Bushra Yasin

θ Defensins Protect Cells from Infection by Herpes Simplex Virus by Inhibiting Viral Adhesion and Entry

ABSTRACT We tested the ability of 20 synthetic θ defensins to protect cells from infection by type 1 and type 2 herpes simplex viruses (HSV-1 and -2, respectively). The peptides included rhesus θ defensins (RTDs) 1 to 3, originally isolated from rhesus macaque leukocytes, and three peptides (retrocyclins 1 to 3) whose sequences were inferred from human θ-defensin (DEFT) pseudogenes. We also tested 14 retrocyclin analogues, including the retro, enantio, and retroenantio forms of retrocyclin 1. Retrocyclins 1 and 2 and RTD 3 protected cervical epithelial cells from infection by both HSV serotypes, but only retrocyclin 2 did so without causing cytotoxicity or requiring preincubation with the virus. Surface plasmon resonance studies revealed that retrocyclin 2 bound to immobilized HSV-2 glycoprotein B (gB2) with high affinity (Kd, 13.3 nM) and that it did not bind to enzymatically deglycosylated gB2. Temperature shift experiments indicated that retrocyclin 2 and human α defensins human neutrophil peptide 1 (HNP 1) to HNP 3 protected human cells from HSV-2 by different mechanisms. Retrocyclin 2 blocked viral attachment, and its addition during the binding or penetration phases of HSV-2 infection markedly diminished nuclear translocation of VP16 and expression of ICP4. In contrast, HNPs 1 to 3 had little effect on binding but reduced both VP16 transport and ICP4 expression if added during the postbinding (penetration) period. We recently reported that θ defensins are miniature lectins that bind gp120 of human immunodeficiency virus type 1 (HIV-1) with high affinity and inhibit the entry of R5 and X4 isolates of HIV-1. Given its small size (18 residues), minimal cytotoxicity, lack of activity against vaginal lactobacilli, and effectiveness against both HSV-2 and HIV-1, retrocyclin 2 provides an intriguing prototype for future topical microbicide development.

Evaluation of the Inactivation of Infectious Herpes Simplex Virus by Host-Defense Peptides

Abstract A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide microplate assay was adapted to screen for the ability of 20 host-defense peptides to inactivate herpes simplex virus type 1 and type 2. The procedure required minimal amounts of material, was reproducible, and was confirmed with standard antiviral testing techniques. In screening tests, with the exception of melittin, a highly cytotoxic and hemolytic peptide found in bee venom, the α-helical peptides in our test panel (magainins, cecropins, clavanins, and LL-37) caused little viral inactivation. Several β-sheet peptides (defensins, tachyplesin, and protegrins) inactivated one or both viruses, sometimes with remarkable selectivity. Two peptides were identified as having antiviral activity against both viruses, indolicidin (a tryptophan-rich peptide from bovine neutrophils) and brevinin-1 (a peptide found in frog skin). The antiviral activity of these two peptides was confirmed with standard antiviral assays. Interestingly, the antiviral activity of brevinin-1 was maintained after reduction and carboxamidomethylation, procedures that abolished its otherwise prominent hemolytic and cytotoxic effects.

Patient Safety in the Clinical Laboratory: A Longitudinal Analysis of Specimen Identification Errors

CONTEXT Patient safety is an increasingly visible and important mission for clinical laboratories. Attention to improving processes related to patient identification and specimen labeling is being paid by accreditation and regulatory organizations because errors in these areas that jeopardize patient safety are common and avoidable through improvement in the total testing process. OBJECTIVE To assess patient identification and specimen labeling improvement after multiple implementation projects using longitudinal statistical tools. DESIGN Specimen errors were categorized by a multidisciplinary health care team. Patient identification errors were grouped into 3 categories: (1) specimen/requisition mismatch, (2) unlabeled specimens, and (3) mislabeled specimens. Specimens with these types of identification errors were compared preimplementation and postimplementation for 3 patient safety projects: (1) reorganization of phlebotomy (4 months); (2) introduction of an electronic event reporting system (10 months); and (3) activation of an automated processing system (14 months) for a 24-month period, using trend analysis and Student t test statistics. RESULTS Of 16,632 total specimen errors, mislabeled specimens, requisition mismatches, and unlabeled specimens represented 1.0%, 6.3%, and 4.6% of errors, respectively. Student t test showed a significant decrease in the most serious error, mislabeled specimens (P < .001) when compared to before implementation of the 3 patient safety projects. Trend analysis demonstrated decreases in all 3 error types for 26 months. CONCLUSIONS Applying performance-improvement strategies that focus longitudinally on specimen labeling errors can significantly reduce errors, therefore improving patient safety. This is an important area in which laboratory professionals, working in interdisciplinary teams, can improve safety and outcomes of care.

Activity of Novispirin G-10, a novel antimicrobial peptide against Chlamydia trachomatis and vaginosis-associated bacteria.

We tested the activity of Novispirin G-10, a novel antimicrobial alpha-helical octadecapeptide structurally related to cathelicidins and other innate immunity peptides, against Chlamydia trachomatis serovars L2, D, and E and three organisms associated with bacterial vaginosis (BV). The peptides activity against C. trachomatis was measured in 48-h shell vial assays with McCoy cell targets. Exposure to 100 micro g/ml of Novispirin G-10 reduced the infectivity of serovars D and E by 99.4-100% and serovar L2 by 91.7-99.1%. At the same concentration of 100 micro g/ml, Novispirin G-10 rapidly killed >99% of Mobiluncus curtisii, Gardnerella vaginalis, and Prevotella bivia, in standard colony-forming unit (CFU) assays. Given its simple structure and relative lack of cytotoxic and hemolytic activity, Novispirin G-10 may be a useful component of microbicide preparations designed to prevent chlamydial infection and/or remediate the abnormal vaginal flora associated with BV.

Examination of Chlamydia trachomatis infection in environments mimicking normal and abnormal vaginal pH.

Background It has long been assumed that a healthy acidic vaginal environment inhibits infection by Chlamydia trachomatis. The research objectives were to evaluate the effect of pH on C trachomatis infection by two in vitro methods, to assess pH effect at different serial dilutions of C trachomatis elementary bodies (EBs), and to examine protection by an antibiotic peptide, protegrin (PG-1), over a pH range. Goals The goals of this study were to test the hypothesis that acidic pH inhibits C trachomatis infection and to determine the ability of PG-1 to provide protection at acidic and neutral pH. Study Design The effect of pH on C trachomatis was examined using two pH-adjusted preincubation shell vial assays. C trachomatis EBs (serovars L2, D, and E) were exposed to pH-adjusted media, with and without PG-1, and infection was assessed by inclusion forming unit (IFU) formation in McCoy cell monolayers. Results Acidic pH in preincubation media markedly decreased IFUs by both in vitro methods. Serial dilution experiments showed a 3- to 10-fold reduction in IFUs for C trachomatis (L2 and E) at pH 5.0, compared with those at pH 7.5. C trachomatis (D) showed a 17- to 23-fold reduction in IFUs (serial dilutions 1:1–1:4). PG-1 protected McCoy cell monolayers from infection by C trachomatis after exposure to varied pH environments. Conclusion Acidic pH exposure significantly reduced C trachomatis infection in vitro. Our results support the hypothesis that a healthy acidic vaginal environment protects women from C trachomatis infection. In addition, antibiotic peptides may provide protection as topical microbicides, regardless of vaginal pH.

Point-of-care testing: Twenty years' experience

Point-of-care testing (POCT) has expanded dramatically in the past 20 years with volume growth of 12% to 15% annually. What have we learned? Three features of POCT essential for a successful POCT program are: 1) a well-defined POCT organization, 2) robust connectivity, and 3) still more information regarding the relationship of POCT to clinical outcomes. The advances and limitations for each of these aspects are discussed in an up-to-date review of the POCT arena.