Robert J. Jacob

Role of EGR-1 in Thapsigargin-Inducible Apoptosis in the Melanoma Cell Line A375-C6

Induction of apoptosis by diverse exogenous signals is dependent on elevation of intracellular Ca2+. This process of cell death can be blocked by actinomycin D, indicating that it requires gene transcription events. To identify genes that are required for apoptosis, we used thapsigargin (TG), which inhibits endoplasmic reticulum-dependent Ca(2+)-ATPase and thereby increases cytosolic Ca2+. Exposure to TG led to induction of the zinc finger transcription factor, EGR-1, and apoptosis in human melanoma cells, A375-C6. To determine the functional relevance of EGR-1 expression in TG-inducible apoptosis, we employed a dominant negative mutant which functionally competes with EGR-1 in these cells. Interestingly, the dominant negative mutant inhibited TG-inducible apoptosis. Consistent with this observation, an antisense oligomer directed against Egr-1 also led to a diminution of the number of cells that undergo TG-inducible apoptosis. These results suggest a novel regulatory role for EGR-1 in mediating apoptosis that is induced by intracellular Ca2+ elevation. We have previously shown that in these melanoma cells, EGR-1 acts to inhibit the growth arresting action of interleukin-1. Together, these results imply that EGR-1 plays inducer-specific roles in growth control.

Molecular Aspects of Herpes Simplex Virus I Latency, Reactivation, and Recurrence

The application of molecular biology in the study of the pathogenesis of herpes simplex virus type 1 (HSV-1) has led to significant advances in our understanding of mechanisms that regulate virus behavior in sensory neurons and epithelial tissue. Such study has provided insight into the relationship of host and viral factors that regulate latency, reactivation, and recurrent disease. This review attempts to distill decades of information involving human, animal, and cell culture studies of HSV-1 with the goal of correlating molecular events with the clinical and laboratory behavior of the virus during latency, reactivation, and recurrent disease. The purpose of such an attempt is to acquaint the clinician/scientist with the current thinking in the field, and to provide key references upon which current opinions rest.

Histone deacetylase inhibitors induce reactivation of herpes simplex virus type 1 in a latency-associated transcript-independent manner in neuronal cells.

Histone acetylation is implicated in the regulation of herpes simplex virus type 1 (HSV-1) latency. However, the role of histone acetylation in HSV-1 reactivation is less clear. In this study, the well-established model system, quiescently infected, neuronally differentiated PC12 (QIF-PC12) cells, was used to address the participation of histone acetylation in HSV-1 reactivation. In this model, sodium butyrate and trichostatin A (TSA), two histone deacetylase inhibitors, stimulated production of infectious HSV-1 progeny from a quiescent state. To identify viral genes responsive to TSA, the authors analyzed representative α, β, and γ viral genes using quantitative real-time polymerase chain reaction. Only the latency-associated transcript (LAT) accumulated in response to TSA treatment, under culture conditions that restricted virus replication and spread. This led the authors to evaluate the importance of LAT expression on TSA-induced reactivation. In QIF-PC12 cells, the LAT deletion mutant virus dLAT2903 reactivated equivalently with its wild-type parental strain (McKrae) after TSA treatment, as well as forskolin and heat stress treatment. Both viruses also reactivated equivalently from latently infected trigeminal ganglia explants from rabbits. In contrast, there was a marked reduction in the recovery of dLAT2903, as compared to wild-type virus, from the eyes of latently infected rabbits following epinephrine iontophoresis. These combined in vitro, ex vivo, and in vivo data suggest that LAT is not required for reactivation from latently infected neuronal cells per se, but may enhance processes that allow for the arrival of virus at, or close to, the site of original inoculation (i.e., recrudescence).

Effect of Prophylactic Valacyclovir on the Presence of Human Herpesvirus DNA in Saliva of Healthy Individuals after Dental Treatment

ABSTRACT Human herpesviruses (HHVs) are ubiquitous pathogens that intermittently reactivate from latency. Transmission is believed to be facilitated by their frequent appearance in saliva. This study sought to understand the factors that influence the appearance of these viruses in saliva by examining the prevalence, pattern, and quantity of all eight HHVs in saliva of immunocompetent adults with a history of recurrent oral herpes simplex virus (HSV) infections following dental treatment and antiviral therapy. Valacyclovir or matched placebo was given (2 g twice on the day of treatment and 1 g twice the following day) to 125 patients in a randomized, double-blind controlled trial. Saliva, collected on the day of dental treatment and 3 and 7 days later, was analyzed using real-time quantitative PCR. At all visits, HHVs coinfected saliva. Over the course of the week, the DNAs of HHV-6 and HHV-7 were detected significantly more often (97% to 99% of patients) than Epstein-Barr virus (EBV; 64.8%), HSV-1 (13.0%), HHV-8 (3.2%), cytomegalovirus (2.4%), HSV-2 (0%), and varicella-zoster virus (0%), irrespective of drug treatment (P < 0.002). Mean genome copy numbers were highest for HSV-1 and HHV-6. Dental treatment did not influence asymptomatic viral shedding patterns. However, valacyclovir treatment resulted in significantly fewer patients shedding EBV at both postoperative visits compared with placebo (P < 0.008). These results suggest that HHVs are simultaneously present in the saliva of healthy adults at levels that could facilitate transmission, and valacyclovir therapy decreases the prevalence of EBV in saliva but has little effect on HHV-6 and HHV-7.

Establishment of a quiescent herpes simplex virus type 1 infection in neurally-differentiated PC12 cells.

Rat pheochromocytoma (PC12) cells differentiated with nerve growth factor (Nd-PC12) were used to investigate the establishment of a non-productive herpes simplex virus type 1 (HSV-1) infection that is reversible. The results of this work are as follows: (i) Nd-PC12 cultures could be maintained as long term (>7 weeks) non-dividing cultures only when plated on collagen-coated dishes in the absence of serum; (ii) Infection of Nd-PC12 with HSV-1 strains KOS and 17 in the transient presence of acycloguanosine (ACV) resulted in all cultures free of detectable levels of infectious virus at the time of ACV removal and ACV was not needed to maintain the non-productive quiescent state in the subsequent 8 weeks; (iii) These persistently infected and quiescent (QIF)-PC12 cultures demonstrated both spontaneous and forskolin-inducible virus production, at low (5%) and high frequencies (92-100%), respectively during the first 2 weeks post-ACV withdrawal. (iv) In contrast to other in vitro models, HSV-1 failed to reactivate following removal of nerve growth factor. (v) A high percentage of QIF-PC12 cultures (50-100%) produced virus in response to forskolin treatment as long as 7 weeks post-ACV withdrawal. (vi) Expression of HSV-1 productive genes (i.e. alpha0, alpha4, alpha27, UL30 and UL18) dropped precipitously in the presence of ACV and remained undetectable or continued to decline following its removal, whereas the levels of LAT and the host gene G3PDH remained relatively constant throughout the 31 day study period as measured by RT-PCR. These results indicate that QIF-PC12 cells offer a novel, neuronal cell culture system that may enhance our ability to study HSV-1 reactivation from a cryptic, latent-like, non-productive state in the absence of replication inhibitors.

Heat stress activates production of herpes simplex virus type 1 from quiescently infected neurally differentiated PC12 cells

We have previously described a novel in vitro model of a non-productive herpes simplex virus type 1 (HSV-1) infection in neurally differentiated (Nd)-PC12 cells that allows for inducible virus replication upon forskolin treatment. In this study, we further characterized the quiescent state of infection and examined the ability of heat stress (HS) to induce virus from this non-productive state. These studies demonstrated that (i) the quiescent state is characterized by the absence of cell-associated virus, capsids, and viral antigens; (ii) HS (43 degrees C, 3 h) efficiently activated virus from quiescently infected Nd-PC12 (QIF-PC12) cells; (iii) the rate of virus production was significantly greater following HS than forskolin treatment, and the rates of both were dependent on MOI; (iv) forskolin and HS appeared to affect pathways of viral activation from a quiescent state as they did not enhance viral growth in Nd-PC12 cells; (v) viral alpha4 gene and host HSP72 gene transcription were rapidly induced in QIF-PC12 as soon as 3 h post-HS initiation; (vi) induction of the viral alpha27 gene followed that of representative beta and gamma genes, U(L)30 and U(L)18, respectively, and (vii) HS induced asynchronous HSV-1 replication from QIF-PC12 cells with 1:400 to 1:22000 positive foci detected as rapid as 24 h post-induction when established at MOIs of 30 and 3, respectively. These findings provide evidence that alpha4 may be involved in the switch from quiescence to productive infection. Furthermore, this model has the potential to advance our understanding of how HS initiates the HSV-1 productive cycle from a cryptic viral genome.

Real-time polymerase chain reaction to determine the prevalence and copy number of epstein-barr virus and cytomegalovirus DNA in subgingival plaque at individual healthy and periodontal disease sites.

BACKGROUND Detection of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in plaque from patients with periodontal disease provides support for the theory that these viruses play a role in the pathogenesis of periodontitis. This study sought to further define this relationship by determining the prevalence of these viruses at individual disease and healthy sites of patients with periodontal disease and to determine whether the presence and amount of viral DNA correlate with disease severity. METHODS Subgingival plaque from three healthy and three disease sites of 65 patients who had chronic periodontitis were evaluated for the presence and amount of EBV, CMV, and Fusobacterium nucleatum DNA using real-time polymerase chain reaction. Patient serum was evaluated for antibodies against EBV and CMV using enzyme-linked immunosorbent assays. RESULTS EBV DNA was detected in 18.5% of subgingival plaque samples (72/390) and in at least one of the six plaque samples in 44.6% (29/65) of the patients. CMV DNA was detected in one plaque sample (0.3%). EBV was significantly more prevalent in disease sites (28.2%; 55/195) than in healthy sites (8.7%; 17/195; P = 0.002). However, neither EBV prevalence nor its amount correlated with increased probing depth >5 mm or attachment loss >2 mm, whereas the amount of F. nucleatum DNA did. Sites positive for EBV had a median copy number of eight. Antibodies against EBV and CMV were detected in 85.7% and 78.6% of persons evaluated, respectively. CONCLUSION EBV was infrequent and CMV was rarely present in individual subgingival sites affected by chronic periodontitis.

Time- and depth-dependent changes in crosslinking and oxidation of shelf-aged polyethylene acetabular liners

Since crosslinking and oxidation of ultrahigh-molecular-weight polyethylene (UHMWPE) have important roles in determining the wear resistance of UHMWPE total joint components, the time and depth dependence of crosslinking and oxidation of new shelf-aged (2-11 years), ready-to-implant acetabular liners were studied by using solvent extraction and Fourier transform infrared spectroscopy. The ultrastructure of these materials also was examined by using low-voltage scanning electron microscopy in an oil-free vacuum. Oxidation levels increased with time and with depth (p < 0.0001) from the surface of the older liners to a maximum value at about 1-2 mm below the surface, then decreased. They were minimal at the midsection of the liners. The crosslinking of these liners decreased with time and depth (p < 0.0001) and was inversely proportional to the level of oxidation. High and depth-dependent oxidation levels were observed in all older liners made from GUR 415 and 412 resins but were distinctly absent from a comparably aged (i.e., 9 years) liner made from 1900 CM-resin. Some liners showed varying degrees of inhomogeneous and discontinuous morphologic ultrastructure in addition to varying amounts of porosity while others had a more homogeneous ultrastructure. Oxidation and crosslinking of polyethylene are time- and depth-dependent processes that are mutually competitive. We suggest that resin choice and perhaps consolidation-related variables lead to differences in polyethylenes ultrastructure. These ultrastructural differences in polyethylenes inhomogeneities, that is, the type (interconnected or closed-cell) or extent may affect the oxidation resistance of polyethylene. While oxygen diffusion to free radicals in polyethylene already is known to explain some of these time- and depth-dependent effects, perhaps such ultrastructural variations also may facilitate or retard oxygen diffusion in this material. Resin-based ultrastructural variability partially may explain the variability in the clinical performance of polyethylene total joint implant components. Thus resin choice or processing modifications related to polyethylenes ultrastructure may increase its oxidation resistance and ultimately improve the clinical wear performance of polyethylene total joint orthopedic implants.

Salivary levels of Epstein‐Barr virus DNA correlate with subgingival levels, not severity of periodontitis

OBJECTIVE The aim of this study was to determine the presence and quantity of human cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNA in the saliva of patients with periodontitis, and investigate the correlation between these factors. METHODS Presence and amounts of viral DNA in saliva and subgingival plaque samples, from healthy and disease sites, of 65 adults diagnosed with chronic periodontitis were determined using quantitative real-time polymerase chain reaction. RESULTS Epstein-Barr virus DNA was detected in saliva of 81.5% (53/65) of patients at a median concentration of 4325 copies ml(-1). CMV DNA was detected in saliva of one individual (1.5%) at low copy number. Patients who had EBV in saliva were 10 times more likely to have EBV in subgingival plaque than patients lacking EBV in saliva (odds ratio = 10.1, 95% confidence interval = 2.6-39.5; P = 0.0009). EBV DNA burden in saliva positively correlated with the amounts detected in plaque and with amounts detected in increasing number of affected sites (P < 0.0001). EBV DNA presence and quantity in saliva did not correlate with increasing severity of disease as measured by periodontal indices. CONCLUSIONS Epstein-Barr virus DNA presence and burden in saliva are associated with its presence and burden in subgingival plaque, but presence and burden in saliva does not correlate with periodontal disease severity.

Shape and size of virgin ultrahigh molecular weight GUR 4150 HP polyethylene powder.

Sizes and shapes of micron- and submicron-sized structures in four lots of virgin GUR 4150 HP ultrahigh molecular weight polyethylene powder were determined by using low-voltage scanning electron microscopy and image analysis. One thousand two hundred micron-sized virgin powder particles and 1200 of their constituent submicron-sized structures were analyzed. The mean maximum diameter of the micron-sized particles was 81.3 microns, and that of the submicron-sized particles was 0.82 micron. Particle shapes, as determined by the aspect ratio (maximum diameter divided by minimum diameter), were remarkably consistent from lot to lot and between micron- and submicron-sized particles (1.55 versus 1.53, respectively). Significant lot to lot variability was observed in the sizes of the micron-sized particles, and the size distribution of the submicron-sized particles closely follows the size distribution of the submicron-sized particles observed in tissue retrievals. This variability leads to questions about variability in polyethylene quality and in vivo wear performance. Size similarity between the submicron-sized particles retrieved from tissues and that observed in virgin powder supports the hypothesis that polyethylene debris has two origins: particles released from structures retained from the virgin powder, and particles generated de novo by friction and wear.