Christine K. Rudy

Imaging flow cytometry elucidates limitations of microparticle analysis by conventional flow cytometry

Microparticles (MPs) are submicron vesicles released from cell membranes in response to activation, cell injury, or apoptosis. The clinical importance of MPs has become increasingly recognized, although no standardized method exists for their measurement. Flow cytometry (FCM) is the most commonly used technique, however, because of the small size of MPs, and the limitations of current FCM instrumentation, accurate identification is compromised by this methodology. We decided to investigate whether the use of FCM combined with imaging, such as is possible with the ImagestreamX imaging FC (ISX), would be a more sensitive approach to characterizing MPs. Combining FCM with imaging eliminates some of the limitations demonstrated by conventional FCM, whereas also providing morphological confirmation and the ability to distinguish true single events from aggregates and cell debris. The detection limit of standard nonspecialized FCM is suboptimal when compared to ISX. Evaluating MPs below 0.200 µm and sizing remain a challenge as some MPs remain below the detection limit of ISX. Standardized calibrators, that more closely reflect the physical characteristics of MPs, need further development.

E2 Quasispecies Specificity of Hepatitis C Virus Association With Allografts Immediately After Liver Transplantation

It is unknown whether all hepatitis C virus (HCV) quasispecies variants found within patient serum have equal capacity to associate with the liver after transplantation; however, in vitro models of HCV infection suggest that variations in the hypervariable region 1 (HVR1) of the second envelope protein (E2) may be important in infectivity. The hypothesis of the current study is that the two hypervariable regions (HVR1 and HVR2) within E2 are important in the initial virus–liver interaction, and, therefore, certain HCV quasispecies variants will be isolated from the liver after reperfusion. In 8 patients with end‐stage liver disease secondary to HCV infection, HCV envelope quasispecies were determined from intraoperative serum samples obtained before the anhepatic phase of transplantation and from liver biopsies 1.5 to 2.5 hours after the transplanted liver was perfused. Explanted (native) liver biopsies were taken as a control. Sequence analysis was performed on clones of specific HCV reverse transcriptase‐polymerase chain reaction products spanning HVR1 and HVR2 of the E2 protein. HVR1 was more variable than HVR2 for all samples. Quasispecies isolated from postperfusion liver differed more from serum than did explanted liver quasispecies at HVR1 (P = 0.03) but not at HVR2 (P = 0.2). Comparison of HVR1 sequences from postperfusion liver versus serum revealed significantly less HVR1 genetic complexity and diversity (P = 0.02 and P = 0.04, respectively). Immediately after transplantation but before actual infection, liver allografts select out from the infecting serum inoculum a less heterogeneous, more closely related population of quasispecies variants. (Liver Transpl 2004;10:208–216.)

Loss of Collectrin, an Angiotensin-Converting Enzyme 2 Homolog, Uncouples Endothelial Nitric Oxide Synthase and Causes Hypertension and Vascular Dysfunction

Background— Collectrin is an orphan member of the renin-angiotensin system and is a homolog of angiotensin-converting enzyme 2, sharing ≈50% sequence identity. Unlike angiotensin-converting enzyme 2, collectrin lacks any catalytic domain. Collectrin has been shown to function as a chaperone of amino acid transporters. In rodents, the renal expression of collectrin is increased after subtotal nephrectomy and during high-salt feeding, raising the question of whether collectrin has any direct role in blood pressure regulation. Methods and Results— Using a susceptible genetic background, we demonstrate that deletion of collectrin results in hypertension, exaggerated salt sensitivity, and impaired pressure natriuresis. Collectrin knockout mice display impaired endothelium-dependent vasorelaxation that is associated with vascular remodeling, endothelial nitric oxide synthase uncoupling, decreased nitric oxide production, and increased superoxide generation. Treatment with Tempol, a superoxide scavenger, attenuates the augmented sodium sensitivity in collectrin knockout mice. We report for the first time that collectrin is expressed in endothelial cells. Furthermore, collectrin directly regulates L-arginine uptake and plasma membrane levels of CAT1 and y+LAT1 amino acid transporters in endothelial cells. Treatment with L-arginine modestly lowers blood pressure of collectrin knockout mice. Conclusions— Collectrin is a consequential link between the transport of L-arginine and endothelial nitric oxide synthase uncoupling in hypertension.

Loss of Collectrin, an ACE2 Homologue, Uncouples Endothelial Nitric Oxide Synthase and Causes Hypertension and Vascular Dysfunction

Background— Collectrin is an orphan member of the renin-angiotensin system and is a homolog of angiotensin-converting enzyme 2, sharing ≈50% sequence identity. Unlike angiotensin-converting enzyme 2, collectrin lacks any catalytic domain. Collectrin has been shown to function as a chaperone of amino acid transporters. In rodents, the renal expression of collectrin is increased after subtotal nephrectomy and during high-salt feeding, raising the question of whether collectrin has any direct role in blood pressure regulation. Methods and Results— Using a susceptible genetic background, we demonstrate that deletion of collectrin results in hypertension, exaggerated salt sensitivity, and impaired pressure natriuresis. Collectrin knockout mice display impaired endothelium-dependent vasorelaxation that is associated with vascular remodeling, endothelial nitric oxide synthase uncoupling, decreased nitric oxide production, and increased superoxide generation. Treatment with Tempol, a superoxide scavenger, attenuates the augmented sodium sensitivity in collectrin knockout mice. We report for the first time that collectrin is expressed in endothelial cells. Furthermore, collectrin directly regulates L-arginine uptake and plasma membrane levels of CAT1 and y+LAT1 amino acid transporters in endothelial cells. Treatment with L-arginine modestly lowers blood pressure of collectrin knockout mice. Conclusions— Collectrin is a consequential link between the transport of L-arginine and endothelial nitric oxide synthase uncoupling in hypertension.

HCV Infection of the Transplanted Liver: Changing CD81 and HVR1 Variants Immediately After Liver Transplantation

The second envelope protein at hypervariable region 1 (HVR1) has been implicated in contributing to hepatitis C virus (HCV)‐host cell interactions and CD81 (a multifunctional protein) has been demonstrated to act as a cell surface receptor for HCV and may interact directly with HVR1. The purpose of the current study was to determine if certain HVR1 quasispecies variants more effectively associate with and infect allografts after liver transplantation than other HVR1 variants and whether CD81 receptor expression changes after transplantation. Blood and allograft samples were obtained from the peritransplant period in seven patients. Clones of RT‐PCR product were directly sequenced to identify HVR1 quasispecies variants. Explanted liver and serial allograft biopsies in recipients with HCV were examined by immunohistochemistry (IHC) for CD81 expression. Examination of HVR1 sequences demonstrated that only a fraction of the quasispecies variants recovered from each patients blood sampled immediately prior to transplantation associated with and infected the allografts. Genetic diversity at HVR1 decreased with reperfusion but did not significantly decrease with infection. Expression of CD81 varied during the immediate post‐transplant period. In conclusion, HVR1 quasispecies variants differentially associate with, and infect allografts, after liver transplantation. Additionally, allografts express variable amounts of CD81 after transplantation.