Macrophage metalloelastase (MME) as adjuvant for intra-tumoral injection of oncolytic adenovirus and its influence on metastases development.

Oncolytic adenoviruses are a promising treatment alternative for many advanced cancers, including colorectal cancer. However, clinical trials have demonstrated that single-agent therapy in advanced tumor masses is rarely curative. Poor spreading of the virus through tumor tissue is one of the major issues limiting efficacy. As oncolytic viruses kill preferentially cancer cells, high extracellular matrix (ECM) content constitutes potential barriers for viral penetration within tumors. In this study, the ECM-degrading proteases relaxin, hyaluronidase, elastase and macrophage metalloelastase (MME) were tested for their antitumor efficacy alone and in combination with oncolytic adenovirus. MME improved the overall antitumor efficacy of oncolytic adenovirus in subcutaneous HCT116 xenografts. In a liver metastatic colorectal cancer model, intra-tumoral treatment of primary tumors from HT29 cells with MME monotherapy or with oncolytic adenovirus inhibited tumor growth. Combination therapy showed no increased mortality in comparison with either monotherapy alone. Contradictory results of effects of MME on tumorigenesis and metastasis formation have been reported in the literature. This study demonstrates for the first time in a metastatic animal model that MME, as a monotherapy or in combination with oncolytic virus, does not increase tumor invasiveness. Co-administration of MME and oncolytic adenovirus may be a suitable approach for further optimization aiming at clinical applications for metastatic colorectal cancer.

Oncolytic adenovirus based on serotype 3.

Oncolytic adenoviruses have been safe in clinical trials but the efficacy has been mostly limited. All published trials have been performed with serotype 5 based viruses. The expression level of the Ad5 receptor CAR may be variable in advanced tumors. In contrast, the Ad3 receptor remains unclear, but is known to be abundantly expressed in most tumors. Therefore, we hypothesized that a fully serotype 3 oncolytic adenovirus might be useful for treating cancer. Patients exposed to adenoviruses develop high titers of serotype-specific neutralizing antibodies, which might compromise re-administration. Thus, having different serotype oncolytic viruses available might facilitate repeated dosing in humans. Ad3-hTERT-E1A is a fully serotype 3 oncolytic adenovirus controlled by the promoter of the catalytic domain of human telomerase. It was effective in vitro on cell lines representing seven major cancer types, although low toxicity was seen in non-malignant cells. In vivo, the virus had anti-tumor efficacy in three different animal models. Although in vitro oncolysis mediated by Ad3-hTERT-E1A and wild-type Ad3 occurred more slowly than with Ad5 or Ad5/3 (Ad3 fiber knob in Ad5) based viruses, in vivo the virus was at least as potent as controls. Anti-tumor efficacy was retained in presence of neutralizing anti-Ad5 antibodies whereas Ad5 based controls were blocked. In summary, we report generation of a non-Ad5 based oncolytic adenovirus, which might be useful for testing in cancer patients, especially in the context of high anti-Ad5 neutralizing antibodies.

No compelling evidence that sibutramine prolongs life in rodents despite providing a dose-dependent reduction in body weight.

OBJECTIVE: The health and longevity effects of body weight reduction resulting from exercise and caloric restriction in rodents are well known, but less is known about whether similar effects occur with weight reduction from the use of a pharmaceutical agent such as sibutramine, a serotonin-norepinephrine reuptake inhibitor. RESULTS AND CONCLUSION: Using data from a 2-year toxicology study of sibutramine in Sprague-Dawley CD rats and CD-1 mice, despite a dose-dependent reduction in food intake and body weight in rats compared with controls, and a body weight reduction in mice at the highest dose, there was no compelling evidence for reductions in mortality rate.

Utility of TK/GCV in the context of highly effective oncolysis mediated by a serotype 3 receptor targeted oncolytic adenovirus.

Arming oncolytic adenoviruses with therapeutic transgenes and enhancing transduction of tumor cells are useful strategies for eradication of advanced tumor masses. Herpes simplex virus thymidine kinase (TK) together with ganciclovir (GCV) has been promising when coupled with viruses featuring low oncolytic potential, but their utility is unknown in the context of highly effective infectivity-enhanced viruses. We constructed Ad5/3-Delta24-TK-GFP, a serotype 3 receptor-targeted, Rb/p16 pathway-selective oncolytic adenovirus, where a fusion gene encoding TK and green fluorescent protein (GFP) was inserted into 6.7K/gp19K-deleted E3 region. Ad5/3-Delta24-TK-GFP killed ovarian cancer cells effectively, which correlated with GFP expression. Delivery of GCV immediately after infection abrogated viral replication, which might have utility as a safety switch. Due to the bystander effect, killing of some cell lines in vitro was enhanced by GCV regardless of timing. In murine models of metastatic ovarian cancer, Ad5/3-Delta24-TK-GFP improved antitumor efficacy over the respective replication-deficient virus with GCV. However, GCV did not further enhance efficacy of Ad5/3-Delta24-TK-GFP in vivo. Simultaneous detection of tumor load and virus replication with bioluminescence and fluorescence imaging provided insight into the in vivo kinetics of oncolysis. In summary, TK/GCV may not add antitumor activity in the context of highly potent oncolysis.

Breast cancer metastasis to bone: evaluation of bioluminescent imaging and microSPECT/CT for detecting bone metastasis in immunodeficient mice.

This study sought to determine if weekly X-ray exposure affected breast cancer cell metastasis to bone and to also evaluate the use of bioluminescent imaging (BLI) and microSPECT for detection of metastatic bone lesions. Five week old nude mice were randomly assigned to the CT exposed (n = 7) and no CT exposure (n = 6) treatment groups. Mice received an intracardiac injection of MDA-MB-435 human breast cancer cells transduced with luciferase, or a sham injection (saline). The CT exposed group of mice received CT irradiation once a week for 5 weeks. All mice underwent weekly BLI and select mice received Tc-99m-MDP followed by microSPECT imaging after 5 weeks. Pathological evaluation and histomorphometry were used to assess the affect of CT X-rays on bone metastasis and to evaluate BLI. BLI results found no significant difference in metastasis between animals that received CT and those that did not (P > 0.05); however, histomorphometry of the knee joints revealed a significant increase (P = 0.029) in tumor area of the leg bones in mice that received CT exposure (60% +/- 7%) compared to animals that did not receive CT scans (33% +/- 8%). Compared to histological analysis, BLI of the leg and spine was determined to have excellent sensitivity (100%), good specificity (80-90%) and accuracy (90-96%), a positive predictive value of 81-93% and a 100% negative predictive value. Thus, multi-modality imaging techniques can be very useful for monitoring bone metastasis, however microCT X-rays should be used judiciously in order to limit irradiation that may stimulate increased metastasis to specific regions of the skeleton. MicroSPECT imaging did not detect metastatic lesions in the legs of these young nude mice.

Luciferase imaging for evaluation of oncolytic adenovirus replication in vivo.

Oncolytic viruses kill cancer cells by tumor-selective replication. Clinical data have established the safety of the approach but also the need of improvements in potency. Efficacy of oncolysis is linked to effective infection of target cells and subsequent productive replication. Other variables include intratumoral barriers, access to target cells, uptake by non-target organs and immune response. Each of these aspects relates to the location and degree of virus replication. Unfortunately, detection of in vivo replication has been difficult, labor intensive and costly and therefore not much studied. We hypothesized that by coinfection of a luciferase expressing E1-deleted virus with an oncolytic virus, both viruses would replicate when present in the same cell. Photon emission due to conversion of D-Luciferin is sensitive and penetrates tissues well. Importantly, killing of animals is not required and each animal can be imaged repeatedly. Two different murine xenograft models were used and intratumoral coinjections of luciferase encoding virus were performed with eight different oncolytic adenoviruses. In both models, we found significant correlation between photon emission and infectious virus production. This suggests that the system can be used for non-invasive quantitation of the amplitude, persistence and dynamics of oncolytic virus replication in vivo, which could be helpful for the development of more effective and safe agents.

A randomized trial of the duration of therapy with metronidazole plus or minus azithromycin for treatment of symptomatic bacterial vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is the most common cause of vaginitis worldwide. Currently recommended treatments have poor efficacy and are associated with high rates of BV recurrence. We examined whether a longer duration of treatment with metronidazole or combination therapy with metronidazole and azithromycin would enhance the cure rates for BV. In addition, we examined factors other than drug therapy associated with cure. METHODS: Women with symptomatic BV (defined by a modified Amsel criteria) were enrolled in a 4-arm study that compared metronidazole for 7 days versus 14 days, plus or minus azithromycin on days 1 and 3. Data regarding interim behaviors were also obtained, as were vaginal specimens for Gram staining. RESULTS: At the first follow-up visit (7 days after the completion of therapy), there was a significant difference in cure rates among patients who received 7 days of metronidazole therapy, compared with those who received 14 days of therapy, combined across azithromycin therapy (P=.0003). There was no effect associated with azithromycin therapy. There were no differences in cure rates between any of the treatment groups at 21 days after completion of therapy. Abstinence or protected sex, refraining from douching, and a lower baseline Nugent score for the vaginal Gram stain were all significantly associated with cure. CONCLUSIONS: Cure rates for BV were significantly improved by 14 days of metronidazole treatment (compared with 7 days of treatment), but the effects were not sustained, suggesting that relapse or reinfection occurred. Combination therapy with the addition of azithromycin had no benefit. Lower baseline Nugent scores--presumably reflecting less complex vaginal flora--were significantly associated with cure, as was refraining from unprotected sex and from douching.

Enteroviral meningitis: natural history and outcome of pleconaril therapy.

Enteroviral meningitis causes appreciable morbidity in adults, including hospitalization, decreased activity, and headache. Limited data define the natural history of disease. No antiviral therapeutic agent has demonstrated improved outcome in controlled clinical trials. Pleconaril, an inhibitor of enterovirus replication, was tested in two placebo-controlled clinical trials. Of 607 randomized patients in a multicenter, double-blind placebo-controlled study of pleconaril (200 mg three times daily versus an identical-appearing placebo), 240 patients were confirmed to have enterovirus infection. The time to headache resolution was evaluated by using Kaplan-Meier survival methodology. A Cox regression model evaluated multivariate factors associated with disease resolution. Resolution of headache in patients with concomitant moderate to severe nausea at baseline occurred at a median of 9.5 days in the absence of therapy and was reduced to 7.0 days for pleconaril recipients (P = 0.009). For a headache score of > 5 alone, treated patients resolved headache significantly more rapidly (P = 0.005). Males resolved headache 50% faster than females. Regardless of randomization group, patients with a baseline headache score of 5 or greater resolved headache 50% more slowly than patients with a baseline headache score of 4. No differences in either clinical or laboratory adverse events were noted. Over 50% of untreated patients had a persistent headache that was greater than 1 week in duration. Pleconaril shortened the course of illness compared to placebo recipients, especially in the early disease course. However, the benefit was achieved only modestly in a subgroup analysis of patients with more severe disease after adjusting for confounding variables.

Combination of gemcitabine and Ad5/3-Delta24, a tropism modified conditionally replicating adenovirus, for the treatment of ovarian cancer.

Conditionally replicating adenoviruses (CRAds) represent a novel approach for the treatment of cancers resistant to conventional therapies. The efficacy of CRAds might be further improved by using chemotherapeutic agents in a multimodal antitumor approach. We have evaluated the use of Ad5/3-Delta24, a serotype 3 receptor targeted Rb/p16 pathway selective CRAd, in combination with gemcitabine against human ovarian adenocarcinoma. The combination of these agents showed synergistic cell killing in vitro compared to single treatments. However, the effect was dependent on dose and sequencing of the agents. Our results also indicate that gemcitabine reduces the initial rate of Ad5/3-Delta24 replication without affecting the total amount of virus produced. Possible reasons for synergy between Ad5/3-Delta24 and gemcitabine include the chemosensitizing activity of E1A and/or altered replication kinetics. In an orthotopic murine model of peritoneally disseminated ovarian cancer, the combination increased the survival of mice over either agent alone, and almost 60% of treated mice were cured. Sequencing of the agents was critical for toxicity versus efficacy. Mice remained free from intraperitoneal disease, but some succumbed to treatment-related hepatic or bone marrow toxicity. This suggests that improved efficacy may uncover treatment-related toxicity, which needs to be monitored closely in clinical trials.

Noninvasive dual modality in vivo monitoring of the persistence and potency of a tumor targeted conditionally replicating adenovirus.

In clinical trials with cancer patients, the safety of conditionally replicating adenoviruses (CRAds) has been good. However, marginal data are available on the persistence or antitumor efficacy of these agents. The oncolytic potency of CRAds is determined by their capacity for entering target cells. Consequently, we constructed a retargeted CRAd featuring a secreted marker protein, soluble human carcinoembryogenic antigen (hCEA), which can be measured in growth medium or plasma. We found that virus replication closely correlated with hCEA secretion both in vitro and in vivo. Further, antitumor efficacy and the persistence of the virus could be deduced from plasma hCEA levels. Finally, using in vivo bioluminescence imaging, we were able to detect effective tumor cell killing by the virus, which led to enhanced therapeutic efficacy.

Mitochondrial DNA mutations in black Americans with hypertension-associated end-stage renal disease.

For the majority of hypertensive cases, no gene or combination of genes and environmental factors clearly leading to hypertension has been identified. Studies to identify "hypertension" genes have focused on the assessment of markers and candidate genes in the nuclear genome. In this study, we have chosen to assess the mitochondrial genome as a site of mutations possibly contributing to susceptibility to hypertension in black Americans who have progressed to end-stage renal disease (H-ESRD). The mitochondrial genomes of 58 H-ESRD and 58 normotensive individuals were systematically analyzed by means of a high-resolution restriction analysis. After stratification by the presence or absence of an African continent-specific HpaI site gain at bp 3,592, differences in the frequencies of mitochondrial DNA (mtDNA) restriction variants in both groups were examined by chi-square analyses. A total of six variants was identified with significant differences in one or both cohorts. An A10398G DdeI mutation in the ND3 gene was significantly increased in the H-ESRD cohort (H-ESRD, P = 0.048; normotensives, P = 0.20), as was an HaeIII T6620C/G6260A double mutation in the CO1 gene (H-ESRD, P = 0.05; normotensives, P = 0.48). The remaining four variants were a G2758A mutation in the 16SrRNA gene (identified by RsaI), T10810C in the ND4 gene (identified by HinfI), a G7028A/T7055C double mutation in the CO1 gene (identified by AluI), and finally, a A10086G mutation in the ND3 gene (identified by TaqI; also causing an Asn-->Asp amino acid change). The RsaI and HinfI variants were in strong linkage disequilibrium with the HpaI site and not amenable to further analysis. After correction of all P values for multiple comparisons, the ND3 A10086G (Asn-->Asp) mutation shown by TaqI remained statistically significant (P = 0.0036) in the H-ESRD cohort, not in the normotensive cohort. To the best of our knowledge, this is the first report of an increased prevalence of mitochondrial gene variants in hypertensive individuals. In addition, we have identified single-nucleotide polymorphisms in flanking regions of these genes. Although replication and further assessment are necessary, the current results support our hypothesis that mtDNA may account for a portion of hypertensive cases in black Americans with ESRD.

Targeting oncolytic adenoviral agents to the epidermal growth factor pathway with a secretory fusion molecule.

Cancer gene therapy with conditionally replicating adenoviruses is a powerful way of overcoming low tumor transduction. However, one of the main remaining obstacles is the highly variable level of the coxsackie-adenovirus receptor expression on human primary cancers. In contrast, the epidermal growth factor receptor (EGFR) is overexpressed in various tumor types, and its expression correlates with metastatic behavior and poor prognosis. We constructed an adenovirus expressing a secretory adaptor capable of retargeting adenovirus to EGFR, resulting in a more than 150-fold increase in gene transfer. A replication-competent dual-virus system secreting the adaptor displayed increased oncolytic potency in vitro and therapeutic gain in vivo. This approach could translate into increased efficacy and specificity in the treatment of EGFR overexpressing human cancers.

Reducing the cost of diagnosis of breast carcinoma: impact of ultrasound and imaging-guided biopsies on a clinical breast practice.

BACKGROUND: The objective of this study was to determine whether the use of ultrasound and percutaneous breast biopsies in patients with screen-detected nonpalpable abnormalities can reduce benign open surgical biopsies of the breast without increasing cost or sacrificing detection of potentially curable breast carcinomas. METHOD: Using a computerized mammography database and consecutive logs of needle localization procedures and fine- and large core needle biopsies of a single university-based breast imaging practice, the authors determined the breast carcinoma yield and cost of diagnosis over a 14-year period and the changes that occurred over time with the sequential introduction of ultrasound, ultrasound-guided biopsies, and stereotactic biopsies. RESULTS: The overall breast carcinoma yield for needle localization biopsies of nonpalpable lesions increased from 21% in 1984 to 68% in 1998 (P < 0.0001). The yield for nonpalpable masses increased from 21% to 87% (P < 0.0001) over the same period. The selective use of ultrasound alone and percutaneous fine- and large core needle biopsy resulted in a substantial reduction in benign open surgical biopsies. A cost analysis showed a 50% reduction in the average expense of discovering breast carcinoma. The breast carcinomas detected after introduction of these methods were prognostically favorable with 88% measuring 1.5 cm or less in size and 66% measuring less than 1 cm. CONCLUSIONS: Selective use of ultrasound and imaging-guided percutaneous biopsies can significantly reduce the number of benign open surgical biopsies generated by mammographic screening. This can result in substantial cost savings without decreasing the sensitivity for detecting small potentially curable lesions.

Migraine aura without headache: prevalence and risk factors in a primary eye care population.

BACKGROUND: Although migraine headache has received substantial attention in the scientific literature, migraine aura without headache (MAWOH) has not been investigated frequently. A general population survey study estimated the lifetime prevalence of MAWOH to be 1% in males and 3% in females. Anecdotal evidence has suggested that MAWOH is encountered by primary eye care practitioners more frequently than these rates suggest. This study is the first investigation of this condition in a primary eye care population. METHODS: A written questionnaire was used to survey 1,000 patients, ages 18 years and older, presenting for a comprehensive eye examination in the Primary Care Optometry Service of the School of Optometry, University of Alabama at Birmingham. Cases were identified by the presence of visual sensations consistent with MAWOH. RESULTS: Of the primary eye care population surveyed, 6.5% reported experiencing visual sensations consistent with MAWOH. The prevalence in males was 2.9% and in females, 8.6%. A multivariate analysis revealed that female gender (odds ratio [OR] = 2.3), history of migraine headaches (OR = 3.2), and history of childhood motion sickness (OR = 2.7) were significantly related to MAWOH. CONCLUSIONS: The prevalence of MAWOH is higher in an adult primary eye care population than previously reported in a general population study.