Few studies have compared the quantification of mRNA by DNA microarray to the results obtained by reverse transcription PCR (RT-PCR). In this study, mRNA was collected from the healing femoral fracture callus of adult and juvenile rats at various times after fracture. Ten samples were measured by both methods for 26 genes. For RT-PCR, mRNA was reverse transcribed, amplified, electrophoresed, blotted, and probed with 32P-labeled internal oligonucleotides, which were quantified. For DNA microarray, the mRNA was processed to biotin-labeled cRNA, hybridized to 10 Affymetrix Rat U34A microarrays, and quantified. Correlation coefficients (r) for each gene for the agreement between RT-PCR and microarray ranged from -0.48 to +0.93. This variation made the interpretation gene-specific. Genes with moderate expression levels gave the highest r values. Increased numbers of absent calls by the microarray software and increased separation between the location of the PCR primers and the microarray probes both led to reduced agreement. Microarray analysis suggested a floor effect in expression levels measured by RT-PCR for two genes. In conclusion, moderate mRNA expression levels with overlap in the location of PCR primers and microarray probes can yield good agreement between these two methods.

Metastatic spread is the mechanism in more than 90 percent of cancer deaths and current therapeutic options, such as systemic chemotherapy, are often ineffective. Here we provide a proof of principle for a novel two-pronged modality referred to as Synergistic Immuno Photothermal Nanotherapy (SYMPHONY) having the potential to safely eradicate both primary tumors and distant metastatic foci. Using a combination of immune-checkpoint inhibition and plasmonic gold nanostar (GNS)–mediated photothermal therapy, we were able to achieve complete eradication of primary treated tumors and distant untreated tumors in some mice implanted with the MB49 bladder cancer cells. Delayed rechallenge with MB49 cancer cells injection in mice that appeared cured by SYMPHONY did not lead to new tumor formation after 60 days observation, indicating that SYMPHONY treatment induced effective long-lasting immunity against MB49 cancer cells.

We employed a newly developed genotyping technique with direct representational detection of LMP-1 gene sequences to study the molecular epidemiology of Epstein-Barr virus (EBV) infection in healthy individuals. Infections with up to five different EBV genotypes were found in two of nine individuals studied. These results support the hypothesis that multiple EBV infections of healthy individuals are common. The implications for the development of an EBV vaccine are discussed.Multiple Epstein-Barr virus (EBV) infections are common among immunocompromised individuals (21,29,31,39,41,43,44,46), but the origin of the multiple EBV strains remains a mystery. Multiple EBV strains could accumulate as superinfections in individuals who have lost previous protective immunity to EBV. Alternatively, they could represent the reactivation of latent EBV strains that were acquired prior to the onset of immunodeficiency. The reported prevalence of multiple EBV infections in healthy individuals ranges broadly between 0 and 100% (Table 1) (4,7,13,16,19,20,23,30,32,35,38,45; M. L. Lung and R. S. Chang, Letter, J. Infect. Dis. 162:994-995, 1990), but differences among these studies in the molecular detection and definition of an EBV strain confound the interpretation of their results.Molecular epidemiologic studies requiring EBV isolation by B-lymphocyte transformation (16,19,23,45; Lung and Chang, letter) suffer from selection bias toward transformation-competent EBV isolates (10,27,33). PCR amplification directly detects the EBV genome and avoids transformation selection bias, but the genetic definition of an EBV strain has been inconsistent across studies. Restriction fragment length polymorphisms detect either point mutations within restriction enzyme cleavage sites or variations of large repetitive regions within genome fragments (19,23,35; Lung and Chang, letter). Similarly, size variation in EBNA proteins ("EBNotype" or "EBNAprint") (16, 45) and size variation in specific gene PCR products (LMP-1, BZLF1, EBNA-6) (13, 35) reflect variations in repetitive and other genome sequences. However, many EBV genome sequences are susceptible to intrastrain homologous and nonhomologous recombination during productive replication and the number of repeat units present may vary in different isolates of the same EBV strain (12,38,(41)(42)(43). Studies examining the major sequence divergence between EBV types 1 and 2 have reported EBV coinfection rates ranging from 0 to 53% (4,13,20,34,35,45). However, EBV types 1 and 2 can both be further subdivided into different strains (1,24,41) and only three studies to date have utilized EBV gene nucleotide sequence variation to define EBV strains in healthy individuals (7,30,38).EBV genotyping assay. A consistent approach is needed for the definition and nomenclature of EBV genomes. It is impractical or even impossible to physically isolate (culture) and fully characterize the EBV genome(s) in clinical infections. A reasonable goal would be to identify an EBV genetic marker that represents the broad...

Interferons (IFN) inhibit the growth of tumor cells by blocking the progression of their cell cycle. Recently, we showed that this cell cycle inhibition correlates with the ability of IFN to upregulate the cyclin-dependent kinase inhibitor p21(WAF1). This, however, is not proof of a causal relationship. Using p21(WAF1)-deficient cells derived from the HCT116 colon adenocarcinoma cell line, we now show that p21(WAF1) is indeed responsible for the antiproliferative effects of the type II IFN, IFN-gamma. IFN-gamma upregulated p21(WAF1) expression in a p53-independent manner, decreased cyclin-dependent kinase 2 activity, and inhibited entry into the S phase of the cell cycle in p21+/+ but not in p21-/- HCT116 cells. We additionally found that the lack of p21(WAF1) expression resulted in an increase in the ability of IFN-gamma to induce apoptosis, as reflected by an earlier induction of DNA fragmentation and caspase 3 activity in p21-/- cell. Our results indicate that p21(WAF1) expression is necessary for IFN-gamma-mediated cell cycle inhibition and suppression of IFN-gamma-induced apoptosis.

The kinematic viscosity of human urine is temperature dependent and higher than water. Urine specific gravity was not a good predictor of viscosity. Of factors that might affect urine viscosity, only proteinuria appeared to be clinically relevant. Estimates of urine viscosity provided in this manuscript may be useful for temperature modelling of bladder hyperthermia treatments with regard to correct prediction of the thermal conduction effects.

Purpose This paper describes a preclinical investigation of the feasibility of thermotherapy treatment of bladder cancer with Magnetic Fluid Hyperthermia (MFH), performed by analyzing the thermal dosimetry of nanoparticle heating in a rat bladder model. Materials and Methods The bladders of twenty-five female rats were instilled with magnetite-based nanoparticles, and hyperthermia was induced using a novel small animal magnetic field applicator (Actium Biosystems, Boulder, CO). We aimed to increase the bladder lumen temperature to 42°C in <10 min and maintain that temperature for 60 min. Temperatures were measured within the bladder lumen and throughout the rat with seven fiberoptic probes (OpSens Technologies, Quebec, Canada). An MRI analysis was used to confirm the effectiveness of the catheterization method to deliver and maintain various nanoparticle volumes within the bladder. Thermal dosimetry measurements recorded the temperature rise of rat tissues for a variety of nanoparticle exposure conditions. Results Thermal dosimetry data demonstrated our ability to raise and control the temperature of rat bladder lumen ≥1°C/min to a steady-state of 42°C with minimal heating of surrounding normal tissues. MRI scans confirmed the homogenous nanoparticle distribution throughout the bladder. Conclusion These data demonstrate that our MFH system with magnetite-based nanoparticles provide well-localized heating of rat bladder lumen with effective control of temperature in the bladder and minimal heating of surrounding tissues.

Type I and type II interferons (IFNs) are known to exert antitumor effects on a variety of tissues and cell types. We have previously shown that the type I IFN IFN␣ induces the expression of the cyclin-dependent kinase inhibitor p21 WAF1 and inhibits the cell cycle of the human prostate adenocarcinoma cell line, DU145, that carries mutations in the tumor suppressor gene products p53 and pRB. We now show that the type II IFN IFN␥ similarly induces the expression of p21 WAF1 and inhibits the cell cycle of DU145 cells. In addition, we show that while both IFNs exert antiproliferative activity, only IFN␥ induced phenotypic changes in these cells that accompanied the antiproliferative effect. For example, IFN␥, but not IFN␣, caused a significant reduction in epidermal growth factor receptor expression as well as an increase in the adhesion molecules intercellular adhesion molecule-1 and integrin ␣3. These phenotypic changes in DU145 cells are suggestive of the acquisition of a non-tumorigenic state. Consistent with these findings, IFN␥ showed a significantly lower invasive ability in in vitro assays using invasion chambers. Thus, IFN␥ inhibits both the cell cycle and the metastatic potential of DU145 cells independent of the p53 and RB status, and our data describe a mechanism for mediating the antitumor capabilities of IFN␥ that bypasses tumor suppressor genes like p53. Int.

This prospective study examined the persistence and transition of Epstein-Barr virus (EBV) in human immunodeficiency virus (HIV)-seropositive subjects with and without oral hairy leukoplakia, a replicative EBV-associated epithelial disease. The intrahost molecular epidemiology of EBV infection was characterized in subjects treated with valacyclovir to suppress EBV replication. Tongue epithelial tissues of HIV-seropositive subjects were found to support not only EBV replication but also persistent, nonproductive EBV infection. EBV appeared to enter the tongue from the blood reservoir of infection and, possibly, from exogenous sources as well. EBV transition from the blood to the tongue appeared to occur even during valacyclovir-mediated suppression of EBV replication, suggesting EBV entry into tongue epithelial tissue as a cell-associated latent infection. In conclusion, these results describe the persistence and transition of EBV as a dynamic interaction between the blood and epithelial reservoirs of EBV infection and suggest a role for entry, persistence, and reactivation of oral epithelial EBV in the pathogenesis of oral hairy leukoplakia.