Anand S. Dighe

Targeted Disruption of the Stat1 Gene in Mice Reveals Unexpected Physiologic Specificity in the JAK–STAT Signaling Pathway

The JAK-STAT signaling pathway has been implicated in mediating biological responses induced by many cytokines. However, cytokines that promote distinct cellular responses often activate identical STAT proteins, thereby raising the question of how specificity is manifest within this signaling pathway. Here we report the generation and characterization of mice deficient in STAT1. STAT1-deficient mice show no overt developmental abnormalities, but display a complete lack of responsiveness to either IFN alpha or IFN gamma and are highly sensitive to infection by microbial pathogens and viruses. In contrast, these mice respond normally to several other cytokines that activate STAT1 in vitro. These observations document that STAT1 plays an obligate and dedicated role in mediating IFN-dependent biologic responses and reveal an unexpected level of physiologic specificity for STAT1 action.

Enhanced in vivo growth and resistance to rejection of tumor cells expressing dominant negative IFNγ receptors

Using a neutralizing monoclonal antibody specific for murine IFN gamma we show that endogenously produced IFN gamma plays an obligate role in mediating LPS-induced rejection of the Meth A fibrosarcoma tumor in syngeneic BALB/c mice. To examine the cellular targets of IFN gamma action, we generated IFN gamma-insensitive tumor cells by stably overexpressing in Meth A a truncated dominant negative form of the murine IFN gamma receptor alpha chain. When implanted in BALB/c mice, IFN gamma-insensitive Meth A cells displayed enhanced tumorigenicity compared with control Meth A cells and were not rejected when tumor-bearing mice were treated with concentrations of LPS that eliminated control tumors. In Meth A immune mice, IFN gamma-insensitive Meth A did not establish tumors while IFN gamma-insensitive tumors grew in a progressive manner. In addition, the IFN gamma-insensitive tumor cells were unable to elicit strong protective immunity to subsequent wild-type tumor challenge. These results show that IFN gamma has direct effects on tumor cell immunogenicity and thus plays an important role in promoting tumor cell recognition and elimination.

Developmental commitment to the Th2 lineage by extinction of IL-12 signaling

Developmental-commitment to Th1 or Th2 responses critically influences host susceptibility to particular pathogens. We describe a novel mechanism governing stable commitment to Th2 differentiation. Naive T cells develop strongly polarized Th1 and Th2 profiles by 7 days after activation. However, commitment of these developing cells differs substantially. Although IL-4 reverses early Th1 differentiation, IL-12 cannot reverse early Th2 differentiation. Th1 reversibility results from maintenance of IL-4 signal transduction, whereas Th2 commitment results from rapid loss of IL-12 signaling. The IL-12 signaling defect in Th2 cells results in failure to phosphorylate Jak2, Stat3, and Stat4. Since Th2 cells express the mRNA for the cloned murine IL-12 receptor beta subunit, the signaling defect may involve expression or function of unidentified receptor components. The rapid extinction of IL-12 signaling in Th2 cells provides a demonstration of a mechanism for the stable commitment to a T helper phenotype.

Ligand-induced autoregulation of IFN-gamma receptor beta chain expression in T helper cell subsets

Interferon γ (IFN-γ) responsiveness in certain cells depends on the state of cellular differentiation or activation. Here an in vitro developmental system was used to show that IFN-γ produced during generation of the CD4+ T helper cell type 1 (TH1) subset extinguishes expression of the IFN-γ receptor β subunit, resulting in TH1 cells that are unresponsive to IFN-γ. This β chain loss also occurred in IFN-γ-treated TH2 cells and thus represents a specific response of CD4+ T cells to IFN-γ rather than a TH1-specific differentiation event. These results define a mechanism of cellular desensitization where a cytokine down-regulates expression of a receptor subunit required primarily for signaling and not ligand binding.

Tissue-specific targeting of gytokine unresponsiveness in transgenic mice

The ubiquitous cellular distribution of certain cytokine receptors has hampered attempts to define the physiologically important cell-specific functions of cytokines in vivo. Herein, we report the generation of transgenic mice that express a dominant-negative IFN gamma receptor alpha chain mutant under the control of either the human lysozyme promoter or the murine lck proximal promoter, which display tissue-specific unresponsiveness in the macrophage or T cell compartments, respectively, to the pleiotropic cytokine, IFN gamma. We utilize these mice to identify previously undefined cellular targets of IFN gamma action in the development of a murine antimicrobial response and the mixed lymphocyte reaction. Moreover, we identify the macrophage as a critical responsive cell in manifesting the effects of IFN gamma in regulating CD4+ T helper subset development. These studies thus represent a novel approach to studying the cell-specific actions of an endogenously produced pleiotropic cytokine in vivo.

Analysis of laboratory critical value reporting at a large academic medical center.

Reporting of laboratory critical values has become an issue of national attention as illustrated by recent guidelines described in the National Patient Safety Goals of the Joint Commission on Accreditation of Healthcare Organizations. Herein, we report the results of an analysis of 37,503 consecutive laboratory critical values at our institution, a large urban academic medical center. We evaluated critical value reporting by test, laboratory specialty, patient type, clinical care area, time of day, and critical value limits. Factors leading to delays in critical value reporting are identified, and we describe approaches to improving this important operational and patient safety issue.

Utilization management in a large urban academic medical center: a 10-year experience.

Management of laboratory test utilization presents an ongoing challenge. Most studies reported in the literature have described efforts to control one or a few tests, but the results cannot be generalized to a broader utilization management strategy. Herein we report our experiences with an organizational utilization management program during a 10-year period. Cumulatively, our program has achieved significant success, saving millions of dollars in blood components and reducing inpatient tests per discharge by 26%. Highlights from our experiences include the importance of implementing an institutional organizational structure to support utilization management, the central role fulfilled by clinical pathologists as leaders of the program, the ability to obtain timely utilization data, and careful selection of the most appropriate implementation tools tailored to the unique circumstances of each utilization management initiative.

“Pre-pre” and “post-post” analytical error: high-incidence patient safety hazards involving the clinical laboratory

Abstract Data from recent studies suggest that the highest incidence of laboratory-related errors occurs in the pre-analytical phase of laboratory testing. However, few studies have examined the frequency of errors in laboratory test selection and interpretation. A survey of physicians who use our clinical laboratory demonstrated that the largest number of test ordering errors appear to involve physicians simply ordering the wrong test. Diagnostic algorithms providing guidance for test selection in specific disorders are also used as the basis for the establishment of reflex protocols in the clinical laboratory. The provision of an expert-driven interpretation by laboratory professionals resulted in improvements both in the time to and the accuracy of diagnosis. A survey of our physician staff has shown that in the absence of such an interpretation, for patients being assessed for a coagulation disorder, approximately 75% of the cases would have involved some level of test result misinterpretation. Clin Chem Lab Med 2007;45:712–9.

Physician Survey of a Laboratory Medicine Interpretive Service and Evaluation of the Influence of Interpretations on Laboratory Test Ordering

CONTEXT Complex coagulation test panels ordered by clinicians are typically reported to clinicians without a patient-specific interpretive paragraph. OBJECTIVES To survey clinicians regarding pathologist-generated interpretations of complex laboratory testing panels and to assess the ability of the interpretations to educate test orderers. DESIGN Surveys were conducted of physicians ordering complex coagulation laboratory testing that included narrative interpretation. Evaluation of order requisitions was performed to assess the interpretations influence on ordering practices. SETTING Physicians ordering coagulation testing at a large academic medical center hospital in Boston, Mass, and physicians from outside hospitals using the academic medical center as a reference laboratory for coagulation testing. OUTCOME MEASURES Physician surveys and evaluation of laboratory requisition slips. RESULTS In nearly 80% of responses, the ordering clinicians perceived that the interpretive comments saved them time and improved the diagnostic process. Moreover, the interpretations were perceived by ordering clinicians to help prevent a misdiagnosis or otherwise impact the differential diagnosis in approximately 70% of responses. In addition, interpretations appeared to be able to train the ordering clinicians as to the standard ordering practices. CONCLUSIONS The results demonstrate physician satisfaction with an innovative information delivery approach that provides laboratory diagnostic interpretation and test-ordering education to clinicians in the context of their daily workflow.

Computerized provider order entry in the clinical laboratory.

Clinicians have traditionally ordered laboratory tests using paper-based orders and requisitions. However, paper orders are becoming increasingly incompatible with the complexities, challenges, and resource constraints of our modern healthcare systems and are being replaced by electronic order entry systems. Electronic systems that allow direct provider input of diagnostic testing or medication orders into a computer system are known as Computerized Provider Order Entry (CPOE) systems. Adoption of laboratory CPOE systems may offer institutions many benefits, including reduced test turnaround time, improved test utilization, and better adherence to practice guidelines. In this review, we outline the functionality of various CPOE implementations, review the reported benefits, and discuss strategies for using CPOE to improve the test ordering process. Further, we discuss barriers to the implementation of CPOE systems that have prevented their more widespread adoption.