Kathleen Kelly

Recruitment of p300/CBP in p53-Dependent Signal Pathways

The products of the p53 and CBP/p300 genes have been individually implicated in control of cell growth and regulation of transcription. p53 is known to act as a positive and negative regulator of gene expression. Here we show that p53, in both wild-type and mutant conformation, forms a specific protein complex with p300. However, in its wild-type but not mutant conformation, p53 inhibits a promoter containing the DNA-binding sequences for the transcription factor AP1, in a p300-dependent manner. p300 stimulates the transcriptional activity of p53 on p53-regulated promoters, and it enhances the responsiveness to a physiological upstream modulator of p53 function, ionizing radiation. A dominant negative form of p300 prevents transcriptional activation by p53, and it counteracts p53-mediated G1 arrest and apoptosis. The data implicate p300 as an important component of p53-signaling, thus providing new insight into the mechanisms of cellular proliferation.

Critical care delivery in the intensive care unit: Defining clinical roles and the best practice model

Patients receiving medical care in intensive care units (ICUs) account for nearly 30% of acute care hospital costs, yet these patients occupy only 10% of inpatient beds (1, 2). In 1984, the Office of Technology Assessment concluded that 80% of hospitals in the United States had ICUs, >20% of hospital budgets were expended on the care of intensive care patients, and approximately 1% of the gross national product was expended for intensive care services (3). With the aging of the U.S. population, greater demand for critical care services will occur. At the same time, market forces are evolving that may constrain both hospitals’ and practitioners’ abilities to provide this increasing need for critical care services. In addition, managed care organizations are requesting justification for services provided in the ICU and for demonstration of both efficiency and efficacy. Hospital administrators are continually seeking methods to provide effective and efficient care to their ICU patients. As a result of these social and economic pressures, there is a need to provide more data about the type and quality of clinical care provided in the ICU. In response, two task forces were convened by the Society of Critical Care Medicine leadership. One task force (models task force) was asked to review available information on critical care delivery in the ICU and to ascertain, if possible, a “best” practice model. The other task force was asked to define the role and practice of an intensivist. The task force memberships were diverse, representing all the disciplines that actively participate in the delivery of health care to patients in the ICU. The models task force membership consisted of 31 healthcare professionals and practitioners, including statisticians and representatives from industry, pharmacy, nursing, respiratory care, and physicians from the specialties of surgery, internal medicine, pediatrics, and anesthesia. These healthcare professionals represented the practice of critical care medicine in multiple settings, including nonteaching community hospitals, community hospitals with teaching programs, academic institutions, military hospitals, critical care medicine private practice, full-time academic practice, and consultative critical care practice. This article is the consensus report of the two task forces. The objectives of this report include the following: (1) to describe the types and settings of critical care practice (2); to describe the clinical roles of members of the ICU healthcare team (3); to examine available outcome data pertaining to the types of critical care practice (4); to attempt to define a “best” practice model; and (5) to propose additional research that should be undertaken to answer important questions regarding the practice of critical care medicine. The data and recommendations contained within this report are sometimes based on consensus expert opinion; however, where possible, recommendations are promulgated based on levels of evidence as outlined by Sacket in 1989 (4) and further modified by Taylor in 1997 (5) (see Appendix 1).

Atrial fibrillation detected by mobile cardiac outpatient telemetry in cryptogenic TIA or stroke

Objective: Atrial fibrillation (AF) may be present within a subset of patients with presumed cryptogenic TIA or stroke and remains undetected by standard diagnostic methods. We hypothesized that AF may be an under-recognized mechanism for cryptogenic TIA/stroke. Methods: A consecutive series of 56 patients with cryptogenic TIA/stroke was analyzed after diagnostic evaluation and Mobile Cardiac Outpatient Telemetry (MCOT) for up to 21 days. Demographic, radiographic, echocardiographic, and MCOT results were reviewed. Predictors of AF detection by MCOT were determined by univariate analysis including Student t test and Fisher exact tests and multivariate analysis. Results: The median MCOT monitoring duration was 21 (range 5–21) days resulting in an AF detection rate of 23% (13/56). AF was first detected after a median of 7 (range 2–19) days of monitoring. Twenty-seven asymptomatic AF episodes were detected in the 13 patients, of which 85% (23/27) were <30 seconds and the remaining 15% (4/27) were 4–24 hours in duration. Diabetes was predictive of AF detection by both univariate (p = 0.024) and multivariate analysis (OR 6.15; 95% CI 1.16 to 32.73; p = 0.033). Conclusions: There is a high rate of atrial fibrillation (AF) detection by Mobile Cardiac Outpatient Telemetry (21 days) in patients with cryptogenic TIA/stroke that may be related to extended monitoring duration, patient selection, and inclusion of all new onset AF episodes. Brief AF episodes (<30 seconds) may be biomarkers of more prolonged and clinically significant AF.

A novel mitogen-inducible gene product related to p50/p105-NF-kappa B participates in transactivation through a kappa B site.

A Rel-related, mitogen-inducible, kappa B-binding protein has been cloned as an immediate-early activation gene of human peripheral blood T cells. The cDNA has an open reading frame of 900 amino acids capable of encoding a 97-kDa protein. This protein is most similar to the 105-kDa precursor polypeptide of p50-NF-kappa B. Like the 105-kDa precursor, it contains an amino-terminal Rel-related domain of about 300 amino acids and a carboxy-terminal domain containing six full cell cycle or ankyrin repeats. In vitro-translated proteins, truncated downstream of the Rel domain and excluding the repeats, bind kappa B sites. We refer to the kappa B-binding, truncated protein as p50B by analogy with p50-NF-kappa B and to the full-length protein as p97. p50B is able to form heteromeric kappa B-binding complexes with RelB, as well as with p65 and p50, the two subunits of NF-kappa B. Transient-transfection experiments in embryonal carcinoma cells demonstrate a functional cooperation between p50B and RelB or p65 in transactivation of a reporter plasmid dependent on a kappa B site. The data imply the existence of a complex family of NF-kappa B-like transcription factors.

Red blood cell transfusion and ventilator-associated pneumonia: A potential link?

ObjectiveTo determine the relationship between packed red blood cell transfusion practice and the development of ventilator-associated pneumonia (VAP). DesignSecondary analysis of a multicentered, prospective observational study of transfusion practice in intensive care units in the United States. SettingA total of 284 intensive care units in the United States were studied from August 2000 to April 2001. PatientsPatients without pneumonia at intensive care unit admission and who then required at least 48 hrs of mechanical ventilation were considered at risk for VAP. Measurements and Main ResultsVAP was diagnosed based on prospectively defined clinical criteria and represented the primary study end point. Late-onset VAP (VAP arising after ≥5 days of mechanical ventilation) represented a secondary end point. Transfusions given during the intensive care unit stay and before the onset of VAP were tracked prospectively. Of 4,892 subjects in the original cohort, 1,518 received mechanical ventilation of ≥48 hrs and did not have preexisting pneumonia. VAP was diagnosed in 311 (20.5%) patients. Multivariate analysis revealed that transfusion independently increased the risk for VAP (odds ratio, 1.89; 95% confidence interval [CI], 1.33–2.68). Other factors increasing the risk for VAP included male sex (odds ratio, 1.54; 95% CI, 1.15–2.07), admission after trauma (odds ratio, 1.68; 95% CI, 1.15–2.47), use of continuous sedation (odds ratio, 1.43; 95% CI, 1.07–1.92), and type of nutritional support (e.g., early enteral nutrition: odds ratio, 2.65; 95% CI, 1.93–3.63; total parenteral nutrition: odds ratio, 3.27; 95% CI, 2.24–4.75). The effect of transfusion on late-onset VAP was more pronounced (odds ratio, 2.16; 95% CI, 1.27–3.66) and demonstrated a positive dose-response relationship (p = .0223 for trend test). ConclusionsTransfusion of packed red blood cells increases the risk of developing VAP. Avoiding the unnecessary use of packed red blood cell transfusions may decrease the occurrence of VAP.

Mechanisms of cancer metastasis to the bone

ABSTRACTSome of the most common human cancers, including breast cancer, prostate cancer, and lung cancer, metastasize with avidity to bone. What is the basis for their preferential growth within the bone microenvironment? Bidirectional interactions between tumor cells and cells that make up bone result in a selective advantage for tumor growth and can lead to bone destruction or new bone matrix deposition. This review discusses our current understanding of the molecular components and mechanisms that are responsible for those interactions.

The GTP binding proteins Gem and Rad are negative regulators of the Rho–Rho kinase pathway

The cytoskeletal changes that alter cellular morphogenesis and motility depend upon a complex interplay among molecules that regulate actin, myosin, and other cytoskeletal components. The Rho family of GTP binding proteins are important upstream mediators of cytoskeletal organization. Gem and Rad are members of another family of small GTP binding proteins (the Rad, Gem, and Kir family) for which biochemical functions have been mostly unknown. Here we show that Gem and Rad interface with the Rho pathway through association with the Rho effectors, Rho kinase (ROK) α and β. Gem binds ROKβ independently of RhoA in the ROKβ coiled-coil region adjacent to the Rho binding domain. Expression of Gem inhibited ROKβ-mediated phosphorylation of myosin light chain and myosin phosphatase, but not LIM kinase, suggesting that Gem acts by modifying the substrate specificity of ROKβ. Gem or Rad expression led to cell flattening and neurite extension in N1E-115 neuroblastoma cells. In interference assays, Gem opposed ROKβ- and Rad opposed ROKα-mediated cell rounding and neurite retraction. Gem did not oppose cell rounding initiated by ROKβ containing a deletion of the Gem binding region, demonstrating that Gem binding to ROKβ is required for the effects observed. In epithelial or fibroblastic cells, Gem or Rad expression resulted in stress fiber and focal adhesion disassembly. In addition, Gem reverted the anchorage-independent growth and invasiveness of Dbl-transformed fibroblasts. These results identify physiological roles for Gem and Rad in cytoskeletal regulation mediated by ROK.

Signal Pathways Which Promote Invasion and Metastasis: Critical and Distinct Contributions of Extracellular Signal-Regulated Kinase and Ral-Specific Guanine Exchange Factor Pathways

ABSTRACT Approximately 50% of metastatic tumors contain Ras mutations. Ras proteins can activate at least three downstream signaling cascades mediated by the Raf–MEK–extracellular signal-regulated kinase family, phosphatidylinositol-3 (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (RalGEFs). Here we investigated the contribution of RalGEF and ERK activation to the development of experimental metastasis in vivo and associated invasive properties in vitro. Each pathway contributes distinct properties to the metastatic phenotype. Following lateral tail vein injection, 3T3 cells transformed by constitutively active Raf or MEK produced lung metastasis that displayed circumscribed, noninfiltrating borders. In contrast, 3T3 cells transformed by Ras(12V,37G), a Ras effector mutant that activates RalGEF but not Raf or P13 kinase, formed aggressive, infiltrative metastasis. Dominant negative RalB inhibited Ras(12V,37G)-activated invasion and metastasis, demonstrating the necessity of the RalGEF pathway for a fully transformed phenotype. Moreover, 3T3 cells constitutively expressing a membrane-associated form of RalGEF (RalGDS-CAAX) formed invasive tumors as well, demonstrating that activation of a RalGEF pathway is sufficient to initiate the invasive phenotype. Despite the fact that Ras(12V,37G) expression does not elevate ERK activity, inhibition of this kinase by a conditionally expressed ERK phosphatase demonstrated that ERK activity was necessary for Ras(12V,37G)-transformed cells to express matrix-degrading activity in vitro and tissue invasiveness in vivo. Therefore, these experiments have revealed a hitherto-unknown but essential interaction of the RalGEF and ERK pathways to produce a malignant phenotype. The generality of the role of the RalGEF pathway in metastasis is supported by the finding that Ras(12V,37G) increased the invasiveness of epithelial cells as well as fibroblasts.

Complexity of the primary genetic response to mitogenic activation of human T cells.

We describe the isolation and characterization of more than 60 novel cDNA clones that constitute part of the immediate genetic response to resting human peripheral blood T cells after mitogen activation. This primary response was highly complex, both in the absolute number of inducible genes and in the diversity of regulation. Although most of the genes expressed in activated T cells were shared with the activation response of normal human fibroblasts, a significant number were more restricted in tissue specificity and thus likely encode or effect the differentiated functions of activated T cells. The activatable genes could be further differentiated on the basis of kinetics of induction, response to cycloheximide, and sensitivity to the immunosuppressive drug cyclosporin A. It is of note that cyclosporin A inhibited the expression of more than 10 inducible genes, which suggests that this drug has a broad genetic mechanism of action.

Simian Immunodeficiency Virus-Infected Macaques Treated with Highly Active Antiretroviral Therapy Have Reduced Central Nervous System Viral Replication and Inflammation but Persistence of Viral DNA

BACKGROUND During the era of highly active antiretroviral therapy (HAART), the prevalence of HIV-associated central nervous system (CNS) disease has increased despite suppression of plasma viremia. METHODS In a simian immunodeficiency virus (SIV) model system in which all animals develop AIDS and 90% develop CNS disease by 3 months after inoculation, pigtailed macaques were treated with a regimen of tenofovir disoproxil fumarate, saquinavir, atazanavir, and an integrase inhibitor starting at 12 days after inoculation and were euthanized at approximately 175 days after inoculation. RESULTS Plasma and cerebrospinal fluid (CSF) viral loads declined rapidly after the initiation of HAART. Brain viral RNA was undetectable at necropsy, but viral DNA levels were not different from those in untreated SIV-infected macaques. CNS inflammation was significantly reduced, with decreased brain expression of major histocompatibility complex class II and glial fibrillary acidic protein and reduced levels of CSF CCL2 and interleukin 6. Brain from treated macaques had significantly lower levels of interferon beta, type 1 interferon-inducible gene myxovirus (influenza) resistance A, and indolamine 2,3-dioxygenase messenger RNA, suggesting that immune hyperactivation was suppressed, and fewer CD4(+) and CD8(+) T cells, suggesting that trafficking of T cells from peripheral blood was reduced. Brain levels of CD68 protein and tumor necrosis factor alpha and interferon gamma RNA were reduced but were not significantly lower, indicating continued CNS inflammation. CONCLUSIONS These data, generated in a rigorous, high-viral-load SIV-infected macaque model, showed that HAART provided benefits with respect to CNS viral replication and inflammation but that no change in the level of viral DNA and continued CNS inflammation occurred in some macaques.