count was 13 cells/100 microns. Data suggest that because the columnar epithelium sloughs intact with the cilia remaining active, toxic smoke may affect their attachment to the basal lamina. Furthermore, the regeneration process involves differentiation of hyperplastic cells in which they elongate down to the basal lamina, thus re-establishing the integrity of tall epithelium in the sheep trachea.