Peak Of Latency Research Articles

T Cell-Specific STAT1 Expression Promotes Lytic Replication and Supports the Establishment of Gammaherpesvirus Latent Reservoir in Splenic B Cells.

ABSTRACTGammaherpesviruses establish lifelong infections in most vertebrate species, including humans and rodents, and are associated with cancers, including B cell lymphomas. While type I and II interferon (IFN) systems of the host are critical for the control of acute and chronic gammaherpesvirus infection, the cell type-specific role(s) of IFN signaling during infection is poorly understood and is often masked by the profoundly altered viral pathogenesis in the hosts with global IFN deficiencies. STAT1 is a critical effector of all classical IFN responses along with its involvement in other cytokine signaling pathways. In this study, we defined the effect of T cell-specific STAT1 deficiency on the viral and host parameters of infection with murine gammaherpesvirus 68 (MHV68). MHV68 is a natural rodent pathogen that, similar to human gammaherpesviruses, manipulates and usurps B cell differentiation to establish a lifelong latent reservoir in B cells. Specifically, germinal center B cells host the majority of latent MHV68 reservoir in the lymphoid organs, particularly at the peak of viral latency. Unexpectedly, T cell-specific STAT1 expression, while limiting the overall expansion of the germinal center B cell population during chronic infection, rendered these B cells more effective at hosting the latent virus reservoir. Further, T cell-specific STAT1 expression in a wild type host limited circulating levels of IFNγ, with corresponding increases in lytic MHV68 replication and viral reactivation. Thus, our study unveils an unexpected proviral role of T cell-specific STAT1 expression during gammaherpesvirus infection of a natural intact host.

Circadian learning and memory changes in Aβ1-42 induced Alzheimer's mice.

Alzheimer disease (AD) is a growing health problem globally, which causes a progressive decline in learning and memory and multiple disturbances of circadian rhythms. Six Alzheimer's mice and six wild type (WT) mice were involved in this study. Morris Water Maze (MWM) tasks were conducted hourly to evaluate their circadian learning and memory performance. We used a single cosinor-based method to evaluate the circadian learning and memory of Alzheimer's mice and WT mice, respectively. An area sensor was used to record locomotor activity for 2weeks continuously, including 7days of 12h light/12h dark (LD) conditions and 7days of 12h dark/12h dark (DD) conditions. All WT mice showed circadian rhythm presence in learning and memory, and the peak of escape latency appeared at circadian time (CT) 12. Only one in six Alzheimer's mice showed a circadian rhythm, but the peak of escape latency was postponed to CT20. Alzheimer's mice showed rhythm absence under LD or DD conditions. Under LD conditions, the WT mice activity was higher than that in the Alzheimer's mice during ZT0-5 (p = 0.007) and ZT18-23 (p = 0.353) but lower during ZT6-11 (p < 0.001) and ZT12-17 (p < 0.001). Learning and memory of wild type mice is proved to have a circadian variation throughout a day. In Alzheimer's mice, rhythmic locomotor activity and circadian learning and memory performance were disrupted. Understanding the role of rhythmic disturbances in the process of AD may assist to identify therapeutic targets.

Murine Gammaherpesvirus M2 Protein Induction of IRF4 via the NFAT Pathway Leads to IL-10 Expression in B Cells

Reactivation of the gammaherpesviruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) from latently infected B cells has been linked to plasma cell differentiation. We have previously shown that the MHV68 M2 protein is important for virus reactivation from B cells and, when expressed alone in primary murine B cells, can drive B cell differentiation towards a pre-plasma cell phenotype. In addition, expression of M2 in primary murine B cells leads to secretion of high levels of IL-10 along with enhanced proliferation and survival. Furthermore, the absence of M2 in vivo leads to a defect in the appearance of MHV68 infected plasma cells in the spleen at the peak of MHV68 latency. Here, employing an inducible B cell expression system, we have determined that M2 activates the NFAT pathway in a Src kinase-dependent manner – leading to induction of the plasma cell-associated transcription factor, Interferon Regulatory Factor-4 (IRF4). Furthermore, we show that expression of IRF4 alone in a B cell line up-regulates IL-10 expression in culture supernatants, revealing a novel role for IRF4 in B cell induced IL-10. Consistent with the latter observation, we show that IRF4 can regulate the IL-10 promoter in B cells. In primary murine B cells, addition of cyclosporine (CsA) resulted in a significant decrease in M2-induced IL-10 levels as well as IRF4 expression, emphasizing the importance of the NFAT pathway in M2- mediated induction of IL-10. Together, these studies argue in favor of a model wherein M2 activation of the NFAT pathway initiates events leading to increased levels of IRF4 – a key player in plasma cell differentiation – which in turn triggers IL-10 expression. In the context of previous findings, the data presented here provides insights into how M2 facilitates plasma cell differentiation and subsequent virus reactivation.

Tracking Murine Gammaherpesvirus 68 Infection of Germinal Center B Cells In Vivo

Infection of mice with murine gammaherpesvirus 68 (MHV68) provides a tractable small animal model to study various aspects of persistent gammaherpesvirus infection. We have previously utilized a transgenic MHV68 that expresses enhanced yellow fluorescent protein (EYFP) to identify infected cells. While this recombinant MHV68 has been useful for identifying infected cell populations by flow cytometry, it has been suboptimal for identification of infected cells in tissue sections due to the high solubility of EYFP. Efficient detection of EYFP expressed from the MHV68 genome in tissue sections requires fixation of whole organs prior to sectioning, which frequently leads to over-fixation of some cellular antigens precluding their detection. To circumvent this issue, we describe the generation and characterization of a transgenic MHV68 harboring a fusion gene composed of the EYFP coding sequence fused to the histone H2B open reading frame. Because the H2bYFP fusion protein is tightly bound in nucleosomes in the nucleus it does not freely diffuse out of unfixed tissue sections, and thus eliminates the need for tissue fixation. We have used the MHV68-H2bYFP recombinant virus to assess the location and distribution of virus infected B cells in germinal centers during the peak of MHV68 latency in vivo. These analyses show that the physical location of distinct populations of infected germinal center B cells correlates well with their surface phenotype. Furthermore, analysis of the distribution of virus infection within germinal center B cell populations revealed that ca. 70% of MHV68 infected GC B cells are rapidly dividing centroblasts, while ca. 20% have a clear centrocyte phenotype. Finally, we have shown that marking of infected cells with MHV68-H2bYFP is extended long after the onset of latency – which should facilitate studies to track MHV68 latently infected cells at late times post-infection.

Gammaherpesvirus-Driven Plasma Cell Differentiation Regulates Virus Reactivation from Latently Infected B Lymphocytes

Gammaherpesviruses chronically infect their host and are tightly associated with the development of lymphoproliferative diseases and lymphomas, as well as several other types of cancer. Mechanisms involved in maintaining chronic gammaherpesvirus infections are poorly understood and, in particular, little is known about the mechanisms involved in controlling gammaherpesvirus reactivation from latently infected B cells in vivo. Recent evidence has linked plasma cell differentiation with reactivation of the human gammaherpesviruses EBV and KSHV through induction of the immediate-early viral transcriptional activators by the plasma cell-specific transcription factor XBP-1s. We now extend those findings to document a role for a gammaherpesvirus gene product in regulating plasma cell differentiation and thus virus reactivation. We have previously shown that the murine gammaherpesvirus 68 (MHV68) gene product M2 is dispensable for virus replication in permissive cells, but plays a critical role in virus reactivation from latently infected B cells. Here we show that in mice infected with wild type MHV68, virus infected plasma cells (ca. 8% of virus infected splenocytes at the peak of viral latency) account for the majority of reactivation observed upon explant of splenocytes. In contrast, there is an absence of virus infected plasma cells at the peak of latency in mice infected with a M2 null MHV68. Furthermore, we show that the M2 protein can drive plasma cell differentiation in a B lymphoma cell line in the absence of any other MHV68 gene products. Thus, the role of M2 in MHV68 reactivation can be attributed to its ability to manipulate plasma cell differentiation, providing a novel viral strategy to regulate gammaherpesvirus reactivation from latently infected B cells. We postulate that M2 represents a new class of herpesvirus gene products (reactivation conditioners) that do not directly participate in virus replication, but rather facilitate virus reactivation by manipulating the cellular milieu to provide a reactivation competent environment.

Identification of Infected B-Cell Populations by Using a Recombinant Murine Gammaherpesvirus 68 Expressing a Fluorescent Protein

Infection of inbred mice with murine gammaherpesvirus 68 (MHV68) has proven to be a powerful tool to study gammaherpesvirus pathogenesis. However, one of the limitations of this system has been the inability to directly detect infected cells harvested from infected animals. To address this issue, we generated a transgenic virus that expresses the enhanced yellow fluorescent protein (YFP), driven by the human cytomegalovirus immediate-early promoter and enhancer, from a neutral locus within the viral genome. This virus, MHV68-YFP, replicated and established latency as efficiently as did the wild-type virus. During the early phase of viral latency, MHV68-YFP efficiently marked latently infected cells in the spleen after intranasal inoculation. Staining splenocytes for expression of various surface markers demonstrated the presence of MHV68 in distinct populations of splenic B cells harboring MHV68. Notably, these analyses also revealed that markers used to discriminate between newly formed, follicular and marginal zone B cells may not be reliable for phenotyping B cells harboring MHV68 since virus infection appears to modulate cell surface expression levels of CD21 and CD23. However, as expected, we observed that the overwhelming majority of latently infected B cells at the peak of latency exhibited a germinal center phenotype. These analyses also demonstrated that a significant percentage of MHV68-infected splenocytes at the peak of viral latency are plasma cells (ca. 15% at day 14 and ca. 8% at day 18). Notably, the frequency of virus-infected plasma cells correlated well with the frequency of splenocytes that spontaneously reactivate virus upon explant. Finally, we observed that the efficiency of marking latently infected B cells with the MHV68-YFP recombinant virus declined at later times postinfection, likely due to shut down of transgene expression, and indicating that the utility of this marking strategy is currently limited to the early stages of virus infection.

Intraoperative Monitoring of Hearing During Cerebellopontine Angle Tumor Surgery Using Transtympanic Electrocochleography

To investigate the use of transtympanic electrocochleography (TT-ECochG) analyzed on-line by a detector strategy software that made possible automatic extraction of TT-ECochG components intraoperatively in real-time domain.Fifteen patients with cerebellopontine angle tumor among 50 patients were included in this study.All subjects were operated on via middle fossa or retrosigmoid approach. Pure-tone average (PTA) was measured at 0.5, 1.0, and 2.0 kHz, and calculations were performed before and after surgery. Auditory function was monitored intraoperatively via TT-ECochG, and analyzed data were displayed on-line.TT-ECochG changes in morphology were described. Postoperative PTA elevation level correlated with TT-ECochG morphology changes occurring intraoperatively.Analyzed on-line, TT-ECochG data were displayed as first negative peak of compound action potential amplitude and latency in time domain every 3 to 5 seconds. A good correlation between postoperative PTA elevation and TT-ECochG morphology changes was showed (Spearman rank test, R = +0.93; t(N-2) = 9.00; p < 0.0001).TT-EcochG seemed to effectively mirror even minimal changes in auditory function during intraoperative monitoring in real-time domain. Developed strategy of on-line analysis makes the intraoperative hearing status assessment faster and easier.

The M2 gene product of murine gammaherpesvirus 68 is required for efficient colonization of splenic follicles but is not necessary for expansion of latently infected germinal centre B cells.

Infection of mice with murine gammaherpesvirus 68 is characterized by a marked transient expansion of latently infected splenic germinal centre (GC) B cells, which is followed by lower levels of persistent infection in GC and memory B cells. Virus transcription within GC B cells is restricted to a number of latency-associated open reading frames, including M2. This gene encodes a structurally unique protein of unknown function, which has been shown to be essential for the transient peak of virus latency during the establishment of latent infection in the spleen. This study shows that upon infection of mice with M2-defective viruses, at 14 days post-infection during the establishment of latency in the spleen, there was a reduction in the number of latently infected follicles when compared with wild-type virus. However, the mean number of latently infected cells within each follicle was equivalent between wild-type and M2-defective viruses. Late in infection, disruption of M2 resulted in sustained and abnormally high levels of virus persistence in splenic GC B cells but not memory B cells. These data indicate that during the establishment of latency in the spleen, the M2 gene product is required for efficient colonization of splenic follicles but is dispensable for the expansion of latently infected GC B cells and that M2 might be a critical modulator of B-cell function.

Development of the Temporal Properties of Visual Evoked Potentials to Luminance and Colour Contrast in Infants

We have studied the development of the temporal characteristics of the pattern visual evoked potentials (P-VEPs) in response to contrast reversal of patterns of low spatial frequency (0.1 c/deg) of either pure luminance contrast (yellow-black plaid patterns) or pure colour contrast (equiluminant red-green plaid patterns) in 15 infants between 6 and 30 weeks of age. High contrast patterns were modulated temporally either sinusoidally at various temporal frequencies to elicit steady-state responses, or abruptly at a low temporal frequency to elicit transient responses. Analysis of both the transient and steady-state responses suggests the existence of three different mechanisms contributing to the infant and adult P-VEP responses at low, medium and high temporal frequencies. The responses at the three different temporal frequency ranges have different time constants, and develop at different rates. The low frequency response predominates at 8 weeks, where it spans the range 1–6 Hz with an apparent latency of about 230 msec, for both colour and luminance stimulation. This response increases in bandwidth and decreases in latency progressively with age, at a similar rate for luminance and colour contrast, up to 14 weeks. After 14 weeks, the luminance response undergoes major changes, with the emergence of a new response with a shorter latency (about 100 msec) and a peak activity near 10 Hz. This mid-frequency response matures further with age, until it dominates the whole response of the adult P-VEP to luminance contrast. It also makes a contribution to the chromatic response at frequencies above 10 Hz, generating the characteristic double-peaked amplitude response in adults. However, its contribution is very limited below 10 Hz, where the response latency is 140 msec in adults, as it was at 14 weeks of age. A third component is evident at very high temporal frequencies of the luminance response as early as 6 weeks, extending up to 15 Hz in 8-week-olds and up to 25 Hz for older infants. It remains apparent up to 18 weeks, thereafter being swamped by the major mid-frequency response. The apparent latency of response over this frequency range is about 70 msec at all ages. The development of transient P-VEPs paralleled that of the steady-state P-VEPs. At all ages there was an early negative component (N70) at about 70 msec, corresponding to the fast steady-state response at high frequencies for luminance contrast. Before 14 weeks, the luminance and chromatic transient response had the same morphology, with a single major peak of similar latency to the apparent latency of the low temporal frequency response. After this age, the morphology of the luminance response changed, particularly in the first 100 msec, consistent with the emergence of the mid-frequency response. We discuss whether the high-frequency component may represent pre- or early post-synaptic cortical activity, already mature by 8 weeks, and how the different maturation rates of the mid and high-frequency components may reflect different intra-cortical circuitry for colour and luminance. Copyright © 1996 Elsevier Science Ltd.

Serotonin modulation of pain responsiveness in the aged rat

This study was designed to examine differences in basal nociceptive responsiveness between young (3 months) and old (25 months) male Fischer-344 rats and also to evaluate the effects of methysergide and fluoxetine on this behavioral paradigm. The results indicate that the aged animals were less sensitive than young animals to pain responsiveness in simple nociceptive tests such as the tail-flick, hot plate (55°C), and hind-paw pressure tests. In both groups of animals, this behavioral response followed a circadian rhythm, with peak of pain latency during the dark phase and trough occuring in the light phase. In all three analgesic tests, treatment with methysergide, which is a serotonin antagonist, resulted in hyperalgesia in both groups of animals within the first hour, followed by a return to basal response level after 2 h. Fluoxetine treatment resulted in a nonsignificant increase in nociceptive response at 30 min posttreatment which returned to the baseline by 1 h. Moreover, in both young and old animals morphine produced moderate analgesia in the hot plate and hind-paw tests, which was potentiated by simultaneous treatment with fluoxetine. This study shows that noxious response was reduced in the aged male Fischer-344 rats, and the data obtained provide evidence that the serotonergic system modulates pain sensitivity similarly in young and old animals.

Development of visual evoked potentials in neonates. A study using light emitting diode goggles.

We used a signal averager with light emitting diode goggles as the photostimulator to study the development of the visual evoked potentials in 40 normal neonates of between 23 and 42 weeks' gestation. All except two infants of less than 24 weeks' gestation had replicable visual evoked potentials. A negative peak of latency (mean (SD), 308 (21) msec) was present in all infants, but the development of the primary positive peak depended on maturity. Only infants of 37 weeks or more had a consistent positive peak of latency (mean (SD), 220 (22) msec). The practical simplicity and reliability of this technique has distinct advantages over previous conventional recording systems. Neonatal visual evoked potentials are shown to change with maturity.