Joseph Latoche

Polycations increase the efficiency of adenovirus-mediated gene transfer to epithelial and endothelial cells in vitro

Recombinant adenoviruses are being developed for gene therapy for cystic fibrosis and other lung diseases, and for prevention and treatment of vascular thrombosis. A major limitation to the clinical utility of adenoviruses is the low efficiency of gene transfer achieved in vivo. In addition, little is known about the initial interactions between adenoviruses and the target cell. To address the hypothesis that the negative charge presented by membrane glycoproteins reduces the efficiency of adenovirus-mediated gene transfer, primary cultures of human airway, Madin–Darby canine kidney cells, an immortalized cystic fibrosis airway epithelial cell line, and primary cultures of sheep pulmonary artery endothelium were infected with recombinant adeno- virus containing the E. coli lacZ reporter gene (Ad2βgal2) in the presence of various polyions. For each cell type, adsorption of Ad2βgal2 in the presence of the polycations polybrene, protamine, DEAE-dextran, and poly-L-lysine significantly increased the percentage of cells that express lacZ. The polyanion heparin did not significantly alter gene transfer efficiency, but completely abrogated the effects of polycations. These data provide evidence that negatively charged moieties on the cell surface reduce the efficiency of adenovirus-mediated gene transfer, and that alteration of the charge interaction between adenoviruses and the cell surface may improve the potential clinical application of these vectors.

Patients with cystic fibrosis have inducible IL-17+IL-22+ memory cells in lung draining lymph nodes.

BACKGROUND IL-17 is an important cytokine signature of the TH differentiation pathway TH17. This T-cell subset is crucial in mediating autoimmune disease or antimicrobial immunity in animal models, but its presence and role in human disease remain to be completely characterized. OBJECTIVE We set out to determine the frequency of TH17 cells in patients with cystic fibrosis (CF), a disease in which there is recurrent infection with known pathogens. METHODS Explanted lungs from patients undergoing transplantation or organ donors (CF samples=18; non-CF, nonbronchiectatic samples=10) were collected. Hilar nodes and parenchymal lung tissue were processed and examined for TH17 signature by using immunofluorescence and quantitative real-time PCR. T cells were isolated and stimulated with antigens from Pseudomonas aeruginosa and Aspergillus species. Cytokine profiles and staining with flow cytometry were used to assess the reactivity of these cells to antigen stimulation. RESULTS We found a strong IL-17 phenotype in patients with CF compared with that seen in control subjects without CF. Within this tissue, we found pathogenic antigen-responsive CD4+IL-17+ cells. There were double-positive IL-17+IL-22+ cells [TH17(22)], and the IL-22+ population had a higher proportion of memory characteristics. Antigen-specific TH17 responses were stronger in the draining lymph nodes compared with those seen in matched parenchymal lungs. CONCLUSION Inducible proliferation of TH17(22) with memory cell characteristics is seen in the lungs of patients with CF. The function of these individual subpopulations will require further study regarding their development. T cells are likely not the exclusive producers of IL-17 and IL-22, and this will require further characterization.

Chaperone displacement from mutant cystic fibrosis transmembrane conductance regulator restores its function in human airway epithelia

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). The most common mutation, ΔF508, omits the phenylalanine residue at position 508 in the first nucleotide binding domain (NBD1) of CFTR. The mutant protein is retained in the endoplasmic reticulum and degraded by the ubiquitin‐proteasome system. We demonstrate that expression of NBD1 plus the regulatory domain (RD) of ΔF508 CFTR (ΔFRD) restores the biogenesis of mature ΔF508 CFTR protein. In addition, ΔFRD elicited a cAMP‐stimulated anion conductance response in primary human bronchial epithelial (HBE) cells isolated from homozygous ΔF508 CF patients. A protein transduction domain (PTD) could efficiently transduce (~90%) airway epithelial cells. When fused to a PTD, direct addition of the ΔFRD peptide conferred a dose‐dependent, cAMP‐stimulated anion efflux to ΔF508 HBE cells. Hsp70 and Hsp90 associated equally with WT and ΔF508 CFTR, whereas nearly twice as much of the Hsp90 cochaperone, Ahal, associated with ΔF508 CFTR. Expression of ΔFRD produced a dose‐dependent removal of Ahal from ΔF508 CFTR that correlated with its functional rescue. These findings indicate that disruption of the excessive association of the cochaperone, Ahal, with ΔF508 CFTR is associated with the correction of its maturation, trafficking and regulated anion channel activity in human airway epithelial cells. Thus, PTD‐mediated ΔFRD fragment delivery may provide a therapy for CF.—Sun, F., Mi, Z., Condliffe, S. B., Bertrand, C. A., Gong, X., Lu, X., Zhang, R., Latoche, J. D., Pilewski, J. M., Robbins, P. D., Frizzell, R. A. Chaperone displacement from mutant cystic fibrosis transmembrane conductance regulator restores its function in human airway epithelia. FASEB J. 22, 3255–3263 (2008)

Innate Immune Activation by Inhaled Lipopolysaccharide, Independent of Oxidative Stress, Exacerbates Silica-Induced Pulmonary Fibrosis in Mice

Acute exacerbations of pulmonary fibrosis are characterized by rapid decrements in lung function. Environmental factors that may contribute to acute exacerbations remain poorly understood. We have previously demonstrated that exposure to inhaled lipopolysaccharide (LPS) induces expression of genes associated with fibrosis. To address whether exposure to LPS could exacerbate fibrosis, we exposed male C57BL/6 mice to crystalline silica, or vehicle, followed 28 days later by LPS or saline inhalation. We observed that mice receiving both silica and LPS had significantly more total inflammatory cells, more whole lung lavage MCP-1, MIP-2, KC and IL-1β, more evidence of oxidative stress and more total lung hydroxyproline than mice receiving either LPS alone, or silica alone. Blocking oxidative stress with N-acetylcysteine attenuated whole lung inflammation but had no effect on total lung hydroxyproline. These observations suggest that exposure to innate immune stimuli, such as LPS in the environment, may exacerbate stable pulmonary fibrosis via mechanisms that are independent of inflammation and oxidative stress.

177 Lu-Labeled Phosphoramidate-Based PSMA Inhibitors: The Effect of an Albumin Binder on Biodistribution and Therapeutic Efficacy in Prostate Tumor-Bearing Mice

Prostate-specific membrane antigen (PSMA) continues to be an active biomarker for small-molecule PSMA-targeted imaging and therapeutic agents for prostate cancer and various non-prostatic tumors that are characterized by PSMA expression on their neovasculature. One of the challenges for small-molecule PSMA inhibitors with respect to delivering therapeutic payloads is their rapid renal clearance. In order to overcome this pharmacokinetic challenge, we outfitted a 177Lu-labeled phosphoramidate-based PSMA inhibitor (CTT1298) with an albumin-binding motif (CTT1403) and compared its in vivo performance with that of an analogous compound lacking the albumin-binding motif (CTT1401). The radiolabeling of CTT1401 and CTT1403 was achieved using click chemistry to connect 177Lu-DOTA-N3 to the dibenzocyclooctyne (DBCO)-bearing CTT1298 inhibitor cores. A direct comparison in vitro and in vivo performance was made for CTT1401 and CTT1403; the specificity and efficacy by means of cellular uptake and internalization, biodistribution, and therapeutic efficacy were determined for both compounds. While both compounds displayed excellent uptake and rapid internalization in PSMA+ PC3-PIP cells, the albumin binding moiety in CTT1403 conferred clear advantages to the PSMA-inhibitor scaffold including increased circulating half-life and prostate tumor uptake that continued to increase up to 168 h post-injection. This increased tumor uptake translated into superior therapeutic efficacy of CTT1403 in PSMA+ PC3-PIP human xenograft tumors.

Metallothionein-induced zinc partitioning exacerbates hyperoxic acute lung injury.

Hypozincemia, with hepatic zinc accumulation at the expense of other organs, occurs in infection, inflammation, and aseptic lung injury. Mechanisms underlying zinc partitioning or its impact on extrahepatic organs are unclear. Here we show that the major zinc-binding protein, metallothionein (MT), is critical for zinc transmigration from lung to liver during hyperoxia and preservation of intrapulmonary zinc during hyperoxia is associated with an injury-resistant phenotype in MT-null mice. Particularly, lung-to-liver zinc ratios decreased in wild-type (WT) and increased significantly in MT-null mice breathing 95% oxygen for 72 h. Compared with female adult WT mice, MT-null mice were significantly protected against hyperoxic lung injury indicated by reduced inflammation and interstitial edema, fewer necrotic changes to distal airway epithelium, and sustained lung function at 72 h hyperoxia. Lungs of MT-null mice showed decreased levels of immunoreactive LC3, an autophagy marker, compared with WT mice. Analysis of superoxide dismutase (SOD) activity in the lungs revealed similar levels of manganese-SOD activity between strains under normoxia and hyperoxia. Lung extracellular SOD activity decreased significantly in both strains at 72 h of hyperoxia, although there was no difference between strains. Copper-zinc-SOD activity was ~4× higher under normoxic conditions in MT-null compared with WT mice but was not affected in either group by hyperoxia. Collectively the data suggest that genetic deletion of MT-I/II in mice is associated with compensatory increase in copper-zinc-SOD activity, prevention of hyperoxia-induced zinc transmigration from lung to liver, and hyperoxia-resistant phenotype strongly associated with differences in zinc homeostasis during hyperoxic acute lung injury.

Prevention of breast cancer-induced osteolytic bone resorption by benzyl isothiocyanate

Osteolytic bone resorption is the primary cause of pain and suffering (e.g. pathological bone fracture) in women with metastatic breast cancer. The current standard of care for patients with bone metastasis for reducing the incidence of skeletal complications includes bisphosphonates and a humanized antibody (denosumab). However, a subset of patients on these therapies still develops new bone metastasis or experiences adverse effects. Moreover, some bisphosphonates have poor oral bioavailability. Therefore, orally-bioavailable and non-toxic inhibitors of breast cancer-induced osteolytic bone resorption are still clinically desirable. We have shown previously that benzyl isothiocyanate (BITC) decreases the incidence of breast cancer in a transgenic mouse model without any side effects. The present study provides in vivo evidence for inhibition of breast cancer-induced osteolytic bone resorption by BITC. Plasma achievable doses of BITC (0.5 and 1 μM) inhibited in vitro osteoclast differentiation induced by co-culture of osteoclast precursor cells (RAW264.7) and breast cancer cells representative of different subtypes. This effect was accompanied by downregulation of key mediators of osteoclast differentiation, including receptor activator of nuclear factor-κB ligand and runt-related transcription factor 2 (RUNX2), in BITC-treated breast cancer cells. Doxycycline-inducible knockdown of RUNX2 augmented BITC-mediated inhibition of osteoclast differentiation. Oral administration of 10 mg BITC/kg body weight, 5 times per week, inhibited MDA-MB-231-induced skeletal metastasis multiplicity by ~81% when compared with control (P = 0.04). The present study indicates that BITC has the ability to inhibit breast cancer-induced osteolytic bone resorption in vivo.

Preclinical Evaluation of 18F-ML-10 to Determine Timing of Apoptotic Response to Chemotherapy in Solid Tumors:

Purpose: We investigated 2-(5-fluoro-pentyl)-2-methyl-malonic acid (18F-ML-10) positron emission tomography (PET) imaging of apoptosis posttherapy to determine optimal timing for predicting chemotherapy response in a mouse head/neck xenograft cancer model. Procedures: BALB/c nude mice (4-8 weeks old) were implanted with UM-SCC-22B tumors. The treatment group received 2 doses of doxorubicin (10 mg/kg, days 0, 2). Small animal 18F-ML-10 PET/computed tomography was performed before and on days 1, 3, and 7 postchemotherapy. Using regions of interest around tumors, 18F-ML-10 uptake change was measured as %ID/g and uptake relative to liver. Terminal Uridine Nick-End Labeling (TUNEL) immunohistochemistry assay was performed using tumor samples of baseline and on days 1, 3, and 7 posttreatment. Results: Treated mice demonstrated increased 18F-ML-10 uptake compared to baseline and controls, and 10 of 13 mice showed tumor volume decreases. All control mice showed tumor volume increases. Tumor-to-liver (T/L) ratios from the control group mice did not show significant change from baseline (P > .05); however, T/L ratios of the treatment group showed significant 18F-ML-10 uptake differences from baseline compared to days 3 and 7 posttreatment (P < .05), but no significant difference at 1 day posttreatment. Conclusion: 2-(5-Fluoro-pentyl)-2-methyl-malonic acid PET imaging has the potential for early assessment of treatment-induced apoptosis. Timing and image analysis strategies may require optimization, depending on the type of tumor and cancer treatment.

Secreted Phosphoprotein 1 and Sex-Specific Differences in Silica-Induced Pulmonary Fibrosis in Mice

Background: Fibrotic lung diseases occur predominantly in males, and reports describe better survival in affected females. Male mice are more sensitive to silica-induced lung fibrosis than silica-treated female mice. Secreted phosphoprotein 1 (SPP1, also known as osteopontin) increases in pulmonary fibrosis, and Spp1 transcription may be regulated by estrogen or estrogen receptor–related receptors. Objective: We determined whether differences in silica-induced SPP1 levels contribute to sex differences in lung fibrosis. Methods: Male and female mice were treated with 0.2 g/kg intratracheal silica, and lung injury was assessed 1, 3, or 14 days post-exposure. Gene-targeted (Spp1–/–) mice, control Spp1+/+ (C57BL/6J) mice, ovariectomized (OVX) female mice, and estrogen-treated male mice were treated with silica, and lung injury was assessed. Results: Silica-induced SPP1 in lung tissue, bronchoalveolar lavage, and serum increased more in male than in female mice. Following silica treatment, bronchoalveolar lavage cell infiltrates decreased in female Spp1–/– mice compared with female Spp1+/+ mice, and lung hydroxyproline decreased in male Spp1–/– mice compared with male Spp1+/+ mice. OVX female mice had increased lung SPP1 expression in response to silica compared with silica-treated sham female mice. Silica-induced lung collagen and hydroxyproline (markers of fibrosis), and SPP1 levels decreased in estrogen-treated males compared with untreated males. Conclusion: These findings suggest that sex-specific differences in SPP1 levels contribute to the differential sensitivity of male and female mice to the development of silica-induced fibrosis. Citation: Latoche JD, Ufelle AC, Fazzi F, Ganguly K, Leikauf GD, Fattman CL. 2016. Secreted phosphoprotein 1 and sex-specific differences in silica-induced pulmonary fibrosis in mice. Environ Health Perspect 124:1199–1207; http://dx.doi.org/10.1289/ehp.1510335