J. Craig Cohen

Reversal of cystic fibrosis phenotype in mice by gene therapy in utero

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Pathophysiologic consequences following inhibition of a CFTR-dependent developmental cascade in the lung

BackgroundExamination of late gestation developmental genes in vivo may be limited by early embryonic lethality and compensatory mechanisms. This problem is particularly apparent in evaluating the developmental role of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the cystic fibrosis (CF) phenotype. A previously described transient in utero knockout (TIUKO) technology was used to address the developmental role of CFTR in the rat lung.ResultsRat fetuses transiently treated with antisense cftr in utero developed pathology that replicated aspects of the human CF phenotype. The TIUKO CF rat developed lung fibrosis, chronic inflammation, reactive airway disease, and the CF Antigen (MRP8/14), a marker for CF in human patients, was expressed.ConclusionsThe transient in utero antisense technology can be used to evaluate genes that exhibit either early lethality or compensating gene phenotypes. In the lung CFTR is part of a developmental cascade for normal secretory cell differentiation. Absence of CFTR results in a constitutive inflammatory process that is involved in some aspects of CF pathophysiology.

Transient in utero knockout (TIUKO) of C-MYC affects late lung and intestinal development in the mouse.

BackgroundDevelopmentally important genes often result in early lethality in knockout animals. Thus, the direct role of genes in late gestation organogenesis cannot be assessed directly. In utero delivery of transgenes was shown previously to result in high efficiency transfer to pulmonary and intestinal epithelial stem cells. Thus, this technology can be used to evaluate late gestation development.ResultsIn utero gene transfer was used to transfer adenovirus with either an antisense c-myc or a C-MYC ubiquitin targeting protein to knockout out c-myc expression in late gestation lung and intestines.Using either antisense or ubiquitin mediated knockout of C-MYC levels in late gestation resulted in similar effects. Decreased complexity was observed in both intestines and lungs. Stunted growth of villi was evident in the intestines. In the lung, hypoplastic lungs with disrupted aveolarization were observed.ConclusionsThese data demonstrated that C-MYC was required for cell expansion and complexity in late gestation lung and intestinal development. In addition they demonstrate that transient in utero knockout of proteins may be used to determine the role of developmentally important genes in the lungs and intestines.

A DNA probe for detecting Mycoplasma genitalium in clinical specimens

Despite decades of careful study, the etiologies of all cases of pelvic inflammatory disease (PID) and non-gonococcal urethritis (NGU) have yet to be described. Mycoplasma genitalium is a newly described organism which has been implicated as a cause of both PID and NGU. Because of fastidious growth requirements, prolonged incubation time and frequent overgrowth in clinical specimens by Mycoplasma hominis, non-culture methods need to be developed for its detection. We have cloned M. genitalium DNA by transfection into Escherichia coli using M13 as the vector. Using these segments as templates, we synthesized radiolabelled cDNAs that were tested for specific hybridization with M. genitalium, and clinically isolated genital mycoplasmas presumptively identified as M. hominis, and Ureaplasma urealyticum. A 256 base-pair segment was found to hybridize with M. genitalium with a sensitivity of 10(2) colour-changing units (CCUs). No cross-hybridization was observed with M. hominis, and cross-hybridization was observed only with large concentrations (greater than 10(6) CCUs) of U. urealyticum. Because of our choice of M13 as the vector, which contains the Lac Operon of E. coli, slight hybridization occurred with E. coli as well. This cDNA can be used against clinical specimens to determine the ecologic niche and spectrum of disease caused by M. genitalium.

Mammary preneoplasia and tumorigenesis in the BALB/c mouse: structure and modification of mouse mammary tumor virus DNA sequences

Mouse mammary tumor virus (MMTV) DNA sequences were examined in mammary tissues from BALB/c mice, including both preneoplastic hyperplastic alveolar nodule (HAN) outgrowth lines, tumors derived from those preneoplastic tissues, and DMBA-induced mammary tumors. Over 60 different HAN and tumors samples were analyzed. The D2 HAN line contained one additional provirus, whereas Cv-2 and Cv-4 HAN lines that are infected with exogenous virus exhibited multiple virus integration events. D2 tumors showed the same provirus pattern as D2 HANs, whereas Cv-2 and Cv-4 tumors exhibited a subset of the acquired proviruses found in the parental HAN populations. Differential methylation patterns of virus-specific sequences were observed that resembled those described for other tissues in which viral DNA replication and integration has occurred, i.e., acquired proviruses were hypomethylated and endogenous proviruses were methylated. In tumors that arose from HAN lines and exhibited only endogenous proviruses, demethylation of the subgenomic Mtv-6 locus was observed. Demethylation of Mtv-6 was not detected in any of the preneoplastic tissues. Altered methylation of Mtv-8 and -9 was observed in both Cv-2 and Cv-4 tumors. Finally, mammary tumors induced by DMBA carried no acquired proviruses and demethylation of endogenous MMTV proviruses was demonstrated.

Simulated transport alters surfactant homeostasis and causes dose-dependent changes in respiratory function in neonatal Sprague-Dawley rats.

Abstract Objective: Forces transmitted to the neonate as a consequence of accelerations during transport have been associated with adverse neonatal outcomes including broncho-pulmonary dysplasia. In this study, we sought to determine the relationship between the duration of transport and respiratory performance in the rat model. Methods: Four groups of Sprague-Dawley rat pups (10–12 pups/groups) were exposed to simulated medical transport on postnatal day of life 11 or 12. Each group was exposed to an average impulse of 27.4 m/s2/min for 0, 30, 60 or 90 min. During the exposure periods, impulse was monitored by computerized sampling using a digital accelerometer. Post-exposure, animals were immediately prepared, placed on mechanical ventilation and analyzed for elastance, tissue damping, airway resistance, ratio of damping to elastance (eta), hysteresivity, and inertance at positive end expiratory pressures (PEEPs) of 0, 3 and 6 cm3 of H2O. Total phospholipid content and surfactant proteins A, B, and C mRNA levels in broncho-alveolar lavage fluid and lung tissue were obtained. Results: Increased transport time resulted in a significant step-wise increase in airway resistance at all levels of PEEP (P<0.01). Static compliance decreased significantly after 60 min at PEEPs of 3 and 6 cm H2O (P<0.01). Eta significantly decreased with greater transport time at a PEEP of 6 cm H2O (P<0.05). Tissue damping increased with duration of transport time across all PEEP levels, but only exhibited statistical significance at a PEEP of 0 cm H2O (P<0.05). No differences were seen in hysteresivity or inertance. Compared with controls, transport was associated with significant reductions in total phospholipid content and mRNA levels of surfactant proteins B and C. Conclusion: Rat pups experienced significant deterioration of respiratory function with increasing duration of simulated transport.