Richard A. McPherson

Human cytomegalovirus completely helps adeno-associated virus replication

Coinfection of adeno-associated virus (AAV) with human cytomegalovirus (HCMV) strain Towne in human embryonic fibroblasts resulted in accumulation of AAV capsid antigen and production of infectious AAV with a lag of 24 hr compared to AAV replication in AAV-adenovirus coinfections. In contrast to previous observation, these findings demonstrated that HCMV is a competent helper virus for the complete replication of AAV. In addition, HCMV and AAV were synergistic in their cytopathic effects on cells, suggesting the possibility that AAV may play a role in the pathogenicity of HCMV infections.

Evidence of a cyclosporine-binding protein in human erythrocytes

To investigate the phenomenon of different erythrocyte saturation capacities for cyclosporine (CsA) in the blood of different individuals, hemolysates of washed red cells were examined for the presence of a CsA-binding protein. Using gel filtration column chromatog-raphy of hemolysates from patients receiving CsA orally, the majority of erythrocyte-associated CsA eluted as a single peak with Mr 15,000–17,000, distinct from hemoglobin and carbonic anhydrase. [3H]CsA added to a hemolysate in vitro eluted similarly. [125I]CsA added to a hemolysate eluted much later in the same position as [3H]CsA mixed with albumin and myoglobin (presumably as free unbound drug). These findings indicate that CsA normally binds to an intraerythrocytic protein similar in molecular size to calf thymus cyclophilin (Mr 15,000). By equilibrium dialysis, the purified erythro-cyte proteins calmodulin (Mr 16,700) and cytochrome b5 (Mr 15,000) failed to bind CsA. By equilibrium dialysis, [3H] CsA did bind to column fractions containing the CsA-binding protein, but [125I]CsA did not, suggesting that attachment to CsA occurs at or near a carbon-carbon double bond in an unusual nine-carbon amino acid of CsA. These results have important implications for CsA therapy with regard to distribution space, phar-macokinetics, and a possible protein-receptor mechanism of action.

Serum albumin and the metabolism of disulfiram

The effectiveness of tetraethylthiuram disulfide (DSF) as a drug used in the treatment of alcohol abuse has been limited by the fact that it is degraded rapidly in the tissues and in the serum. Hence, a useful dose-response curve for this drug cannot be determined easily. The degradation in the tissues has been well characterized; however, its fate in the serum is less well understood. Here we kinetically describe the first steps in the degradation of DSF in the serum which results from a covalent interaction of this drug with the free sulfhydryl of serum albumin. DSF and its cleavage product diethyldithiocarbamate (DDC) both absorb significantly in the ultraviolet region. The reduction of DSF with mercaptoethanol to two molecules of DDC resulted in a large change in absorption in this region. The reaction of serum albumin with DSF produced a similar but much slower change in the ultraviolet absorption. As a result of the existence of this slow spectral change, we have been able to directly and continuously monitor the interaction of serum albumin and DSF and have determined that it is an overall first-order process. A model is proposed wherein DSF and serum albumin rapidly form a noncovalent adduct and, subsequently, in a slow unimolecular process, DSF is reduced to one mole of free DDC and one mole of the serum albumin-DDC mixed disulfide. At pH 9 the half-time for this process was 30 to 40 sec, and at pH 7.4 the half-time for this process was 1 to 1.5 min. These results suggest that degradation of DSF by serum albumin is physiologically and clinically important since the drug is maximally active only many hours after administration.

Widely Used Types and Clinical Applications of D-Dimer Assay

D-dimers are formed by the breakdown of fibrinogen and fibrin during fibrinolysis. D-dimer analysis is critical for the diagnosis of deep vein thrombosis, pulmonary embolism, and disseminated intravascular coagulation. Modern assays for D-dimer are monoclonal antibody based. The enzyme-linked immunosorbent assay (ELISA) is the reference method for D-dimer analysis in the central clinical laboratory, but is time consuming to perform. Recently, a number of rapid, point-of-care D-dimer assays have been developed for acute care settings that utilize a variety of methodologies. In view of the diversity of D-dimer assays used in central laboratory and point-of-care settings, several caveats must be taken to assure the proper interpretation and clinical application of the results. These include consideration of preanalytical variables and interfering substances, as well as patient drug therapy and underlying disease. D-dimer assays should also be validated in clinical studies, have established cut-off values, and reported according to the reagent manufacturers recommendations.

Call Subject Patterns Among On-Call Clinical Pathology Residents in an Academic Institution: How Can Tracking Changes in Patterns Over Time Benefit Resident Education?

CONTEXT On-call responsibility is an important part of residency training in clinical pathology. This task provides important consultative services for the hospital and serves as a valuable learning experience for the resident. OBJECTIVE To identify the types of calls received by residents at a large teaching hospital, to assess how and why these calls have changed over time, and to determine the educational value in tracking such changes. DESIGN A retrospective review of resident on-call records from 2 periods (2005-2006 and 1997-1998) was performed. Calls were classified based on the call subject and the caller. RESULTS Although some general patterns remained similar, several differences were identified between the time periods. Calls regarding mislabeled specimens fell, while calls concerning panic values and the blood bank (specifically therapeutic apheresis) increased. CONCLUSIONS The different patterns identified in calls between the 2 periods reflect the ever-changing role of the clinical pathologist within the hospital system and provide evidence that monitoring these shifting patterns could be a valuable tool in the education of clinical pathology residents.