Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present in the recipient mice after 1 week, but 1-5 months later, the donor cells accounted for 1.5-12% of the cells in bone, cartilage, and lung in addition to marrow and spleen. A PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma, and reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner. The results, therefore, demonstrated that mesenchymal precursor cells from marrow that are expanded in culture can serve as long-lasting precursors for mesenchymal cells in bone, cartilage, and lung. They suggest that cells may be particularly attractive targets for gene therapy ex vivo.

The emergence of multiple drug-resistant bacteria has prompted interest in alternatives to conventional antimicrobials. One of the possible replacement options for antibiotics is the use of bacteriophages as antimicrobial agents. Phage therapy is an important alternative to antibiotics in the current era of drug-resistant pathogens. Bacteriophages have played an important role in the expansion of molecular biology and have been used as antibacterial agents since 1966. In this review, we describe a brief history of bacteriophages and clinical studies on their use in bacterial disease prophylaxis and therapy. We discuss the advantages and disadvantages of bacteriophages as therapeutic agents in this regard.

Nitric oxide (NO) has been implicated as a pathogenic mediator in a variety of central nervous system (CNS) We have reported (1) that Borna disease virus, rabies virus, and herpes simplex virus induce the increased expression of inducible nitric oxide synthase (iNOS) mRNA in the brains of intrathecally infected mice and rats. We and others (2) also found that the induction of experimental allergic encephalomyelitis resulted in a similar increase in iNOS mRNA expression, suggesting that a similar phenomenon may occur in the brains of patients with multiple sclerosis (MS). We investigated this hypothesis by detecting iNOS mRNA in extracts of brains from patients who died with MS and from extracts of brain tissues from patients who died from nonneurological diseases. We have also colocalized the iNOS expressing cells by using immunocytochemical techniques combined with reverse transcriptase (RT) driven-in situ PCR and demonstrated the presence of iNOS protein by the same double labeling procedures. Finally, as a surrogate marker for nitric oxide (NO) presence, we have immunocytochemically localized nitroty-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.rosine adducts in histological sections of brain from MS patients. MATERIALS AND METHODSSources and Selection of Specimens. The samples used in these studies were collected from patients with an antemortem diagnosis of MS based on clinical presentation and reconfirmed by histopathology after death. All MS samples were found to contain pathognomonic MS plaques; control brains were examined and found to be pathologically unremarkable. In most cases, autopsy samples were of frontal lobes, which provided mixed white and gray matter.In two of the MS cases, special efforts were made to preserve the tissue morphology. Small portions of the brain specimens were frozen in liquid nitrogen immediately after sectioning and then stored at -70°C. This procedure preserved the architecture of the brain significantly better than direct freezing of fresh brain specimens at -80°C, which results in ice crystal formation in the tissue and distorts its architecture.Cell Culture and Cytokine Activation. A549 human pulmonary epithelial cells were purchased from the American Type Culture Collection. Cells were grown in Ham's F-12 medium supplemented with 10% (vol/vol) fetal calf serum, penicillin (50 units/ml), streptomycin (50 ,ug/ml), and 5 ,tM L-glutamine. Cells were seeded into 6-well tissue culture clusters (Falcon) for Northern blot analysis or seeded into 8-well Lab-Tek culture slides (Nunc) for RT-in situ PCR. For those cultures stimulated for the expression of iNOS, interleukin 1(3 (IL-1,B at 100 units/ml), tumor necrosis factor a (TNF-a at 10 ng/ml), and interferon y (IFN-,y at 500 units/ml) were added for 8 h.For use in Northern blot analysis, the cells were scraped from the culture substrate, washed three ti...

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In this study we provide further evidence associating activated cells of the monocyte lineage with the lesions of multiple sclerosis (MS). Using a combination of immunohistochemistry and reverse transcriptase-dependent in situ polymerase chain reaction analysis, we have identified monocytes expressing inducible nitric oxide synthase (iNOS) to be prevalent in the plaque areas of post mortem brain tissue from patients with MS. In addition, we have obtained evidence of the nitration of tyrosine residues in brain areas local to accumulations of iNOS-positive cells. In parallel studies we have assessed the effects of inhibitors of iNOS induction, as well as scavengers of nitric oxide and peroxynitrite in the experimental allergic encephalomyelitis model. Significant therapeutic effects were seen with the inhibitor of iNOS induction, tricyclodecan-9-xyl-xanthogenate, a nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and a peroxynitrite scavenger, uric acid. In particular, treatment with high doses of uric acid virtually prevented clinical symptoms of the disease. Together with our demonstration of the presence of activated macrophages expressing high levels of iNOS and evidence of peroxynitrite formation in brain tissue from patients with MS, these findings are of importance in the development of approaches to treat this disease.

Background: The genetic and molecular mechanisms responsible for and associated with the development and progression of prostate malignancy are largely unidentified. The peripheral zone is the major region of the human prostate gland where malignancy develops. The normal peripheral zone glandular epithelium has the unique function of accumulating high levels of zinc. In contrast, the ability to accumulate zinc is lost in the malignant cells. The lost ability of the neoplastic epithelial cells to accumulate zinc is a consistent factor in their development of malignancy. Recent studies identified ZIP1 (SLC39A1) as an important zinc transporter involved in zinc accumulation in prostate cells. Therefore, we investigated the possibility that down-regulation of hZIP1 gene expression might be involved in the inability of malignant prostate cells to accumulate zinc. To address this issue, the expression of hZIP1 and the depletion of zinc in malignant versus non-malignant prostate glands of prostate cancer tissue sections were analyzed. hZIP1 expression was also determined in malignant prostate cell lines.

Background: Zinc is vital to normal prostate function and uniquely concentrates in healthy prostate. A hallmark of prostate cancers is diminished zinc levels. Results: The miR-183 family is overexpressed in prostate cancer and regulates intracellular zinc via suppression of zinc transporters. Conclusion: Prostatic zinc homeostasis is regulated by microRNAs. Significance: The miR-183 family regulates zinc and may contribute to prostate carcinogenesis.

The presence of herpes simplex virus (HSV-1) DNA in the trigeminal ganglia of latently infected mice was detected by an in situ DNA polymerase chain reaction (PCR), which includes a DNA:DNA hybridization step (indirect in situ PCR). These results were compared to the number of neurons possessing the HSV-1 latency associated transcript (LAT), as detected by in situ RNA hybridization with LAT probes. Sensitivity assays were shown to detect a single copy cellular gene in 48% of neuronal cell bodies. The results suggest that in situ PCR is an effective method to locate and detect HSV-1 within latently infected neurons. Moreover, the number of neurons found to be harboring HSV-1, by the method of in situ PCR, which does not depend upon virus gene expression, is within threefold of the number detected by in situ hybridization for LAT. Therefore, this report describes the first detection of HSV-1 DNA in latently infected murine trigeminal ganglia by the method of indirect in situ PCR, and compares the findings to the number of neurons expressing LAT, as assessed by conventional in situ hybridization.