Antibiotic effects on bacterial profile in osteonecrosis of the jaw.

OBJECTIVE: Oral infection is considered to play a critical role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ), and antibiotic therapy has become a mainstay of BRONJ therapy. This study was aimed to investigate the effect of antibiotics on bacterial diversity in BRONJ tissues. MATERIALS AND METHODS: The bacterial profile from soft tissues associated with the BRONJ lesion was determined using 16S rRNA-based denaturing gradient gel electrophoresis (DGGE) and sequencing. Twenty BRONJ subjects classified as stage 0-2 were enrolled in this study, and patient groups were divided into an antibiotic cohort (n=10) treated with systemic antibiotic and a non-antibiotic cohort (n=10) with no prior antibiotic therapy. RESULTS: The DGGE fingerprints indicated no significant differences in bacterial diversity of BRONJ tissue samples. Patients on antibiotics had higher relative abundance of phylum Firmicutes with bacterial species, Streptococcus intermedius, Lactobacillus gasseri, Mogibacterium timidum, and Solobacterium moorei, whereas patients without antibiotics had greater amounts of Parvimonas micra and Streptococcus anginosus. Thirty percent of bacterial populations were uncultured (yet-to be cultured) phylotypes. CONCLUSION: This study using limited sample size indicated that oral antibiotic therapy may have a limited efficacy on the bacterial population associated with BRONJ lesions.

Optical characterization of melanin.

The optical properties of melanin have been characterized for a number of laser wavelengths in the visible region. The index of refraction of melanin is measured by the conventional method of minimum deviation using a hollow quartz prism at these wavelengths. The inverse adding doubling method based on the diffusion approximation and radiative transport theory have been employed to determine the absorption, scattering, and scattering anisotropy coefficients of melanin from the measurements of diffuse transmission, diffuse reflection and collimated transmission using double integrating spheres. The results obtained by the use of inverse adding doubling method have been compared to the Monte Carlo simulation technique.

A model to evaluate bone substitutes for immediate implant placement.

A calcified alloplast was evaluated as a gap-filling material around implants placed immediately into fresh extraction sockets. Periodontal measurements and computed tomography scans were obtained to evaluate the clinical effectiveness of the alloplast when compared with demineralized freeze-dried bone. To determine whether this alloplast would be a suitable grafting material, 14 patients were selected to evaluate the extraction socket as a model for routine histologic confirmation of the efficacy and biocompatibility of bone substitutes. The results of this study showed the following: (1) human extraction sockets can be models for the study of bone/implant interaction; (2) the alloplast was well tolerated and demonstrated no inflammation through histologic evaluation of core biopsies; (3) the alloplast was a suitable material when used as a gap-filling graft in sockets around immediately placed implants; and (4) dental computed tomography scans and periodontal measurements around grafted implants 6 months after the procedure provide valuable clinical information about graft healing and osteointegration.

Chronic sinusitis complicating sinus lift surgery.

Sinusitis has been reported as a complication of sinus lift surgery with antral bone augmentation. The procedure involves the creation of a submucoperiosteal pocket in the floor of the maxillary sinus for placement of a graft consisting of autogenous, allogenic, or alloplastic material. This can result in inadvertent tearing of the mucoperiosteal flap with extrusion of graft material into the antrum. Obstruction of the sinus outflow tract by mucosal edema and particulate graft material may result in sinusitis. We will discuss the clinical presentation and management of 14 cases of chronic sinusitis following sinus lift surgery with alloplastic hydroxyapatite (HA) augmentation of the maxillary antrum.

Induction of cytotoxic T lymphocyte and antibody responses to enhanced green fluorescent protein following transplantation of transduced CD34(+) hematopoietic cells.

Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However, the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34(+) bone marrow cells transduced with a retroviral vector expressing eGFP. CD34(+) cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow--derived colony-forming units at periods of 5 to 18 weeks after transplantation. However, 5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood, as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted, mediated by CD8(+) lymphocytes, and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34(+) cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34(+) hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol. (Blood. 2001;97:1951-1959)

Erbium: YAG versus holmium:YAG lithotripsy.

PURPOSE: We test the hypothesis that erbium:YAG (Er:YAG) lithotripsy is more efficient than holmium:YAG (Ho:YAG) lithotripsy. MATERIALS AND METHODS: Human calculi composed of greater than 97% calcium oxalate monohydrate and cystine were studied. Calculi were irradiated in water using Er:YAG or Ho:YAG lasers. Er:YAG lithotripsy was done with a 425 microm sapphire optical fiber at a pulse energy of 50 mJ at 10 Hz. Ho:YAG lithotripsy was performed with a 365 microm low hydroxy optical fiber at a pulse energy of 500 mJ at 10 Hz or a 425 microm sapphire optical fiber at a pulse energy of 50 mJ at 10 Hz. Fragmentation was defined as the initial stone mass minus the final dominant fragment mass and normalized for incident laser fluence (energy per unit area of fiber tip). RESULTS: Mean fragmentation plus or minus standard deviation for calcium oxalate monohydrate was 38 +/- 27 mg for Er:YAG and 22 +/- 6 for Ho:YAG (low hydroxy silica fiber) versus 5 +/- 1 for Ho:YAG (sapphire fiber, p = 0.001). When fragmentation was normalized for incident laser fluence given different optical fiber sizes, mean fragmentation efficiency was 53.6 +/- 38.7 g-microm2/J for Er:YAG lithotripsy compared with 22.6 +/- 6.4 for Ho:YAG (low hydroxy silica fiber) lithotripsy (p = 0.04). Mean cystine fragmentation was 15 +/- 3 mg for Er:YAG versus 9 +/- 1 for Ho:YAG (sapphire fiber, p = 0.0005). CONCLUSIONS: Er:YAG lithotripsy is more efficient than Ho:YAG lithotripsy.

In search of Winnicott's aggression.

Going beyond Winnicott's widely known ideas about creativity, in this paper the authors ask why some people are able to live creatively while others suffer recurrent feelings of anger, futility, and depression. Examining Winnicott's reframing of aggression as a life force, it attempts to answer this question by tracing the evolution of his thinking on the nature and origin of aggression. It argues that because he saw aggression as inherent and as central to emotional development, interference in its expression compromises psychic maturation. The paper explores how Winnicott arrived at the conception of a combined love-strife drive and demonstrates that for him, there is no love without aggression, no subject, no object, no reality, and no creativity. That is, for Winnicott, aggression is an achievement that leads to the capacity to live creatively and to experience authenticity. Clinical vignettes illustrate the therapeutic use of these conclusions and their value for psychoanalytic theory.

Selenium and immunocompetence in patients with head and neck cancer.

This randomized double-blind placebo-controlled study aimed to determine whether oral intake of 200 microg/d of sodium selenite, a dose within the safe and adequate daily intake (50-200 microg/d) recommended by the U.S. Food and Nutrition Board, will abrogate depressed or enhance normal-level immune functions of patients receiving therapy for squamous cell carcinoma of the head and neck. Subjects were given one selenium/placebo tablet/d for 8 wk, beginning on the day of their first treatment for the disease (e.g., surgery, radiation, or surgery and radiation) and their immune functions were monitored. Supplementation with selenium (Se) during therapy resulted in a significantly enhanced cell-mediated immunue responsiveness, as reflected in the ability of the patient's lymphocytes to respond to stimulation with mitogen, to generate cytotoxic lymphocytes, and to destroy tumor cells. The enhanced responsiveness was evident during therapy and following conclusion of therapy. In contrast, patients in the placebo arm of the study showed a decline in immune responsiveness during therapy, which was followed, in some patients, by an enhancement, but the responses of the group remained significantly lower than baseline values. The data also show that at baseline, patients entered in the study had significantly lower plasma Se levels than healthy individuals, and patients in stage I or II of disease had significantly higher plasma selenium levels than patients in stage III or IV of disease.

Effective induction of simian immunodeficiency virus-specific systemic and mucosal immune responses in primates by vaccination with proviral DNA producing intact but noninfectious virions.

We report a pilot evaluation of a DNA vaccine producing genetically inactivated simian immunodeficiency virus (SIV) particles in primates, with a focus on eliciting mucosal immunity. Our results demonstrate that DNA vaccines can be used to stimulate strong virus-specific mucosal immune responses in primates. The levels of immunoglobulin A (IgA) detected in rectal secretions of macaques that received the DNA vaccine intradermally and at the rectal mucosa were the most striking of all measured immune responses and were higher than usually achieved through natural infection. However, cytotoxic T lymphocyte responses were generally low and sporadically present in different animals. Upon rectal challenge with cloned SIVmac239, resistance to infection was observed, but some animals with high SIV-specific IgA levels in rectal secretions became infected. Our results suggest that high levels of IgA alone are not sufficient to prevent the establishment of chronic infection, although mucosal IgA responses may have a role in reducing the infectivity of the initial viral inoculum.

Induction of mucosal homing virus-specific CD8(+) T lymphocytes by attenuated simian immunodeficiency virus.

Induction of virus-specific T-cell responses in mucosal as well as systemic compartments of the immune system is likely to be a critical feature of an effective AIDS vaccine. We investigated whether virus-specific CD8(+) lymphocytes induced in rhesus macaques by immunization with attenuated simian immunodeficiency virus (SIV), an approach that is highly effective in eliciting protection against mucosal challenge, express the mucosa-homing receptor alpha4beta7 and traffic to the intestinal mucosa. SIV-specific CD8(+) T cells expressing alpha4beta7 were detected in peripheral blood and intestine of macaques infected with attenuated SIV. In contrast, virus-specific T cells in blood of animals immunized cutaneously by a combined DNA-modified vaccinia virus Ankara regimen did not express alpha4beta7. These results demonstrate the selective induction of SIV-specific CD8(+) T lymphocytes expressing alpha4beta7 by a vaccine approach that replicates in mucosal tissue and suggest that induction of virus-specific lymphocytes that are able to home to mucosal sites may be an important characteristic of a successful AIDS vaccine.

Laser lithotripsy and cyanide.

BACKGROUND AND PURPOSE: Holmium:YAG lithotripsy of uric acid calculi produces cyanide. The laser and stone parameters required to produce cyanide are poorly defined. In this study, we tested the hypotheses that cyanide production: (1) varies with holmium:YAG power settings; (2) varies among holmium:YAG, pulsed-dye, and alexandrite lasers; and (3) occurs during holmium:YAG lithotripsy of all purine calculi. MATERIALS AND METHODS: Holmium:YAG lithotripsy of uric acid calculi was done using various optical fiber diameters (272-940 microm) and pulse energies (0.5-1.5 J) for constant irradiation (0.25 kJ). Fragmentation and cyanide were quantified. Cyanide values were divided by fragmentation values, and fragment sizes were characterized. To test the second hypothesis, uric acid calculi were irradiated with Ho:YAG, pulsed-dye, and alexandrite lasers. Fragmentation and cyanide were measured, and cyanide per fragmentation was calculated. Fragment sizes were characterized. Finally, Ho:YAG lithotripsy (0.25 kJ) of purine and nonpurine calculi was done, and cyanide production was measured. RESULTS: Fragmentation increased as pulse energy increased for the 550- and 940-microm optical fibers (P < 0.05). Cyanide increased as pulse energy increased for all optical fibers (P < 0.002). Cyanide per fragmentation increased as pulse energy increased for the 272-microm optical fiber (P = 0.03). Fragment size increased as pulse energy increased for the 272-microm, 550-microm, and 940-microm optical fibers (P < 0.001). The mean cyanide production from 0.25 kJ of optical energy was Ho:YAG laser 106 microg, pulsed-dye 55 microm, and alexandrite 1 microg (P < 0.001). The mean cyanide normalized for fragmentation (microg/mg) was 1.18, 0.85, and 0.02, respectively (P < 0.001). The mean fragment size was 0.6, 1.1, and 1.9 mm, respectively (P < 0.001). After 0.25 kJ, the mean amount of cyanide produced was monosodium urate stones 85 microg, uric acid 78 microg, xanthine 17 microg, ammonium acid urate 16 microg, calcium phosphate 8 microg, cystine 7 microg, and struvite 4 microg (P < 0.001). CONCLUSIONS: Cyanide production varies with Ho:YAG pulse energy. To minimize cyanide and fragment size, Ho:YAG lasertripsy is best done at a pulse energy < or = 1.0 J. Cyanide production from laser lithotripsy of uric acid calculi varies among Ho:YAG, pulsed-dye, and alexandrite lasers and is related to pulse duration. Cyanide is produced by Ho:YAG lasertripsy of all purine calculi.

Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation.

PURPOSE: This study compared two methods of preparing platelet-rich plasma (PRP) gel and the levels of PDGF and TGFbeta in each preparation. MATERIALS AND METHODS: Platelet-rich plasma gel was prepared by centrifugation and clotted using the ITA gelling agent (Natrex Technologies Inc, Greenville, NC) or by the addition of thrombin and calcium chloride. The levels of platelet-derived growth factor (PDGF) and transforming growth factor beta (TGFbeta) generated by clot formation were assayed by enzyme-linked immunoassay (ELISA). RESULTS: Both methods of preparation yielded PRP gel in less than 30 minutes. However, the ITA preparation did not require thrombin to achieve adequate gel formation. The levels of PDGF and TGFbeta were similar regardless of which method was used for initiation of clot formation. CONCLUSION: Use of ITA for gel preparation is equivalent to using calcium chloride and thrombin, without the need for special equipment and the risk of coagulopathy.

Holmium: YAG lithotripsy: optimal power settings.

PURPOSE: We tested the hypothesis that holmium:YAG laser lithotripsy speed is best maximized by using low pulse energy at high pulse frequency. MATERIALS AND METHODS: To demonstrate that optical fiber damage increases with pulse energy and irradiation, the 365-microm optical fiber irradiated calcium hydrogen phosphate dihydrate (CHPD), calcium oxalate monohydrate (COM), cystine, magnesium ammonium phosphate hexahydrate (MAPH), and uric acid calculi at pulse energies of 0.5 to 2.0 J. Optical energy output was measured with an energy detector after 10 J to 200 J of total energy. To demonstrate that lithotripsy efficiency varies with power, fragmentation was measured at constant power settings at total energies of 200 J and 1 kJ with the 365-microm optical fiber. Fragmentation was measured for the 272-microm optical fiber at pulse energies of 0.5 J to 1.5 J at 10 Hz. To demonstrate that low pulse energy produces smaller fragments than high pulse energy, fragment size was characterized for COM and uric acid calculi after 0.25 kJ of irradiation using the 272-microm to 940-microm optical fibers at 0.5 J to 1.5 J. RESULTS: Damage to the 365-microm optical fiber was greatest for irradiation of CHPD, followed by MAPH, and COM (P<0.001). There was no significant optical fiber damage after cystine and uric acid lithotripsy. For the 365-microm optical fiber and CHPD, fragmentation after 200 J was greatest for pulse energies < or =1.0 J (P< 0.001). For other compositions, fragmentation was not statistically different among the power settings for constant irradiation. No significant difference was noted in fragmentation for any composition at different pulse energies (1.0 v. 2.0 J) for 1-kJ irradiation. However, for all compositions, the calculated lithotripsy speed was greatest at high power settings (P<0.001). For the 272-microm optical fiber, CHPD fragmentation was greatest for the 1.0-J pulse energy. The mean fragment size and relative quantity of fragments > or =2 mm both increased as pulse energy increased. CONCLUSIONS: Optical fiber degradation varies with stone composition, irradiation, and pulse energy. Holmium:YAG lithotripsy speed is maximized with higher power (either increased pulse energy or higher pulse frequency). Because low pulse energy may be safer and yields smaller fragments than high pulse energy, holmium:YAG lithotripsy speed is best increased by using pulse energies < or =1.0 J at a high repetition rate.

Characterization of SIV-specific CD4+ T-helper proliferative responses in macaques immunized with live-attenuated SIV.

Analysis of immune responses generated by live-attenuated simian immunodeficiency virus (SIV) strains may provide clues to the mechanisms of protective immunity induced by this approach. We examined SIV-specific T-helper responses in macaques immunized with the live-attenuated SIV strains SIVmac239deltanef and SIVmac239delta3. Optimization of the concentration and duration of antigenic stimulation resulted in the detection of relatively strong SIV-specific proliferative responses, with peak stimulation indices of up to 84. SIV-specific proliferative responses were mediated by CD4+ T cells and were major histocompatibility (MHC) class II restricted. Limiting dilution analysis revealed SIV-specific T-helper precursor frequencies of up to 96 per 10(6) peripheral blood mononuclear cells (PBMC). Intracellular flow-cytometric analysis demonstrated the production of interleukin (IL)-2, interferon (IFN)-gamma, RANTES and macrophage inhibitory protein-1alpha (MIP-1alpha) by T lymphocytes from SIVmac239deltanef-vaccinated animals following SIV p55 stimulation. Induction of strong SIV-specific T-helper responses by live-attenuated SIV vaccines may play a role in their ability to induce protective immunity.

Immunization with live attenuated simian immunodeficiency virus induces strong type 1 T helper responses and beta-chemokine production.

Immunization with live attenuated simian immunodeficiency virus (SIV) strains has proved to be one of the most effective strategies to induce protective immunity in the SIV/macaque model. To better understand the role that CD4(+) T helper responses may play in mediating protection in this model, we characterized SIV-specific proliferative and cytokine responses in macaques immunized with live attenuated SIV strains. Macaques chronically infected with live attenuated SIV had strong proliferative responses to SIV proteins, with stimulation indices of up to 74. The magnitude of the proliferative response to SIV Gag varied inversely with the degree of attenuation; Gag-specific but not envelope-specific responses were lower in animals infected with more highly attenuated SIV strains. SIV-specific stimulation of lymphocytes from vaccinated macaques resulted in secretion of interferon-gamma, IL-2, regulated-upon-activation, normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta but not IL-4 or IL-10. Intracellular flow cytometric analysis documented that, in macaques vaccinated with SIVmac239Deltanef, up to 2% of all CD4(+)T cells were specific for SIV p55. The ability of live attenuated SIV to induce a strong, sustained type 1 T helper response may play a role in the success of this vaccination approach to generate protection against challenge with wild-type SIV.

Proteus mirabilis viability after lithotripsy of struvite calculi.

PURPOSE: We tested the hypotheses that Proteus mirabilis viability of struvite calculi differs after exposure to different lithotripsy modalities and that the photothermal mechanism of holmium:YAG lithotripsy is antibacterial. MATERIALS AND METHODS: Human calculi of known struvite composition (greater than 90% magnesium ammonium phosphate hexohydrate) were incubated with P. mirabilis. Calculi were randomly distributed and fragmented with no lithotripsy (controls), or shock wave, intracorporeal ultrasonic, electrohydraulic, pneumatic, holmium:YAG or pulsed dye laser lithotripsy. After lithotripsy fragments were sonicated and specimens were serially plated for 48 hours at 38C. Bacterial counts and the rate of bacterial sterilization were compared. RESULTS: Median bacterial counts (colony-forming units per ml.) were 8 x 10(6) in controls and 3 x 10(6) in shock wave, 3 x 10(7) in ultrasonic, 4 x 10(5) in electrohydraulic, 8 x 10(6) in pneumatic, 5 x 10(4) in holmium:YAG and 1 x 10(6) in pulsed dye laser lithotripsy cases (p <0.001). The rate of bacterial sterilization was 50% for holmium:YAG lithotripsy treated stones versus 0% for each of the other cohorts (p <0.01). CONCLUSIONS: P. mirabilis viability varies among lithotrites. The photothermal mechanism of holmium:YAG lithotripsy is antibacterial.

Efficient and durable gene marking of hematopoietic progenitor cells in nonhuman primates after nonablative conditioning.

Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 microgram/kg) and G-CSF (20 microgram/kg) for 7 days and autologous CD34(+) peripheral blood stem cells harvested by leukapheresis. CD34(+) cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% +/- 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% +/- 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naïve (CD45RA(+) and CD62L(+)) CD4(+) and CD8(+) cells were the predominant phenotype of the marked CD3(+) T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naïve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.

Inhibition of antigen presentation by the glycine/alanine repeat domain is not conserved in simian homologues of Epstein-Barr virus nuclear antigen 1.

Most humans and Old World nonhuman primates are infected for life with Epstein-Barr virus (EBV) or closely related gammaherpesviruses in the same lymphocryptovirus (LCV) subgroup. Several potential strategies for immune evasion and persistence have been proposed based on studies of EBV infection in humans, but it has been difficult to test their actual contribution experimentally. Interest has focused on the EBV nuclear antigen 1 (EBNA1) because of its essential role in the maintenance and replication of the episomal viral genome in latently infected cells and because EBNA1 endogenously expressed in these cells is protected from presentation to the major histocompatibility complex class-I restricted cytotoxic T-lymphocyte (CTL) response through the action of an internal glycine-alanine repeat (GAR). Given the high degree of biologic conservation among LCVs which infect humans and Old World primates, we hypothesized that strategies essential for viral persistence would be well conserved among viruses of this subgroup. We show that the rhesus LCV EBNA1 shares sequence homology with the EBV and baboon LCV EBNA1 and that the rhesus LCV EBNA1 is a functional homologue for EBV EBNA1-dependent plasmid maintenance and replication. Interestingly, all three LCVs possess a GAR domain, but the baboon and rhesus LCV EBNA1 GARs fail to inhibit antigen processing and presentation as determined by using three different in vitro CTL assays. These studies suggest that inhibition of antigen processing and presentation by the EBNA1 GAR may not be an essential mechanism for persistent infection by all LCV and that other mechanisms may be important for immune evasion during LCV infection.