Antibiotic-mediated chemoprotection enhances adaptation of E. coli PNP for herpes simplex virus-based glioma therapy.

The E. coli PNP suicide gene sensitizes solid tumors to nucleoside prodrugs, such as 6-methylpurine-2'-deoxyriboside (MeP-dR). In this study using lentiviral, MuLv, and HSV-based gene transfer, we quantified thresholds for inhibition of tumor growth and bystander killing by E. coli PNP and tested the role of intestinal flora in this process. Regressions of human glioma tumors following retroviral transduction exhibited dose dependence on both the level of PNP expression and the dose of MeP-dR administered, including strong tumor inhibition when 90-99% bystander cells comprised the tumor mass. A replication competent, non-neurovirulent herpes simplex virus (HSV) deficient in both copies of the gamma-1 34.5 gene was next engineered to express E. coli PNP under the egr-1 promoter (HSV-PNP). HSV-PNP injected intratumorally (17 million pfu/0.05 ml) in nude mice bearing 300 mg human glioma flank tumors produced a delay in tumor growth (approximately 24 days delay to one doubling). MeP-dR treatment after antibiotic therapy (to eliminate enteric flora encoding PNP enzymes) resulted in antitumor enhancement, with arrest of tumor growth (delay to doubling >50 days). Bystander killing of the magnitude described here has been difficult to accomplish with other suicide genes, such as HSV-tk or cytosine deaminase. The results establish a model for applying E. coli PNP to HSV treatment of glioma.

Poliovirus replicons encoding the B subunit of Helicobacter pylori urease protect mice against H. pylori infection.

We developed a novel vaccine for Helicobacter pylori based on a poliovirus vector in which capsid genes were replaced with the gene for the B subunit of H. pylori urease (UreB). Mice were vaccinated with UreB or control (L1) replicon and challenged with H. pylori. Twenty percent of mice vaccinated prophylactically with UreB, but 80% vaccinated with L1, and then challenged with H. pylori became infected (P = 0.003). Seventy-three percent of mice with established H. pylori infection vaccinated therapeutically with UreB replicon cleared their infection compared to 33% vaccinated with L1 (P = 0.067). In therapeutically vaccinated mice with residual infection, UreB-vaccinated animals had fewer H. pylori than L1-vaccinated mice (P < 0.05). Anti-urease antibody titres in prophylactically, but not therapeutically, vaccinated mice were markedly higher in animals that received UreB versus L1 replicon (P = 0.01). Vaccination with poliovirus vector containing the gene for the B subunit of H. pylori urease provides significant prophylactic and strong therapeutic protection against H. pylori in mice.

Development of dermatitis in CD18-deficient PL/J mice is not dependent on bacterial flora, and requires both CD4+ and CD8+ T lymphocytes.

CD18-deficient PL/J mice develop dermatitis characterized by hyperkeratosis, and a mixed dermal and epidermal inflammatory infiltrate. The development of this disease requires low-level CD18 expression and at least two PL/J loci. Currently, the mechanisms by which decreased beta(2) integrin expression on leukocytes promotes skin inflammation in PL/J mice are unknown. In these studies, we investigated the role of microbial infection and T lymphocytes in the pathogenesis of this disease. We found that germ-free CD18(-/-) PL/J mice developed dermatitis indistinguishable from that of mice raised in pathogen-free conditions. Adoptive transfer of CD18(-/-) PL/J splenocytes into skin disease-resistant CD18(+/-) PL/J mice failed to induce skin inflammation. However, transfer of CD18(+/-) splenocytes blocked the progression and ultimately led to resolution of skin disease in the majority of CD18(-/-) recipients. Depletion of both CD4(+) and CD8(+) T cells mice prior to onset of the disease significantly delayed the appearance of inflammatory skin disease. In contrast, single depletions of these T cells did not inhibit disease development. These studies show that dermatitis in CD18-deficient PL/J mice is not the consequence of infection, does not require bacterial superantigens, and is mediated by both CD4(+) and CD8(+) T lymphocytes. Furthermore, they suggest that one possible mechanism for skin disease development in these mice may involve the absence or dysfunctional activity of a regulatory T cell population. These mice may therefore be useful in identifying potential mechanisms of pathogenesis and genetic predisposition in human inflammatory skin diseases.

Nucleotide-mediated inhibition of alveolar fluid clearance in BALB/c mice after respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract disease in infants and children worldwide. Intranasal infection of BALB/c mice with RSV strain A2, but not ultraviolet-inactivated RSV, for 2 or 4 days reduced basal alveolar fluid clearance (AFC), a seminal function of bronchoalveolar epithelium, and caused loss of AFC sensitivity to amiloride inhibition. Reduced AFC was temporally associated with increased lung water content but was not a consequence of increased epithelial permeability or cell death. Reduced AFC was also not due to decreased transcription of epithelial Na+ channel subunit genes in lung tissue. RSV-mediated inhibition of AFC 2 days after infection was rapidly prevented by addition to the instillate of P2Y receptor antagonists (suramin and XAMR-0721) or enzymes that degrade UTP, but not those that degrade ATP. After UTP degradation, AFC returned to control levels but was no longer sensitive to amiloride. UTP at nanomolar concentrations recapitulated the AFC inhibitory effect of RSV in normal mice and mice infected with RSV for 6 days, indicating that normalization of AFC at this time point is a consequence of cessation of UTP release, rather than P2Y receptor desensitization. We conclude that RSV infection of the bronchoalveolar epithelium results in reduced AFC as a consequence of autocrine feedback inhibition mediated by UTP. These studies are the first to demonstrate AFC inhibition by an important pulmonary viral pathogen. Reduced AFC may result in formation of an increased volume of fluid mucus, airway congestion, and rhinorrhea, all features of severe RSV disease.

Role of surfactant protein-A in nitric oxide production and mycoplasma killing in congenic C57BL/6 mice.

We generated congenic surfactant protein A (SP-A)-deficient (SP-A[-/-]) mice on the mycoplasma resistant C57BL/6 background (B6.SP-A[-/-]) and characterized their response to mycoplasma infection in comparison to C57BL/6 (B6) mice. B6.SP-A(-/-) mice infected with 10(6) colony-forming units (cfu) of Mycoplasma pulmonis had significantly higher bacterial lung loads than B6 mice at 72 h postinfection (p.i.). At the higher infection dose of 10(7), B6.SP-A(-/-) mice had significantly higher lung cfu at 24 h; however, no difference in mycoplasma cfu was observed between B6 and B6.SP-A(-/-) mice at 48 and 72 h p.i. We found that uninfected B6 mice had lower bronchoalveolar lavage nitrite (NO(2)(-)) and nitrate (NO(3)(-)) levels as compared with B6.SP-A(-/-) mice. On the other hand, infection of B6 mice with mycoplasmas resulted in significantly higher bronchoalveolar lavage NO(2)(-) and NO(3)(-) as compared with B6.SP-A(-/-) mice. These data indicate that SP-A may help regulate NO production in response to a specific stimulus, i.e., suppression of NO in the absence of bacteria and increased NO in the presence of bacteria. These data indicate that the contribution of SP-A to mycoplasma killing may be limited to lower doses of pathogens.

Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Since approximately 5% of all mutant CF alleles are stop mutations, it can be calculated that approximately 10% of CF patients carry a premature stop mutation in at least one copy of the CFTR gene. Certain ethnic groups, such as the Ashkenazi Jewish population, carry a much higher percentage of CF stop mutations. Consequently, a therapeutic strategy aimed at suppressing this class of mutation would be highly desirable for the treatment of this common genetic disease. We have shown previously that aminoglycoside antibiotics can suppress premature stop mutations in the CFTR gene in a bronchial epithelial cell line [Nat Med (1997) 3:1280]. To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr-/-) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. We then investigated whether the daily administration of the aminoglycoside antibiotics gentamicin or tobramycin could restore the expression of a detectable level of CFTR protein. Immunofluorescence staining of intestinal tissues from Cftr-/- hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Weaker staining was also observed in the intestinal glands following tobramycin treatment. Short-circuit current measurements made on intestinal tissues from these mice demonstrated that a significant number of positive cAMP-stimulated transepithelial chloride current measurements could be observed following gentamicin treatment (P=0.008) and a near significant number following tobramycin treatment (P=0.052). When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo.

Anti-tumor efficacy of a novel antisense anti-MDM2 mixed-backbone oligonucleotide in human colon cancer models: p53-dependent and p53-independent mechanisms.

BACKGROUND: The MDM2 oncogene is amplified or overexpressed in many human cancers and MDM2 levels are associated with poor prognosis. MDM2 not only serves as a negative regulator of p53 but also has p53-independent activities. This study investigates the functions of the MDM2 oncogene in colon cancer growth and the potential value of MDM2 as a drug target for cancer therapy, by inhibiting MDM2 expression with an antisense anti-human-MDM2 oligonucleotide. MATERIALS AND METHODS: The selected antisense mixed-backbone oligonucleotide was evaluated for its in vitro and in vivo antitumor activity in human colon cancer models: LS174T cell line containing wild-type p53 and DLD-1 cell line containing mutant p53. The levels of MDM2, p53 and p21 proteins were quantified by Western blot analysis. RESULTS: In vitro antitumor activity was found in both cell lines, resulting from specific inhibition of MDM2 expression. In vivo antitumor activity of the oligonucleotide occurred in a dose-dependent manner in both models and synergistically or additive therapeutic effects of MDM2 inhibition and the cancer chemotherapeutic agents 10-hydroxycamptothecin and 5-fluorouracil were also observed. CONCLUSIONS: These results suggest that MDM2 have a role in tumor growth through both p53-dependent and p53- independent mechanisms. We speculate that MDM2 inhibitors have a broad spectrum of antitumor activities in human cancers regardless of p53 status. This study should provide a basis for future development of anti-MDM2 antisense oligonucleotides as cancer therapeutic agents used alone or in combination with conventional chemotherapeutics.

Role of bacteriophage MAV1 as a mycoplasmal virulence factor for the development of arthritis in mice and rats.

The lysogenic bacteriophage MAV1 has been shown to be a virulence factor for the development of arthritis in rats infected with Mycoplasma arthritidis. In the present study, arthritis was evaluated by histopathologic examination to demonstrate that MAV1 is a virulence factor not only in the rat but also in the mouse. Specifically, the MAV1 lysogen 158L3-1 was more virulent than the nonlysogen strain 158 in DBA/2NCr, C3H/HeNCr, C3H/HeJ, and C3Smn.CB17-Prkdc(scid)/J mice, as well as in LEW rats.