Epstein-Barr virus infection of Langerhans cell precursors as a mechanism of oral epithelial entry, persistence, and reactivation.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with many malignant and nonmalignant human diseases. Life-long latent EBV persistence occurs in blood-borne B lymphocytes, while EBV intermittently productively replicates in mucosal epithelia. Although several models have previously been proposed, the mechanism of EBV transition between these two reservoirs of infection has not been determined. In this study, we present the first evidence demonstrating that EBV latently infects a unique subset of blood-borne mononuclear cells that are direct precursors to Langerhans cells and that EBV both latently and productively infects oral epithelium-resident cells that are likely Langerhans cells. These data form the basis of a proposed new model of EBV transition from blood to oral epithelium in which EBV-infected Langerhans cell precursors serve to transport EBV to the oral epithelium as they migrate and differentiate into oral Langerhans cells. This new model contributes fresh insight into the natural history of EBV infection and the pathogenesis of EBV-associated epithelial disease.

Prevalence and risk factors for polyomavirus reactivation in solid-organ transplantation

Background. Polyomavirus reactivation is common in solid-organ transplant recipients who are given immunosuppressive medications as standard treatment of care. Previous studies have shown that polyomavirus infection can lead to allograft failure in as many as 45% of the affected patients. Hypothesis. Ubiquitous polyomaviruses when reactivated by post-transplant immunosuppressive medications may lead to impaired renal function and possibly lower survival prospects. Study Overview. Secondary analysis of data was conducted on a prospective longitudinal study of subjects who were at least 18 years of age and were recipients of liver and/or kidney transplant at Mayo Clinic Scottsdale, Arizona. Methods. DNA extractions of blinded urine and blood specimens of transplant patients collected at Mayo Clinic during routine transplant patient visits were performed at Baylor College of Medicine using Qiagen kits. Virologic assays included testing DNA samples for specific polyomavirus sequences using QPCR technology. De-identified demographic and clinical patient data were merged with laboratory data and statistical analysis was performed using Stata10. Results. 76 patients enrolled in the study were followed for 3.9 years post transplantation. The prevalence of BK virus and JC virus urinary excretion was 30% and 28%. Significant association was observed between JC virus excretion and kidney as the transplanted organ (P = 0.039, Pearson Chi-square test). The median urinary JCV viral loads were two logs higher than those of BKV. Patients that excreted both BKV and JCV appeared to have the worst renal function with a mean creatinine clearance value of 71.6 millimeters per minute. A survival disadvantage was observed for dual shedders of BKV and JCV, log-rank statistics, p = 0.09; 2/5 dual-shedders expired during the study period. Liver transplant and male sex were determined to be potential risk factors for JC virus activation in renal and liver transplant recipients. All patients tested negative for SV40 and no association was observed between polyomavirus excretion and type of immunosuppressive medication (tacrolimus, mycophenolate mofetil, cyclosporine and sirolimus). Conclusions. Polyomavirus reactivation was common after solid-organ transplantation and may be associated with impaired renal function. Male sex and JCV infection may be potential risk factors for viral reactivation; findings should be confirmed in larger studies.^

Mechanism of methylation gene reactivation in human cancer cells

Epigenetic silencing of tumor suppressor genes by DNA hypermethylation at promoter regions is a common event in carcinogenesis and tumor progression. Abrogation of methylation and reversal of epigenetic silencing is a very potent way in cancer treatment. However, the reactivation mechanisms are poorly understood. In this study, we first developed a cell line model system named YB5, derived from SW48 cancer cell line, which bears one copy of stably integrated EGFP gene on Chromosome 1p31.1 region. The GFP gene expression is transcriptionally silenced due to the hypermethylated promoter CMV. However, the GFP expression can be restored using demethylating agent 5-aza-2' deoxycytidine (DAC), and detected by FACS and fluorescent microscopy. Using this system, we observed the heterogeneous reactivation induced by DAC treatment. After flow sorting, GFP negative cells exhibited similar level of incomplete demethylation compared to GFP positive cells on repetitive LINE1 element, tumor suppressor genes such as P16, CDH13, and RASSF1a, and CMV promoter as well. However, the local chromatin of CMV-GFP locus altered to an open structure marked by high H3 lysine 9 acetylation and low H3 lysine 27 tri-methylation in GFP positive cells, while the GFP negative cells retained mostly the original repressive marks. Thus, we concluded that DAC induced DNA hypomethylation alone does not directly determine the level of re-expression, and the resetting of the local chromatin structure under hypomethylation environment is required for gene reactivation. Besides, a lentivirus vector-based shRNA screening was performed using the YB5 system. Although it is the rare chance that vector lands in the neighboring region of GFP, we found that the exogenous vector DNA inserted into the upstream region of GFP gene locus led to the promoter demethylation and reactivated the silenced GFP gene. Thus, epigenetic state can be affected by changing of the adjacent nucleic acid sequences. Further, this hypermethylation silenced system was utilized for epigenetic drug screening. We have found that DAC combined with carboplatin would enhance the GFP% yield and increase expression of other tumor suppressor genes than DAC alone, and this synergistic effect may be related to DNA repair process. In summary, these studies reveal that reversing of methylation silencing requires coordinated alterations of DNA methylation, chromatin structure, and local microenvironment. ^

Humoral immunity in tuberculin skin test anergy and its role in high-risk persons exposed to active tuberculosis.

The most common test to identify latent tuberculosis is the tuberculin skin test that detects T cell responses of delayed type hypersensitivity type IV. Since it produces false negative reactions in active tuberculosis or in high-risk persons exposed to tuberculosis patients as shown in this report, we studied antibody profiles to explain the anergy of such responses in high-risk individuals without active infection. Our results showed that humoral immunity against tuberculin, regardless of the result of the tuberculin skin test is important for protection from active tuberculosis and that the presence of high antibody titers is a more reliable indicator of infection latency suggesting that latency can be based on the levels of antibodies together with in vitro proliferation of peripheral blood mononuclear cells in the presence of the purified protein derivative. Importantly, anti-tuberculin IgG antibody levels mediate the anergy described herein, which could also prevent reactivation of disease in high-risk individuals with high antibody titers. Such anti-tuberculin IgG antibodies were also found associated with blocking and/or stimulation of in vitro cultures of PBMC with tuberculin. In this regard, future studies need to establish if immune responses to Mycobacterium tuberculosis can generate a broad spectrum of reactions either toward Th1 responses favoring stimulation by cytokines or by antibodies and those toward diminished responses by Th2 cytokines or blocking by antibodies; possibly involving mechanisms of antibody dependent protection from Mtb by different subclasses of IgG.

MECHANISMS OF SPECIES DIFFERENCES IN BRAIN ACETYLCHOLINESTERASE SENSITIVITY TO ORGANOPHOSPHATE INHIBITION

In vitro incubation of acetylcholinesterase from brain tissue of several species with organophosphate compounds indicated that the concentrations required to inhibit 50% of acetylcholinesterase activity (IC(,50)) differed from species to species for the same compound (Murphy, et al., 1968; Andersen, et al., 1972, 1977 and 1978).^ The hypothesis that non-specific binding proteins (Lauwerys and Murphy, 1969a,b) exerts a protective effect on acetylcholinesterase, and thus cause the differences observed in IC(,50) studies was tested by a ('3)H-DFP binding experiment. It was found that differences in the amount of non-specific binding protein cannot explain the observed differences observed in IC(,50) studies.^ An alternative hypothesis, that acetylcholinesterase from different species have different affinities for binding and/or different rates of phosphorylation by organophosphate insecticides was tested by determining the apparent affinity constant (k(,a)) and apparent rate of phosphorylation (k(,p)). Kinetic studies indicated that acetylcholinesterases from different species have different sensitivities to inhibition by organophosphate insecticides, and the differences are due to different affinities for binding and/or different rates of phosphorylation by the same organophosphate compound.^ Studies of the spontaneous reactivation of acetylcholinesterase after inhibition by organophosphate insecticides also indicated that acetylcholinesterases from different species have different rates and extents of spontaneous reactivation. This further substantiates the hypothesis that acetylcholinesterases from different species have different kinetic characteristics with respect to organophosphate insecticides inhibition.^ Eleven paraoxon analogs were synthesized for a quantitative structure-activity relationship study. It was found that the electron-withdrawing power ((sigma)) and hydrophobicity ((PARAGR)) of the substituent are important in determining the anti-cholinesterase activity of paraoxon analogs. Thus, predictions of species differences in acetylcholinesterase sensitivities to paraoxon analogs can be made if the physicochemical parameters ((sigma) and (PARAGR)) of the substituents are known.^ In another approach, i.e. enzyme modeling, the sensitivity of rat brain acetylcholinesterase to organophosphate insecticides was used as the independent variable to predict the sensitivities of acetylcholinesterases from other species to the same compound. Regression equations were derived for each species based on nineteen organophosphate insecticides studied. It was found, that in addition to paraoxon analogs, this method is also applicable to other organophosphate compounds with wide variations in structure. Thus, the sensitivities of acetylcholinesterases from other species can also be predicted from the sensitivity of rat brain acetylcholinesterase. ^

Spatial and temporal aspects of synaptic plasticity

The notion that changes in synaptic efficacy underlie learning and memory processes is now widely accepted even if definitive proof of the synaptic plasticity and memory hypothesis is still lacking. When learning occurs, patterns of neural activity representing the occurrence of events cause changes in the strength of synaptic connections within the brain. Reactivation of these altered connections constitutes the experience of memory for these events and for other events with which they may be associated. These statements summarize a long-standing theory of memory formation that we refer to as the synaptic plasticity and memory hypothesis. Since activity-dependent synaptic plasticity is induced at appropriate synapses during memory formation, and is both necessary and sufficient for the information storage, we can speculate that a methodological study of the synapse will help us understand the mechanism of learning. Random events underlie a wide range of biological processes as diverse as genetic drift and molecular diffusion, regulation of gene expression and neural network function. Additionally spatial variability may be important especially in systems with nonlinear behavior. Since synapse is a complex biological system we expect that stochasticity as well as spatial gradients of different enzymes may be significant for induction of plasticity. ^ In that study we address the question "how important spatial and temporal aspects of synaptic plasticity may be". We developed methods to justify our basic assumptions and examined the main sources of variability of calcium dynamics. Among them, a physiological method to estimate the number of postsynaptic receptors as well as a hybrid algorithm for simulating postsynaptic calcium dynamics. Additionally we studied how synaptic geometry may enhance any possible spatial gradient of calcium dynamics and how that spatial variability affect plasticity curves. Finally, we explored the potential of structural synaptic plasticity to provide a metaplasticity mechanism specific for the synapse. ^

The Influence of Immunization Route on the Adjuvant Effect of alpha-Galactosylceramide

Potent vaccine formulations ideally include adjuvants to activate innate immune responses and enhance antigen-specific adaptive immunity. The synthetic glycolipid alpha-Galactosylceramide (α-GalCer) effectively activates the innate immune mediating NKT cells to produce cytokines and activate downstream immune cells, resulting in development of humoral and cell mediated immune responses to co-administered antigens. While a single intravenous immunization of α-GalCer strongly activates NKT cells, multiple doses by this route are well documented to induce anergy in NKT cells. Anergy is defined as the deficiency in NKT proliferation and cytokine production, including IL-4 and IFNγ. However, our studies have shown that two doses of α-GalCer administered intranasally by the intranasal route leads to reactivation of NKT cells and improved adaptive immune responses after each subsequent dose. I therefore investigated the role of multiple routes of immunization in activation of NKT cells, i.e. anergy versus repeated activation. Specifically, I hypothesized that the differential capacity of NKT cells to produce IFNγ, as a result of route of immunization with α-GalCer, influences the induction of adaptive immune responses to co-administered antigen. Our experimental design utilizes the observation that intranasal immunization primarily induces immune responses in the lungs while intravenous immunization induces responses in the liver. Using intracellular cytokine staining for IFNγ production and Elispot analyses for determining NKT and T cell activation, respectively, it was determined that administering two consecutive intravenous doses resulted in anergy to NKT cells (no IFNγ production) in the liver and lack of adaptive immunity while second immunization by the intranasal route overcame anergy in the lung. The outcome in the other tissues analyzed was mixed and could be the result of tissue microenvironment among others possible reasons. When intranasal dosing preceded systemic, NKT cells were reactivated to produce IFNγ and induced positive adaptive immune responses in the responding lung tissue. These results indicate that the mechanism by which mucosal and systemic immunization routes activate NKT cells may differ in that there is a differential tissue-specific effect induced by each route. Future studies are necessary to determine the reason for these tissue-specific effects and how they relate to NKT cell activation.

Characterization of Jak, STAT, and Src interactions in Head and Neck Squamous Cell Carcinoma

Recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is common; thus, it is essential to improve the effectiveness and reduce toxicity of current treatments. Proteins in the Src/Jak/STAT pathway represent potential therapeutic targets, as this pathway is hyperactive in HNSCC and it has roles in cell migration, metastasis, proliferation, survival, and angiogenesis. During short-term Src inhibition, Janus kinase (Jak) 2, and signal transducer and activator of transcription (STAT) 3 and STAT5 are dephosphorylated and inactivated. Following sustained Src inhibition, STAT5 remains inactive, but Jak2 and STAT3 are reactivated following their early inhibition. To further characterize the mechanism of this novel feedback pathway we performed several experiments to look at the interactions between Src, Jak2, STAT5 and STAT3. We attempted to develop a non-radioactive kinase assay using purified recombinant Jak2 and Src proteins, but found that phospho-tyrosine antibodies were non-specifically binding to purified recombinant proteins. We then performed in vitro kinase assays (IVKAs) using purified recombinant Jak2, Src, STAT3, and STAT5 proteins with and without Src and Jak2 pharmacologic inhibitors. We also examined the interactions of these proteins in intact HNSCC cells. We found that recombinant Jak2, STAT3, and STAT5 are direct substrates of Src and that recombinant Src, STAT3, and STAT5 are direct substrates of Jak2 in the IVKA. To our knowledge, the finding that Src is a Jak substrate is novel and has not been shown before. In intact HNSCC cells we find that STAT3 can be reactivated despite continuous Src inhibition and that STAT5 continues to be inhibited despite Jak2 reactivation. Also, Jak2 inhibition did not affect Src or STAT5 activity but it did cause STAT3 inhibition. We hypothesized that the differences between the intact cells and the IVKA assays were due to a potential need for binding partners in intact HNSCC cells. One potential binding partner that we examined is the epidermal growth factor receptor (EGFR). We found that EGFR activation caused increased activation of Src and STAT5 but not Jak2. Our results demonstrate that although STAT3 and STAT5 are capable of being Src and Jak2 substrates, in intact HNSCC cells Src predominantly regulates STAT5 and Jak2 regulates STAT3. Regulation of STAT5 by Src may involve interactions between Src and EGFR. This knowledge along with future studies will better define the mechanisms of STAT regulation in HNSCC cells and ultimately result in an ideal combination of therapeutic agents for HNSCC.