Julianne L. Baron

The DREAM complex mediates GIST cell quiescence and is a novel therapeutic target to enhance imatinib-induced apoptosis

Gastrointestinal stromal tumors (GIST) can be successfully treated with imatinib mesylate (Gleevec); however, complete remissions are rare and patients frequently achieve disease stabilization in the presence of residual tumor masses. The clinical observation that discontinuation of treatment can lead to tumor progression suggests that residual tumor cells are, in fact, quiescent and, therefore, able to re-enter the cell-division cycle. In line with this notion, we have previously shown that imatinib induces GIST cell quiescence in vitro through the APC(CDH1)-SKP2-p27(Kip1) signaling axis. Here, we provide evidence that imatinib induces GIST cell quiescence in vivo and that this process also involves the DREAM complex, a multisubunit complex that has recently been identified as an additional key regulator of quiescence. Importantly, inhibition of DREAM complex formation by depletion of the DREAM regulatory kinase DYRK1A or its target LIN52 was found to enhance imatinib-induced cell death. Our results show that imatinib induces apoptosis in a fraction of GIST cells while, at the same time, a subset of cells undergoes quiescence involving the DREAM complex. Inhibition of this process enhances imatinib-induced apoptosis, which opens the opportunity for future therapeutic interventions to target the DREAM complex for more efficient imatinib responses.

Shift in the microbial ecology of a hospital hot water system following the introduction of an on-site monochloramine disinfection system.

Drinking water distribution systems, including premise plumbing, contain a diverse microbiological community that may include opportunistic pathogens. On-site supplemental disinfection systems have been proposed as a control method for opportunistic pathogens in premise plumbing. The majority of on-site disinfection systems to date have been installed in hospitals due to the high concentration of opportunistic pathogen susceptible occupants. The installation of on-site supplemental disinfection systems in hospitals allows for evaluation of the impact of on-site disinfection systems on drinking water system microbial ecology prior to widespread application. This study evaluated the impact of supplemental monochloramine on the microbial ecology of a hospital’s hot water system. Samples were taken three months and immediately prior to monochloramine treatment and monthly for the first six months of treatment, and all samples were subjected to high throughput Illumina 16S rRNA region sequencing. The microbial community composition of monochloramine treated samples was dramatically different than the baseline months. There was an immediate shift towards decreased relative abundance of Betaproteobacteria, and increased relative abundance of Firmicutes, Alphaproteobacteria, Gammaproteobacteria, Cyanobacteria and Actinobacteria. Following treatment, microbial populations grouped by sampling location rather than sampling time. Over the course of treatment the relative abundance of certain genera containing opportunistic pathogens and genera containing denitrifying bacteria increased. The results demonstrate the driving influence of supplemental disinfection on premise plumbing microbial ecology and suggest the value of further investigation into the overall effects of premise plumbing disinfection strategies on microbial ecology and not solely specific target microorganisms.

Evaluation of a New Monochloramine Generation System for Controlling Legionella in Building Hot Water Systems

OBJECTIVE To evaluate the efficacy of a new monochloramine generation system for control of Legionella in a hospital hot water distribution system. SETTING A 495-bed tertiary care hospital in Pittsburgh, Pennsylvania. The hospital has 12 floors covering approximately 78,000 m(2). METHODS The hospital hot water system was monitored for a total of 29 months, including a 5-month baseline sampling period prior to installation of the monochloramine system and 24 months of surveillance after system installation (postdisinfection period). Water samples were collected for microbiological analysis (Legionella species, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter species, nitrifying bacteria, heterotrophic plate count [HPC] bacteria, and nontuberculous mycobacteria). Chemical parameters monitored during the investigation included monochloramine, chlorine (free and total), nitrate, nitrite, total ammonia, copper, silver, lead, and pH. RESULTS A significant reduction in Legionella distal site positivity was observed between the pre- and postdisinfection periods, with positivity decreasing from an average of 53% (baseline) to an average of 9% after monochloramine application (P<0.5]). Although geometric mean HPC concentrations decreased by approximately 2 log colony-forming units per milliliter during monochloramine treatment, we did not observe significant changes in other microbial populations. CONCLUSIONS This is the first evaluation in the United States of a commercially available monochloramine system installed on a hospital hot water system for Legionella disinfection, and it demonstrated a significant reduction in Legionella colonization. Significant increases in microbial populations or other negative effects previously associated with monochloramine use in large municipal cold water systems were not observed.

Fungal diversity and presence of potentially pathogenic fungi in a hospital hot water system treated with on-site monochloramine.

Currently, our knowledge of fungal ecology in engineered drinking water systems is limited, despite the potential for these systems to serve as a reservoir for opportunistic pathogens. In this study, hot water samples were collected both prior to and following the addition of monochloramine as an on-site disinfectant in a hospital hot water system. Fungal ecology was then analyzed by high throughput sequencing of the fungal ITS1 region. The results demonstrate that the genera Penicillium, Aspergillus, Peniophora, Cladosporium and Rhodosporidium comprised the core fungal biome of the hospital hot water system. Penicillium dominated the fungal community with an average relative abundance of 88.89% (±6.37%). ITS1 sequences of fungal genera containing potential pathogens such as Aspergillus, Candida, and Fusarium were also detected in this study. No significant change in fungal community structure was observed before and after the initiation of on-site monochloramine water treatment. This work represents the first report of the effects of on-site secondary water disinfection on fungal ecology in premise plumbing system, and demonstrates the necessity of considering opportunistic fungal pathogens during the evaluation of secondary premise plumbing disinfection systems.

Diagnostic testing for Legionnaires’ disease

Legionnaires’ disease is commonly diagnosed clinically using a urinary antigen test. The urinary antigen test is highly accurate for L. pneumophila serogroup 1, however other diagnostic tests should also be utilized in conjunction with the urinary antigen as many other Legionella species and serogroups are pathogenic. Culturing of patient specimens remains the gold standard for diagnosis of Legionnaires’ disease. Selective media, BYCE with the addition of antibiotics, allows for a high sensitivity and specificity. Culturing can identify all species and serogroups of Legionella. A major benefit of culturing is that it provides the recovery of a patient isolate, which can be used to find an environmental match. Other diagnostic tests, including DFA and molecular tests such as PCR and LAMP, are useful tests to supplement culturing. Molecular tests provide much more rapid results in comparison to culture, however these tests should not be a primary diagnostic tool given their lower sensitivity and specificity in comparison to culturing. It is recommended that all laboratories develop the ability to culture patient specimens in-house with the selective media.

Effect of monochloramine treatment on the microbial ecology of Legionella and associated bacterial populations in a hospital hot water system

Opportunistic pathogens, including Legionella spp. and non-tuberculous mycobacteria, can thrive in building hot water systems despite municipal and traditional on-site chlorine disinfection. Monochloramine is a relatively new approach to on-site disinfection, but the microbiological impact of on-site chloramine use has not been well studied. We hypothesized that comparison of the microbial ecology associated with monochloramine treatment versus no on-site treatment would yield highly dissimilar bacterial communities. Hot water samples were collected monthly from 7 locations for three months from two buildings in a Pennsylvania hospital complex supplied with common municipal water: (1) a hospital administrative building (no on-site treatment) and (2) an adjacent acute-care hospital treated on-site with monochloramine to control Legionella spp. Water samples were subjected to DNA extraction, rRNA PCR, and 454 pyrosequencing. Stark differences in the microbiome of the chloraminated water and the control were observed. Bacteria in the treated samples were primarily Sphingomonadales and Limnohabitans, whereas Flexibacter and Planctomycetaceae predominated in untreated control samples. Serendipitously, one sampling month coincided with dysfunction of the on-site disinfection system that resulted in a Legionella bloom detected by sequencing and culture. This study also demonstrates the potential utility of high-throughput DNA sequencing to monitor microbial ecology in water systems.

Abstract 1647: Imatinib mesylate leads to modulation of regulators of tumor cell quiescence in gastrointestinal stromal tumors (GIST) cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Gastrointestinal stromal tumors (GISTs) are caused by activating mutations in the KIT or PDGFRA receptor tyrosine kinase genes. While GISTs can successfully be treated with imatinib mesylate, many patients retain detectable tumor mass during treatment and experience relapse following termination of treatment. We wanted to determine whether these clinical observations result from direct, imatinib-induced effects on GIST cells. We have shown that imatinib can directly induce tumor cell quiescence in a proportion of GIST cells through an increase of nuclear CDH1, an activator of the anaphase-promoting complex (APC). This event was associated with downregulation of the F-box protein SKP2 and subsequent upregulation of its target, the CDK inhibitor p27(Kip1). Exit from the cell division cycle was reflected by reduced BrdU incorporation and reduced cyclin A protein expression. Quiescent tumor cells were able to re-enter the cell division cycle five days after removal of imatinib as indicated by enhanced BrdU incorporation, decrease in p27(Kip1) expression and increase of cyclin A expression. Furthermore, high SKP2 and low p27(Kip1) levels correlated with an increased risk of progression. Mechanistically, imatinib was found to modulate components of the human DREAM complex, a key regulator of quiescence. Imatinib treatment resulted in upregulation of p130 and downregulation of E2F4 as well as LIN37 and LIN9. After removal of imatinib, levels of p130 decreased, whereas E2F4, LIN37 and LIN9 levels increased, as a reflection of the cells re-entering the cell cycle. Our results indicate that GIST cells that do not undergo apoptosis in response to imatinib enter quiescence and are hence temporarily removed from the proliferative pool However, cells were found to readily re-enter the cell division cycle in the absence imatinib. Our results encourage further studies to target the quiescence pathway in GISTs therapeutically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1647.