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Control of virus infection is mediated in part by major histocompatibility complex (MHC) Class Ia presentation of viral peptides to conventional CD8 T cells. Although important, the absolute requirement for MHC Class Ia–dependent CD8 T cells for control of chronic virus infection has not been formally demonstrated. We show here that mice lacking MHC Class Ia molecules (Kb−/−xDb−/− mice) effectively control chronic γ-herpesvirus 68 (γHV68) infection via a robust expansion of β2-microglobulin (β2-m)-dependent, but CD1d-independent, unconventional CD8 T cells. These unconventional CD8 T cells expressed: (1) CD8αβ and CD3, (2) cell surface molecules associated with conventional effector/memory CD8 T cells, (3) TCRαβ with a significant Vβ4, Vβ3, and Vβ10 bias, and (4) the key effector cytokine interferon-γ (IFNγ). Unconventional CD8 T cells utilized a diverse TCR repertoire, and CDR3 analysis suggests that some of that repertoire may be utilized even in the presence of conventional CD8 T cells. This is the first demonstration to our knowledge that β2-m–dependent, but Class Ia–independent, unconventional CD8 T cells can efficiently control chronic virus infection, implicating a role for β2-n–dependent non-classical MHC molecules in control of chronic viral infection. We speculate that similar unconventional CD8 T cells may be able to control of other chronic viral infections, especially when viruses evade immunity by inhibiting generation of Class Ia–restricted T cells.
In this paper the authors identify a β2-microglobulin–dependent but major histocompatibility complex (MHC) Class Ia– and CD1-independent class of CD8 T cells that effectively control chronic γ-herpesvirus infection in mice. The important point that should be of general interest to the readers of PLoS Pathogens is that an effective CD8 T cell response develops during chronic infection of mice lacking MHC Class Ia molecules. Enormous efforts have gone into characterizing the role of conventional CD8 T cells that recognize viral peptides together with MHC Class Ia molecules during chronic viral infection, and many vaccine approaches focus solely on this response. This paper shows that additional types of CD8 T cells can operate during chronic infection, and that indeed, conventional MHC Class Ia–restricted T cells may be dispensable for control of chronic herpesvirus infection. The authors believe this is a fundamentally important point because it raises the question of whether unconventional CD8 T cells are important for control of other chronic viral infections such as infection with HIV, Hepatitis C virus, Hepatitis B virus, or human herpesviruses.
Citation: Braaten DC, McClellan JS, Messaoudi I, Tibbetts SA, McClellan KB, Nikolich-Zugich J, et al. (2006) Effective Control of Chronic γ-Herpesvirus Infection by Unconventional MHC Class Ia–Independent CD8 T Cells. PLoS Pathog 2(5): e37. doi:10.1371/journal.ppat.0020037
Editor: Donald Ganem, University of California San Francisco, United States of America
Received: January 9, 2006; Accepted: March 23, 2006; Published: May 19, 2006
Copyright: © 2006 Braaten et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grants RO1 CA74730 and RO1 CA96511 to HWV. JNZ and IM were supported by RO1 AI066096. DCB was a Leukemia and Lymphoma Society Fellow. JSM was supported by a pre-doctoral fellowship from the Cancer Research Institute. SAT is a Leukemia and Lymphoma Society Special Fellow (#3460–04).
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: γHV68, γ-herpesvirus 68 ; bp, base pair; FACS, fluorescence-activated cell sorter; IFNγ, interferon-γ; MHC, major histocompatibility complex; NK, natural killer; PFU, plaque forming unit; PMA, phorbol myristate acetate; SEM, standard error of the mean; TNFα, tumor necrosis factor-α; β2-m, β2-microglobulin
A defining characteristic of herpesviruses is their ability to persist for the life of the infected host by establishing latent infection after acute infection is cleared. Herpesviruses can reactivate from latency, generating new infectious virus that can either re-initiate lytic replication (a process termed herein "persistent replication," to distinguish it from replication during acute infection) or spread to a new host. Many γ-herpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), and the murine pathogen γ-herpesvirus 68 (γHV68), establish life-long latent infections within hematopoietic cells [1–6]. Consequently, chronic γ-herpesvirus infections are frequently associated with the development of B cell malignancies, especially in immunocompromised individuals [7–12]. γ-Herpesvirus-associated diseases are particularly common in immunocompromised hosts, a fact indicating that the immune system normally controls chronic γ-herpesvirus infection and thereby prevents disease [10,11,13–16].
Immune control of chronic γHV68 infection has been studied extensively as a model for defining viral and host mechanisms that are responsible for maintaining latency as a stable equilibrium between virus and host [15–28]. After either intranasal or intraperitoneal infection [1,23], cells that harbor latent γHV68 can be found in hematopoietic organs such as the bone marrow and spleen, and in body cavities such as the peritoneum [1–5,29]. Latent γHV68 infection of splenocytes and peritoneal cells has been extensively characterized in wild-type mice, and two forms of γHV68 latency have been observed [23,25,30,31]. An early form of latency occurs by 16 d after infection that is characterized by a high efficiency of reactivation from latency (i.e., frequency of reactivation per number of latently infected cells) in an ex vivo assay, with the majority of genome-bearing cells reactivating [23,30]. A second, long-term form of latency occurs 28–42 d after infection and is characterized by a lower efficiency of reactivation ex vivo, with approximately 10% of genome-bearing cells reactivating [23,25,30]. This long-term form of latency reflects a stable relationship between the virus and the host that is independent of both dose and route of virus infection [23], but which can be perturbed by immunodeficiency [15,25,30,31]. During this equilibrium phase of latency, sensitive assays can detect a very small amount of infectious virus in some mice, consistent with reactivation from latency and persistent replication [29].
The immune system normally controls latency and chronic infection at several levels, including immune-mediated decreases in the number of latently infected cells [17,19,20,25] and regulation of the efficiency of reactivation from latency [25]. Different components of the immune system have specific roles in regulating γHV68 latency at different sites; for example, the absence of interferon-γ (IFNγ) increases the efficiency of reactivation of latently infected peritoneal but not spleen cells [25,31]. In addition to regulating latency, the immune system normally prevents most persistent lytic replication, which flares in immunocompromised mice, especially those lacking CD8 or IFNγ [15,16,25,29,32–34]. In addition to its role in limiting persistent replication, IFNγ (as well as IFNαβ) regulates latent γHV68 gene expression in vivo, and IFNγ can directly inhibit the reactivation of γHV68 from latency [32,33]. Persistent replication requires genes such as the v-bcl2 and v-cyclin that are not required for replication during acute infection, which indicates that persistent replication occurs by a distinct mechanism from that required for acute replication [31,35]. Persistent replication is likely critical for the maintenance of a constant pool of latently infected cells [27,34,36] by infection and establishment of latency in uninfected target cells.
A large body of evidence indicates that CD8 T cells are important for control of acute, latent, and persistent γHV68 infection. CD8 T cell responses directed against Class Ia–presented peptides derived from lytic cycle proteins are detectable as early as 10 d after infection [21,37]. These CD8 T cells actively cycle for many months after infection [22,37,38], which is consistent with a role for CD8 T cells in the control of persistent replication [25]. TCR transgenic CD8 T cells that recognize the SIINFEKL peptide of ovalbumin can control infection of a recombinant γHV68 virus that expresses ovalbumin [19]. Furthermore, CD8 T cells that recognize the latency-associated γHV68 antigen M2 control the early form of γHV68 latency [28,39]. These data demonstrate that both lytic and latent γHV68 antigens are presented by Class Ia on infected cells during chronic infection. These data are supported by published studies demonstrating that either CD8 deficiency or β2-microglobulin (β2-m) deficiency is associated with increased latency and persistent replication [25,40–42], and by studies showing that antibody-mediated depletion of CD8 T cells from wild-type mice results in increased latent infection [20,43].
That classical CD8 T cells are involved in control of γHV68 infection is consistent with a critical role for Class Ia molecules in presenting γHV68-encoded antigens; however, the requirement for Class Ia molecules for control of infection has not been formally tested. This is of interest because some published studies suggest that unconventional T cells are stimulated during γHV68 infection. For example, T cell hybridomas isolated from γHV68-infected mice can respond to γHV68-infected cells in the absence of TAP1 or β2-m [42,44]. However, the use of β2-m−/− mice and cells as models for Class Ia deficiency is complicated by the fact that β2-m is also an important accessory molecule for the cell surface expression of multiple non-classical major histocompatibility complex (MHC) Class Ib molecules, including CD1d, M3, FcRn, Qa-1, and HFE (reviewed in [45,46]). It has also been reported that β2-m−/− mice are not completely Class Ia deficient [47–49].
To circumvent these limitations, we evaluated γHV68 infection in Class Ia–deficient (Class Ia) mice (Kb−/−xDb−/−) [50,51] and in mice deficient in both Class Ia and β2-m molecules (Kb−/−xDb−/−xβ2-m−/−) [52]; Kb−/−xDb−/−xβ2-m−/− mice lack expression of both Class Ia and β2-m–dependent Class Ib molecules. Because the non-classical Class Ib molecule CD1d has been implicated in control of herpesvirus infection ([53], reviewed in [54]), we also analyzed mice deficient in both Class Ia and CD1d (Kb−/−xDb−/−xCD1d−/−). We found that mice lacking Class Ia molecules mount a substantial effector CD8 T cell response that requires β2-m but not CD1d. These unconventional effector CD8 T cells effectively controlled latent γHV68 infection. These data suggest that unconventional T cells could play an important role in control of other chronic viral infections, with important implications for the study of antigens recognized by CD8 T cells during chronic infection and, especially, for viruses that evade immunity by inhibiting MHC Class Ia expression.
Class Ia–Deficient Mice Effectively Control Acute γHV68 Infection
To begin to evaluate the role of Class Ia molecules during γHV68 infection, acute virus replication was compared in strains of mice 9 d after intraperitoneal injection of 106 plaque forming units (PFU) of wild-type γHV68. Spleens from B6, CD8−/−, β2-m−/−, Kb−/−xDb−/−, Kb−/−xDb−/−xβ2-m−/−, and Kb−/−xDb−/−xCD1d−/− mice were harvested, and the titer of virus present was determined (Figure 1A, Table 1). There were no significant differences in titers in the spleens from wild-type, Kb−/−xDb−/−, and Kb−/−xDb−/−xCD1d−/− mice, whereas mice lacking either CD8 or β2-m demonstrated splenic titers that were 8.7-fold (p = 0.03) and 5.1-fold (p = 0.04) higher, respectively, as compared to wild-type mice. We noted that Kb−/−xDb−/−xβ2-m−/− mice failed to control infection compared to either Kb−/−xDb−/− or wild-type mice. Furthermore, lack of CD8 resulted in decreased control of γHV68 replication even though lack of Class Ia molecules did not. Although there are several potential explanations for these findings, these data could be explained if a β2-m–dependent, but Class Ia–independent, population of CD8 T cells was important for the control of infection observed in the Kb−/−xDb−/− mice.
(A) Log viral titers in spleen of B6, CD8α−/−, β2-m−/−, Kb−/−xDb−/−, Kb−/−xDb−/−xCD1d−/−, and Kb−/−xDb−/−xβ2-m−/− mice 9 d after intraperitoneal infection with 106 PFU of virus. Data were pooled from two experiments consisting of two to three mice each. An asterisk (*) indicates a significant increase in viral titer relative to B6 (p < 0.05).
(B) Frequency of cells reactivating from latency ex vivo (left), frequency of cells bearing viral genome (middle), and persistent replication (right, open symbols) at day 42 post-infection in peritoneal cells from B6, β2-m−/−, Kb−/−xDb−/−, Kb−/−xDb−/−xCD1d−/−, and Kb−/−xDb−/−xβ2-m−/− mice. On the y-axis is the percentage of wells positive for viral cytopathic effect (left and right) or viral genome (middle). The horizontal line within the graph indicates the 63.2% Poisson distribution line used to calculate the frequency of cells reactivating virus. For each group, cells were pooled from three to five mice. Data are the mean of three to four independent experiments ± SEM.
(C) Frequency of latent infection in splenocytes from B6, β2-m−/−, Kb−/−xDb−/−, Kb−/−xDb−/−xCD1d−/−, and Kb−/−xDb−/−xβ2-m−/− mice. For each group, cells were pooled from three to five mice. Data are the mean of three to four independent experiments ± SEM.
(D) γHV68 latency at day 16 post-infection in peritoneal cells from Kb−/−xDb−/−xCD1d−/− mice either untreated, treated with a control antibody (Ab), or depleted of CD8 T cells beginning 1 d prior to infection. Left: ex vivo reactivation of peritoneal cells from control-treated and CD8-depleted groups. Middle: frequency of peritoneal cells that contain viral genome in control-treated and CD8-depleted groups. Right: persistent replication in peritoneal cells from control-treated and CD8-depleted groups.
(E) γHV68 latency at day 16 post-infection in splenocytes from Kb−/−xDb−/−xCD1d−/− mice either untreated, treated with a control antibody, or depleted of CD8 T cells beginning 1 d prior to infection. For each group in D and E, cells were pooled from three to five mice. Data are the mean of three independent experiments ± SEM.
Viral Titer in Spleen 9 Days after Infection
Class Ia–Deficient Mice Effectively Control Chronic γHV68 Infection
We next evaluated the role of Class Ia molecules in control of chronic γHV68 infection in peritoneal cells and splenocytes from B6, Kb−/−xDb−/−, and Kb−/−xDb−/−xCD1d−/− mice (Figure 1B and 1C, Tables 2 and 3). As read-outs for chronic infection we quantified the frequency of latently infected cells bearing viral genome, the frequency of latently infected cells that reactivated from latency when explanted onto murine embryonic fibroblast (MEF) monolayers, and persistent virus replication [23,25]. Mice lacking Class Ia or both Class Ia and CD1d were able to control chronic γHV68 infection in both the peritoneum and spleen as well as wild-type mice, as measured by either the frequency of cells that reactivate from latency or the frequency of latently infected cells carrying viral genome (Figure 1B and 1C). Thus, as observed for acute infection, mice lacking Class Ia or both Class Ia and CD1d molecules exhibited a significant capacity to control chronic γHV68 infection.
Peritoneal Exudate Cells, Day 42 Post-Infection
Splenocytes, Day 42 Post-Infection
Control of Chronic γHV68 Infection in Class Ia–Deficient Mice Is Dependent on β2-m and CD8 T Cells
Having shown that MHC Class Ia molecules are not required for control of either acute or chronic γHV68 infection, we next determined if control of chronic γHV68 infection in the absence of Class Ia required β2-m or CD8 T cells. B6, β2-m−/−, Kb−/−xDb−/−, and Kb−/−xDb−/−xβ2-m−/− mice were evaluated at 42 d post-infection. For both peritoneal cells and splenocytes, all measures of latent infection in β2-m−/− and Kb−/−xDb−/−xβ2-m−/− mice indicated significant loss of control compared to wild-type and Kb−/−xDb−/− mice (Figure 1B and 1C, Tables 2 and 3). Together, these data strongly indicated that control of γHV68 latency and persistent replication is β2-m dependent in either the presence or absence of Class Ia molecules.
Since CD8 T cells were important for control of chronic γHV68 infection ([25], and Figure S1), even when Class Ia molecules were not (Figure 1), these data were consistent with the hypothesis that a Class Ia–independent, but β2-m–dependent, CD8 T cell response was able to control γHV68 infection. We therefore determined if CD8 T cells were important for control of chronic γHV68 infection in the absence of Class Ia molecules. Since dendritic cells are latently infected with γHV68 [4] and can express CD8α [55–57], we wished to deplete CD8αβ+ T cells without altering dendritic cells. Furthermore, we wished to avoid depleting unconventional CD8αα+ T cells that develop in mice lacking Class Ia [58]. We therefore selected a depleting antibody specific for CD8β that is not expressed on dendritic cells or CD8αα+ CD8 T cells [55,57,59,60]. Kb−/−xDb−/−xCD1d−/− mice were either mock treated or treated with a CD8β+ cell–depleting antibody during infection. The extent of CD8 T cell depletion was assessed by fluorescence-activated cell sorter (FACS) assay and was found to be equal to or greater than 94% effective in these experiments (Figure S2).
Depletion of CD8β+ T cells resulted in loss of control of chronic infection in the Class Ia–deficient mice (Figure 1D and 1E, Tables 4 and 5). Loss of control was especially evident in peritoneal cells in which the majority of latently infected cells are macrophages [1], because the frequency of cells reactivating virus, the frequency of cells positive for viral genome, and the amount of persistent replication were all increased (Figure 1D). These data showed that CD8 T cells were required to control chronic γHV68 infection in mice lacking Class Ia molecules, consistent with the hypothesis that these CD8 T cells were stimulated by a Class Ia–independent mechanism.
Peritoneal Cells, Day 16 Post-Infection.
Splenocytes, Day 16 Post-Infection
Control of Chronic γHV68 Infection in Class Ia–Deficient Mice Is Associated with Stimulation and Expansion of Unconventional CD8 T Cells
To more directly assess our hypothesis that CD8 T cells were present in the Class Ia–deficient mice, splenocytes from mock infected and infected B6, CD8−/−, β2-m−/−, Kb−/−xDb−/−, Kb−/−xDb−/−xCD1d−/−, and Kb−/−xDb−/−xβ2-m−/− mice were characterized for the presence of CD4 and CD8 T cells and for the absolute number and percentage of CD8 T cells. In agreement with previously reported observations, uninfected mice lacking Class Ia molecules (Kb−/−xDb−/− and Kb−/−xDb−/−xCD1d−/− mice) had a significantly lower percentage of CD8 T cells compared to B6 mice (Figure 2A and 2B). This phenotype was even more pronounced in mice lacking both MHC Class Ia and β2-m, which was expected because these mice lack expression of all Class Ia and β2-m–dependent Class Ib molecules. In infected B6, Kb−/−xDb−/−, and Kb−/−xDb−/−xCD1d−/− mice, however, the percentages and absolute numbers of both CD4 and CD8 T cells were similar (Figure 2A and 2B), indicating that viral infection stimulated a massive expansion of CD8 T cells in mice lacking the Class Ia and CD1d molecules. To confirm that the CD8 T cells observed in the spleens of infected Kb−/−xDb−/− mice were T cells, CD8+ splenocytes from Kb−/−xDb−/− mice were further characterized by flow cytometry and shown to express TCRβ, CD3, and CD8β (Figure 2C). These data demonstrated that the unconventional CD8+ cells that expand in Kb−/−xDb−/− mice after γHV68 infection were indeed CD8 T cells.
Splenocytes were harvested from B6, CD8α−/−, β2-m−/−, Kb−/−xDb−/−, Kb−/−xDb−/−xβ2-m−/−, and Kb−/−xDb−/−xCD1d−/− mice 42 d after mock infection or γHV68 infection and analyzed by flow cytometry.
(A) Representative flow cytometric analysis of CD4 and CD8 expression on splenocytes from B6, Kb−/−xDb−/−, and Kb−/−xDb−/−xβ2-m−/− mice that were either mock infected or γHV68 infected for 42 d.
(B) Absolute number of splenocytes expressing CD8α in mice that were mock infected or infected with γHV68 for 42 d. Data are the mean of four to eight independent experiments using three to five mice per group, ± SEM.
(C) Representative flow cytometric analysis of TCRβ, CD3, CD8β, and CD8α expression on splenocytes from B6, Kb−/−xDb−/− mice that were either mock infected or γHV68 infected for 42 d.
We compared the expansion of CD8 T cells in Class Ia–deficient mice to that observed in mice lacking both Class Ia and β2-m (Figure 2A and 2B). There was an increase in CD8 T cells in mice lacking both Class Ia and β2-m, and a significant part of the CD8 T cell expansion in Class Ia–deficient mice was β2-m dependent. We speculate that the T cells that develop in the absence of both β2-m and Class I may be related to cells previously shown to respond to γHV68 infection in the absence of β2-m [42,44].
Unconventional CD8 T Cells Induced by γHV68 Infection in Class Ia–Deficient Mice Demonstrate an Activated Phenotype
We further evaluated the CD8 T cells that expand during infection of Class Ia–deficient mice by determining whether they express cell surface markers observed on conventional Class Ia–dependent effector/memory CD8 T cells. Using flow cytometry, we compared expression of cell surface markers between mock-infected Class Ia–deficient mice and mice that had been infected for 42 d (Figure 3). The results were consistent with the cells being effector/memory cells (CD43+, CD69+/−, CD62L-low, CD122+/− [IL2Rβ chain], CD127-low [IL7Rα chain]) [61–63]. We also evaluated the cells for the pan–natural killer (NK) cell surface marker DX5 (CD49b) and antibodies specific for the γδT cell receptor; all of which were negative on both mock-infected and infected unconventional CD8 T cells. Finally, the cells were evaluated for expression of known CD8 T cell–associated NK cell markers characteristic of activated cytotoxic CD8 T cells [63]. The unconventional CD8 T cells were highly positive for NKG2A and expressed elevated amounts of NKG2D. Together, these results were consistent with the unconventional CD8 T cells being effector/memory cells and, therefore, with the cells being anti-viral T cells.
Splenocytes were harvested from Kb−/−xDb−/− mice 42 d after mock infection or γHV68 infection. CD8α+ splenocytes from these mice were analyzed by flow cytometry for cell surface expression of markers of effector/memory phenotype and expression of NK and T cell markers. Panels are representative of the flow cytometric analysis from two independent experiments.
Unconventional CD8 T Cells Are Vβ4-Biased after γHV68 Infection
In wild-type, β2-m−/−, and TAP1−/− mice, the CD8 T cell response to γHV68 is Vβ4 biased [26,42]. We therefore used flow cytometry to characterize Vβ usage in infected versus control B6, Kb−/−xDb−/−, and Kb−/−xDb−/−xβ2-m−/− mice (Figure 4A and 4B). For CD4 T cells, Vβ usage was not significantly skewed by γHV68 infection; for CD8 T cells, Vβ usage was predominantly Vβ4 biased after infection. This contrasts with uninfected Class Ia–deficient mice, which have previously been shown not to have biased Vβ TCR usage compared to wild-type mice [64]. The percentage of total splenic CD8 T cells expressing Vβ4 increasing from 6% to approximately 40% by 42 d after infection (Figure 4A). In infected Kb−/−xDb−/− mice, the percentage of Vβ4 T cells (about 70%) was much higher than that observed in B6 mice. These results confirmed that Class Ia molecules are not required for stimulating the Vβ4 T cells [42] and that the majority of CD8 T cells in Class Ia–deficient mice were biased toward Vβ4 usage. In infected Kb−/−xDb−/−xβ2-m−/− mice, however, the percentage of CD8 T cells that express Vβ4 was significantly less (p < 0.01) than that seen in Kb−/−xDb−/− mice (Figure 4A), which was consistent with the stimulation of Vβ4 CD8 T cells in Kb−/−xDb−/− mice being significantly but not completely dependent on β2-m.
Splenocytes were harvested from B6, β2-m−/−, Kb−/−xDb−/−, Kb−/−xDb−/−xβ2-m−/−, and Kb−/−xDb−/−xCD1d−/− mice 42 d after mock infection or γHV68 infection. TCR Vβ usage by CD4 and CD8 T cells from these mice was assessed by flow cytometry.
(A) Percentage of total CD8α+ splenocytes that co-express Vβ4. Data are the mean of four to eight independent experiments using three to five mice per group, ± SEM.
(B) Absolute number of CD8α+Vβ4− and CD8α+Vβ4+ splenocytes. Data are the mean of four to eight independent experiments using three to five mice per group, ± SEM.
(C) TCR Vβ analysis of CD4 and CD8 T cells obtained from the spleens of Kb−/−xDb−/− mice 42 d after mock-infection or γHV68 infection.
Data are the mean ± SEM of three independent experiments.
In addition to the increase in Vβ4 usage, we observed that both Vβ3 and Vβ10 usage were maintained in infected as compared to uninfected Class Ia–deficient mice (Figure 4C). Given the large increase in total CD8 T cells stimulated by γHV68 infection, this indicated that both Vβ3- and Vβ10-expressing T cells expanded in infected Class Ia–deficient mice, although to a lesser extent than Vβ4-expressing T cells. Together these data demonstrated significant Vβ bias in CD8 T cells that develop during chronic γHV68 infection of Class Ia–deficient mice.
T Cell Receptor Diversity in Unconventional CD8 T Cells that Control Chronic γHV68 Infection
The above bias could be due to a very limited repertoire of T cells selected on and restricted by Class Ib MHC molecules; in which case only a few clones could account for the expansion of Vβ4 and other families. To assess clonal complexity of unconventional CD8 T cells that develop during γHV68 infection, we compared CDR3 length complexity in CD8 T cells from infected and uninfected mice. CDR3 regions are the most polymorphic parts of the TCR, and their diversity is representative of T cell diversity within a given population. CDR3 length analysis determines in a population of cells the relative abundance of mRNAs encoding TCRs with specific CDR3 lengths (usually between 18–39 base pairs (bp) encoding CDR3 regions of 6–13 amino acids) [65]. In young, non-immunized mice, this analysis reveals within each TCR Vβ family a pattern of CDR3 length peaks spaced 3 bp apart in a Gaussian distribution around the most frequent length (27–30 bp encoding 9–10 amino acids), representative of a diverse T cell repertoire [65]. Antigenic challenge induces expansion of a handful of Ag-specific T cell clones, leading to relative or absolute dominance of one (monoclonal response) or a few (oligoclonal response) peaks in the profile [66,67]. During the contraction phase of the T cell response, expanded T cell clones die by apoptosis, restoring the Gaussian CDR3 length profile. Therefore, distorted profiles, with a few, or only a single, peak, are strongly suggestive of restricted TCR utilization, whereas a Gaussian distribution of CDR3 lengths within proliferating T cells is indicative of diverse TCR utilization.
CDR3 length analysis revealed that the CD8 T cells from a broad range of Vβ families retained CDR3 length diversity in γHV68-infected wild-type mice (unpublished data and Figure 5), suggesting that numerous T cell clones from a variety of Vβ families respond to chronic γHV68 infection. This included the dominant Vβ4+ population of T cells that showed signs of polyclonal response (Figure 5). In contrast, the CDR3 length profiles of unconventional CD8 T cells that developed in infected Kb−/−xDb−/− mice revealed two patterns (Figure 5, and unpublished data). The dominant Vβ4 and subdominant Vβ10 families, comprising more than 80% of the total CD8 response of Kb−/−xDb−/− mice, displayed diversity similar to that seen in wild-type mice. This suggests that much of the response in both wild-type and MHC Class Ia–deficient mice to γHV68 is diverse. Of note, the subdominant Vβ10 response in both the wild-type and Kb−/−xDb−/− mice included an asymmetric peak to the right of the center (Figure 5, arrows), suggesting that the response in normal animals may include Class Ia–independent clones. A second pattern was seen in the case of Vβ3 and Vβ20 TCR families, where peaks suggestive of oligoclonal or monoclonal expansions are seen in Class Ia–deficient, but not wild-type, animals (Figure 5). Sequencing analysis is in progress to ascertain that some of the Class Ia–independent clones are invoked in the response of wild-type animals. Together, these data indicated that the Class Ia–independent anti-γHV68 CD8 T cell response encompasses substantial diversity, indicating that the development of these cells is not restricted to a limited and highly specialized subset of T cells.
TCR CDR3 length analysis of CD8 T cells obtained from the spleens of B6 and Kb−/−xDb−/− mice 42 d after mock-infection or γHV68 infection. Shown on the x-axis is CDR3 length within the CD8 T cell populations expressing Vβ3, Vβ4, Vβ10, or Vβ20. The y-axis represents the relative abundance of mRNA encoding TCR chains of individual CDR3 lengths. Data are from one of two representative experiments. Arrows indicate an asymmetric peak to the right of the center observed in the subdominant Vβ10 response from both the wild-type and Kb−/−xDb−/− mice.
Unconventional CD8 T Cells Express Effector Molecules in Infected Class Ia–Deficient Mice
Because unconventional CD8 T cells were capable of controlling γHV68 infection in Class Ia–deficient mice (Figure 1), we examined their capacity to express effector molecules known to be important for control of chronic γHV68 infection. We focused on expression of IFNγ, which is of proven importance for control of latent γHV68 infection, reactivation of γHV68 from latency, and persistent γHV68 replication [15,16,25,31–33]. The unconventional CD8 T cells in splenocytes from Kb−/−xDb−/− mice were compared to CD8 T cells in splenocytes from B6 mice. Detection of the expression of cytokines by effector CD8 T cells in other viral systems often requires re-stimulation of cells in vitro. As we do not know the antigen specificity of the unconventional CD8 T cells that develop in γHV68-infected Class Ia–deficient mice, we restimulated cells with phorbol myristate acetate (PMA) and ionomycin prior to staining for expression of IFNγ and tumor necrosis factor-α (TNFα). Unstimulated cells expressed only low levels of IFNγ and TNFα in these experiments (unpublished data).
CD8 T cells from mock-infectedB6 mice produced little TNFα and no IFNγ even after stimulation with PMA and ionomycin. In contrast, approximately 11% of CD8 T cells from uninfected Kb−/−xDb−/− mice produced both TNFα and IFNγ after PMA and ionomycin stimulation, whereas smaller populations of cells produced either TNFα or IFNγ alone (Figure 6A). These data demonstrated a functional difference between unstimulated conventional and unconventional CD8 T cells, and indicate that even in naive Kb−/−xDb−/− mice, the CD8 T cells were in a partially activated state. These findings are consistent with previous data obtained in characterizing these cells [58,68].
Splenocytes were harvested from B6 and Kb−/−xDb−/− mice 42 d after mock infection or γHV68 infection, and analyzed by flow cytometry.
(A) Representative flow cytometric analysis of intracellular IFNγ and TNFα expression by CD8 T cells from B6 and Kb−/−xDb−/− mice that were either mock infected or γHV68 infected for 42 d.
(B) Absolute number of total CD8 T cells and Vβ4 CD8 T cells expressing intracellular IFNγ and TNFα in mice that were mock infected or γHV68 infected for 42 d. Data are the mean of two independent experiments using five to ten mice per group, ± SEM.
(C) Representative flow cytometric analysis of intracellular IFNγ, intracellular TNFα versus Vβ4 expression by CD8 T cells from B6 and Kb−/−xDb−/− mice that were either mock-infected or γHV68-infected for 42 d. Representative from one of two experiments.
In B6 mice 42 d after infection, PMA and ionomycin treatment induced a significant percentage of CD8 T cells to express IFNγ, TNFα, or both cytokines (Figure 6A). Similarly, infection of Kb−/−xDb−/− mice resulted in a significant increase in the proportion of PMA and ionomycin–stimulated cells that expressed IFNγ (Figure 6A). Because the majority of CD8 T cells that develop in γHV68-infected Class Ia–deficient mice express Vβ4, we also examined cytokine expression in Vβ4 CD8 T cells (Figure 6B and 6C). In both wild-type and Class Ia–deficient mice, γHV68 infection induced a significant increase in CD8 T cells expressing IFNγ (Figure 6B and 6C). It would be of interest to examine these cells for the expression of IFNαβ, which also can control γHV68 latency [33]. These data were consistent with CD8 T cells in both B6 and Kb−/−xDb−/− mice acquiring an activated phenotype after γHV68 infection, confirming our conclusions from analysis of cell surface molecule expression on these cells (Figure 3). Because IFNγ is important for control of γHV68 latency and persistent replication, these data indicated that γHV68-induced CD8 T cells that develop in Class Ia–deficient mice express important anti-viral effector molecules.
We present data in this paper demonstrating that unconventional CD8 T cells arise in infected Class Ia–deficient mice. We show furthermore that these unconventional CD8 T cells express surface markers and cytokines consistent with memory and effector function and that they are required for control of chronic γHV68 infection in the absence of classical Class Ia molecules. The unconventional CD8 T cells are β2-m–dependent, but CD1d-independent, and predominantly express Vβ4, Vβ3, and Vβ10, as well as NK cell markers such as NKG2A and NKG2D. They exhibit a diverse TCR repertoire, consistent with a potential to respond to different viral antigens. This is the first demonstration of the development of a diverse and functionally important Class Ia–independent CD8 T cell response during chronic viral infection.
Several previously published studies have demonstrated that CD8 T cells are important for control of γHV68 infection [19,20,25,43], and that Class Ia–restricted CD8 T cells specific for both lytic and latent γHV68 antigens can mediate protection [21,39]. Furthermore, vaccination with immunodominant Class Ia–binding γHV68 peptides can decrease acute titers and transiently decrease the early form of latency [69–71], and adoptive transfer of latent virus antigen-specific Class Ia–restricted CD8 T cells decreases latency early after infection [28]. Thus, there is no question that conventional Class Ia–restricted CD8 T cells are an important part of the response to γHV68 infection. The data presented here add unconventional CD8 T cells to the host's anti-viral armamentarium for control of chronic infection, and indicate that the role of unconventional CD8 T cells needs to be systematically explored in chronic viral infections. These T cells might be particularly important for control of viruses that actively inhibit expression of host cell MHC Class I as a mechanism of immune evasion.
Our experiments did not address the extent to which unconventional CD8 T cells function as effector cells in wild-type mice. We speculate that these cells do play a role in normal hosts based on the presence of T cells with similar markers in normal hosts and the diverse TCR repertoire used by these unconventional CD8 T cells. Moreover, CDR3 repertoire analysis suggests that some CDR3 lengths (and, perhaps, T cell clones) may be shared between the responses in wild-type and Kb−/−xDb−/− mice. However, the proof that such cells play a role when Class Ia molecules are present must await identification of specific mechanisms of activation and selection for these cells and loss of function approaches based on that information, or identification of cell surface molecules that are specific to this interesting set of CD8 T cells, followed by depletion studies.
The observation that β2-m deficiency disrupts immunity to chronic γHV68 infection was initially attributed to decreased Class Ia expression on β2-m–deficient cells [40]. This interpretation is not correct because data presented here demonstrate that effective control of long-term latency does not require Class Ia molecules. Moreover, since β2-m deficiency resulted in a more-severe loss of control of latency than did Class Ia deficiency alone, we hypothesize that β2-m has an important functional role in controlling infection in addition to its more commonly known function as the essential light chain for Class Ia proteins. Consistent with this, Kb−/−xDb−/−xβ2-m−/− mice have much higher levels of latency and persistent infection in both splenocytes and peritoneal cells than Kb−/−xDb−/− mice. The simplest explanation for this Class Ia–independent role for β2-m in control of chronic viral infection is that β2-m–dependent, but Class Ia–independent, unconventional CD8 T cells are important for control of γHV68 infection. We speculate that these cells are restricted by β2-m–dependent MHC Class Ib molecules.
T cells restricted to MHC Class Ib proteins are often mono- or oligoclonal with regard to TCRα and/or TCRβ usage. This characteristic has been observed in HLA-E-restricted CD8 T cells in humans [72], MR1-restricted mucosal-associated T cells [73], and CD1d-restricted NKT cells [74]. Thus, the Vβ4-, Vβ3-, and Vβ10-biased nature of the CD8 T cell response to γHV68 in Class Ia–deficient mice is consistent with the presence of one or more populations of MHC Class Ib–restricted CD8 T cells. An important role for one or more MHC Class Ib molecules in γHV68 immunity could therefore explain both the importance of β2-m for efficient expansion of CD8 T cells in Class Ia–deficient mice, and the Vβ bias of the unconventional CD8 T cells demonstrated herein.
We ruled out a contribution for CD1d in control of chronic γHV68 infection, but other candidates remain. CD8 T cells restricted to the Class Ib molecules Qa-1 and H2-M3 contribute to resistance to intracellular bacteria such as Listeria monocytogenes and Mycobacterium tuberculosis [75–78]. In humans, H2-M3 has been shown to present influenza virus peptides to a human CD8 T cell clone [79]. In addition, a population of CD8 T cells that recognizes peptides derived from human beta- and γ-herpesviruses in the context of HLA-E, the human homolog of Qa-1, has been identified [72]. This latter observation is of particular interest since the CD8 T cells that developed during chronic γHV68 infection express high levels of NKG2A, which interacts with Qa1.
In summary, we demonstrate here that γHV68 infection triggers a robust β2-m–dependent, Class Ia–independent, and CD1d-independent unconventional CD8 T cell response that can control γHV68 latency and persistent replication. These findings make two important points regarding immunity to chronic viral infection: (1) Class Ia–independent non-classical CD8 T cells can control latent herpesvirus infection, and (2) the role of β2-m in immunity to chronic viral infection is not limited to its function as a light chain for Class Ia molecules. It is possible that similar Class Ia–independent CD8 T cells are required for control of other chronic viral infections, such as human herpesviruses, HIV, hepatitis C virus, hepatitis B virus, and lymphocytic choriomeningitis virus (LCMV). Furthermore, since Class Ia proteins are not essential for anti-viral CD8 T cell responses, CD8 T cell–directed vaccination strategies that target only Class Ia–presented antigens may not induce a maximal antiviral response.
Materials and Methods
C57BL/6J [B6, Jackson# 000664] mice and mice deficient in either the CD8α chain (CD8−/−, Jackson# 002665, [80]) or β2-m (β2-m−/−, Jackson# 002087, [81,82]) were obtained from The Jackson Laboratories (Bar Harbor, Maine, United States). Kb−/−xDb−/− mice [51] were a generous gift from both Dr. Ted Hansen and Dr. Albert Bendelac. Kb−/−xDb−/−x β2-m−/− (Kb−/−xDb−/− x β2-m−/−) mice were a generous gift of Dr. Ted Hansen [52]. CD1d−/− [83] and Kb−/−xDb−/−xCD1d−/− (Kb−/−xDb−/−x CD1d−/−) mice were a generous gift of Dr. Albert Bendelac. All mice were on the B6 background and were bred and housed at Washington University School of Medicine in accordance with all Federal and University guidelines. Eight- to 12-wk-old mice were used for all experiments.
Cell culture and virus infection.
NIH 3T12 and B6 mouse embryonic fibroblasts were maintained as described [40]. γHV68 clone WUMS (ATCC VR1465) was passed and titered by plaque assay on NIH 3T12 cells [40]. Mice were infected intraperitoneally with 106 PFU in 0.5 ml Dulbecco modified Eagle's Medium with 1% fetal calf serum (DMEM-1) [23]. Mock-infected animals were injected intraperitoneally with NIH 3T12 cell lysate diluted in DMEM-1.
Preparation of splenocytes, antibodies, and flow cytometry.
Single-cell suspensions of splenocytes were prepared for flow cytometry as described [40]. Splenocytes, 106 per condition, were incubated in FACS-blocking buffer (1× PBS with 1.0% BSA, 0.1% sodium azide plus rat anti-mouse CD16/CD32 [clone 2.4G2, ATCC#HB-197] and 5% rat serum) for 30 min on ice. Five percent mouse serum was added for NKG2A/C/E staining. Antibody staining for cell surface markers was performed in 100 μl of FACS buffer (1× PBS with 1.0% BSA, 0.1% sodium azide) on ice for 45 min, followed by three washes of (200 μl each) of FACS buffer. The cells were then fixed in 1% paraformaldehyde (in PBS) and stored in FACS buffer until analyzed. For intracellular staining, cells were activated with 20-ng/ml PMA plus1 μM ionomycin for 4–6 h in the presence of GolgiPlug from the Cytofix/Cytoperm kit from BD Pharmingen (San Diego, California, United States). After staining the cells for surface markers, cells were fixed and permeabilized using the Cytofix/Cytoperm kit according to the manufacturer's instructions. Next, the cells were stained with antibodies specific to IFNγ and TNFα, and then washed and resuspended in FACS buffer prior to analysis. PE anti-CD43, CD62L, CD122, CD127, CD44, CD69, DX5, CD4, αβTCR, TNFα, PE-labeled strepavidin, CyChrome anti-CD3, fluorescein isothiocyanate (FITC) anti-CD4, PE, allophycocyanin (APC) and Alexa 488 anti- IFNγ, biotin anti-NKG2A/C/E, and FITC anti-TCRβ (pan β, clone H57–597) were obtained from BD Pharmingen. TCR V Beta usage of T cells from mock-infected and infected Kb−/−xDb−/− mice was performed using the mouse Vβ TCR Screening Panel (BD Pharmingen, Catalog# 557004) according to the manufacturer's instructions. Labeled isotype–matched control antibodies were obtained from BD Pharmingen. Stained cell populations were processed with a FACSCaliber flow cytometer (BD Pharmingen), and the data were analyzed with either CellQuest (BD Pharmingen) or FCS Express Version 2 software (De Novo Software, Ontario, Canada).
CDR3 length analysis.
The PCR conditions and the primers were described previously [84]. CDR3 length polymorphism profiles of Vβ families were obtained using the Immunoscope software, depicting the relative intensity of bands migrating to the same CDR3 nucleotide length, as previously described [84].
Plaque assays.
Plaque assays were performed as previously described [40]. Briefly, organs were harvested into sterile, screw-top 2-ml tubes containing 1 ml of DMEM and 100 μl of 1-mm zirconia/silica beads (BioSpec Products, Bartlesville, Oklahoma, United States) and stored at −80 °C. Samples were thawed on ice and homogenized using a Mini BeadBeater (BioSpec Products), then further diluted in DMEM prior to infecting NIH 3T12 monolayers. Infected monolayers were overlaid with Noble agar, and plaques were visualized at 7 d post-harvest using neutral red staining. The limit of detection was 50 PFU.
In vivo depletion of lymphocyte subsets.
Monoclonal antibodies were produced in INTEGRA Celline CL1000 flasks (Integra Biosciences, Ijamsville, Maryland, United States) as described [20]. Beginning 1 d prior to virus infection, 2 mg of CD8-depleting antibody (H35, [85]) or an isotype-matched control antibody (SFR3-DR5, IgG2b, ATCC HB-151, [86]) were administered to each mouse by intraperitoneal injection. Monoclonal antibodies (1 mg) were administered every fourth day thereafter. The efficacy of CD8 depletion was monitored by flow cytometric analysis of splenocytes and was 94% or greater in each case.
Ex vivo limiting dilution reactivation analysis.
The frequency of cells reactivating from latency was assayed as described [40]. Briefly, serial 2-fold dilutions of harvested cells (24 wells/dilution starting at 1 × 105 cells/well for splenocytes and 4 × 104 cells/well for peritoneal cells) were plated onto permissive mouse embryonic fibroblast monolayers (MEFs) for 21 d, then scored for cytopathic effect that are due to reactivating virus. To determine if samples contained preformed infectious virus at harvest due to persistent replication, replicate cell aliquots were mechanically disrupted prior to limiting dilution and plating.
Limiting dilution PCR analysis.
To determine the frequency of cells carrying the γHV68 genome, single-copy sensitivity nested PCR for the γHV68 v-cyclin (gene 72), was performed on serial dilutions of cells as described [1,24]. Primers for v-cyclin were 5′gagatctgtactcaggcacctgt3′ and 5′ggatttcttgacagctccctgt3′ for round 1, and 5′tgtcagctgttgttgctcct3′ and 5′ctccgtcaggataacaacgtct3′ for round 2. One false positive was detected in 246 reactions across all experiments. Positive control reactions containing ten, one, or 0.1 copies of v-cyclin plasmid DNA were positive in 99%, 42%, and 5% of all reactions, respectively.
Statistical analysis.
All data points represent the mean ± the standard error of the mean (SEM) for two to three experiments with three to five mice per condition per experiment. To quantify the frequency of cells from which the virus reactivated or that carried latent viral genome, data were subjected to nonlinear regression using GraphPad Prism (GraphPad, San Diego, California, United States). Frequencies were determined using the Poisson distribution assuming that the cell number at which 63.2% of the wells scored positive for reactivation or viral genome represented a single event. To calculate significance, data were statistically analyzed by unpaired t test. For limiting dilution analyses, the calculated frequencies were compared by unpaired t test.
Supporting Information
Figure S1. Control of Chronic γHV68 Infection Is Dependent on CD8 T Cells
(A) Frequency of cells reactivating from latency ex vivo (left), frequency of cells bearing viral genome (middle), and persistent replication (right) at day 42 post-infection in peritoneal cells from B6, Kb−/−xDb−/−, and CD8−/− mice. On the y-axis is the percentage of wells positive for viral cytopathic effect (left and right) or viral genome (middle). The horizontal line within the graph indicates the 63.2% Poisson distribution line used to calculate the frequency of cells reactivating virus. Data are the mean of three to four independent experiments ± SEM.
(B) Frequency of latent infection in splenocytes from B6, Kb−/−xDb−/−, and CD8−/− mice. Data are the mean of three to four independent experiments ± SEM.
Figure S2. Efficacy of CD8 T Cell Depletion as Assessed by Flow Cytometry
Splenocytes from Kb−/−xDb−/−xCD1d−/− mice that were either treated with a CD8-depleting antibody (Ab) or an irrelevant control antibody were harvested 16 d after γHV68 infection and analyzed by flow cytometry. Panels show representative flow cytometric analysis of CD4 and CD8 expression on splenocytes.
We would like to thank Albert Bendelac and Ted Hansen for providing mice. We thank Ted Hansen for many helpful discussions.
Author Contributions
All the authors conceived and designed the experiments. DCB, JSM, IM, and KBM performed the experiments. All the authors analyzed the data. JNZ and HWV contributed reagents/materials/analysis tools. DCB, JSM, and HWV wrote the paper.
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- 76. Soloski MJ, Szperka ME, Davies A, Wooden SL (2000) Host immune response to intracellular bacteria: A role for MHC-linked class-Ib antigen-presenting molecules. Proc Soc Exp Biol Med 224: 231–239.
- 77. Chun T, Serbina NV, Nolt D, Wang B, Chiu NM, et al. (2001) Induction of M3-restricted cytotoxic T lymphocyte responses by N-formylated peptides derived from Mycobacterium tuberculosis. J Exp Med 193: 1213–1220.
- 78. Lo WF, Ong H, Metcalf ES, Soloski MJ (1999) T cell responses to gram-negative intracellular bacterial pathogens: A role for CD8(+) T cells in immunity to Salmonella infection and the involvement of MHC class Ib molecules. J Immunol 162: 5398–5406.
- 79. Byers DE, Lindahl KF (1998) H2-M3 presents a nonformylated viral epitope to CTLs generated in vitro. J Immunol 161: 90–96.
- 80. Fung-Leung WP, Schilham MW, Rahemtulla A, Kundig TM, Vollenweider M, et al. (1991) CD8 is needed for development of cytotoxic T cells but not helper T cells. Cell 65: 443–449.
- 81. Koller BH, Marrack P, Kappler JW, Smithies O (1990) Normal development of mice deficient in beta 2M, MHC class I proteins, and CD8+ T cells. Science 248: 1227–1230.
- 82. Zijlstra M, Bix M, Simister NE, Loring JM, Raulet DH, et al. (1990) Beta 2-microglobulin deficient mice lack CD4–8+ cytolytic T cells. Nature 344: 742–746.
- 83. Chen YH, Chiu NM, Mandal M, Wang N, Wang CR (1997) Impaired NK1(+) T cell development and early IL-4 production in CD1-deficient mice. Immunity 6: 459–467.
- 84. LeMaoult J, Messaoudi I, Manavalan JS, Potvin H, Nikolich-Zugich D, et al. (2000) Age-related dysregulation in CD8 T cell homeostasis: Kinetics of a diversity loss. J Immunol 165: 2367–2373.
- 85. Smith SC, Allen PM (1991) Myosin-induced acute myocarditis is a T cell-mediated disease. J Immunol 147: 2141–2147.
- 86. Radka SF, Machamer C, Cresswell P, Kostyu DD, Ward FE, et al. (1983) SFR3-DR5, a monoclonal antibody with HLA-DR5 specificity. J Immunol 130: 1863–1866.


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Pain Management Center - Santa Barbara, CA
- WebMD Physician Directory of Santa Barbara Pain Management Physician Doctors
- Related Pain Management Articles
- Santa Barbara Emergency Contacts
- Nearby Santa Barbara Hospitals
- Find a Local Pharmacy on RxList - Santa Barbara
Santa Barbara Pain Management Physician Doctors for Pain Management
Type of Physician: Pain Management Physician
What is a Pain Management Physician? An interdisciplinary subspecialty certification by the Boards of Anesthesiology, Psychiatry & Neurology, and Physical Medicine & Rehabilitation; practitioners provide a high level of care either as a primary physician or consultant for patients experiencing problems with acute or chronic pain in both inpatient and outpatient settings, and coordinate a multidisciplinary approach toward pain management.
Specialty: Pain Management
Common Name:
Pain Management Physician Doctors in Santa Barbara * results
Don't see your city? Use the WebMD Physician Directory to expand your search.
Pain Management
Introduction to pain management
Pain management can be simple or complex, depending on the cause of the pain. An example of pain that is typically less complex would be nerve root irritation from a herniated disc with pain radiating down...
Other Related Pain Management Articles
Emergency Contact for Santa Barbara
- In case of Emergency, call 911
Nearby Santa Barbara Hospitals results
- 1Santa Barbara Cottage Hospital
400 W Pueblo St
Santa Barbara, California 93105
(805) 682-711
- 2Cottage Rehabilitation Hospital
2415 De La Vina St
Santa Barbara, California 93105
(805) 682-711
- 3Psychiatric Health Facility
315 Camino Del Remedio
Santa Barbara, California 93110
(805) 681-524
- 4Goleta Valley Cottage Hospital
351 S Patterson Ave
Santa Barbara, California 93111
(805) 967-341
- 5Devereux California
6980 Falberg Way
Santa Barbara, California 93117
(805) 968-252
- 6Ojai Valley Community Hospital
1306 Maricopa Hwy
Santa Barbara, California 93023
(805) 646-140
- 7Aurora Vista Del Mar Hospital
801 Seneca St
Santa Barbara, California 93001
(805) 653-643
- 8Community Memorial Hospital
147 N Brent St
Santa Barbara, California 93003
(805) 652-501
- 9Ventura County Medical Center
3291 Loma Vista Rd
Santa Barbara, California 93003
(805) 652-600
- 10Santa Ynez Valley Cottage Hosp
2050 Viborg Rd
Santa Barbara, California 93463
(805) 688-643
- 11Pacific Shores Hospital
2130 N Ventura Rd
Santa Barbara, California 93036
(805) 604-121
- 12Saint John's Regional Med Ctr
1600 N Rose Ave
Santa Barbara, California 93030
(805) 988-250
* Provider Directory Terms of Use:
The WebMD 'Provider Directory' is provided by WebMD for use by the general public as a quick reference of information about Providers. The Provider Directory is not intended as a tool for verifying the credentials, qualifications, or abilities of any Provider contained therein. Inclusion in the Provider Directory does not imply recommendation or endorsement nor does omission in the Provider Directory imply WebMD disapproval.
You are prohibited from using, downloading, republishing, selling, duplicating, or "scraping" for commercial or any other purpose whatsoever, the Provider Directory or any of the data listings or other information contained therein, in whole or in part, in any medium whatsoever.
The Provider Directory is provided on an "AS-IS" basis. WebMD disclaims all warranties, either express or implied, including but not limited to the implied warranties of merchantability and fitness for particular purpose. Without limiting the foregoing, WebMD does not warrant or represent that the Provider Directory or any part thereof is accurate or complete. You assume full responsibility for the communications with any Provider you contact through the Provider Directory. WebMD shall in no event be liable to you or to anyone for any decision made or action taken by you in the reliance on information provided in the Provider Directory.
The use of WebMD Provider Directory by any entity or individual to verify the credentials of Providers is prohibited. The database of Provider information which drives WebMD Provider Directory does not contain sufficient information with which to verify Provider credentials under the standards of the Joint Commission on Accreditation of Healthcare Organizations (JCAHO), National Committee for Quality Assurance (NCQA) of the Utilization Review Accreditation Committee (URAC).
By using the WebMD Provider Directory, you agree to these Terms and Conditions.

subset: commoncrawl


text:
Would You Rather Work For Jeff Lewis or Jenni Pulos' Daughter?
July 14, 2016 • 10:01 PM ET
This season on Flipping Out brings us an extra dose of cute with adorable daughter Alianna. Jenni shares that, despite Alianna being in her tantrum stage, working for has fully prepared her to deal with anything and everything the...Read more
Shop Jeff Lewis' Favorite Designs
July 13, 2016 • 4:37 PM ET
If you're anything like us, you're left drooling at the design perfection every time takes on a room. The colors, the art, the furniture, all come together in a way that only Jeff Lewis Design could. If only our spaces could be so...Read more
The 12 Best (And Snarkiest) Jeff Lewis Quotes Ever
July 11, 2016 • 3:16 PM ET
Jeff Lewis doesn't sugarcoat anything unless it's a cocktail. We've come to know and love his hilarious honesty over the past eight seasons of Flipping Out and boy, are we ready for more. While we patiently wait to hear what will come out of his...Read more
Jenni: Dreams Do Come True
August 19, 2015 • 10:01 PM ET
Miracles Happen and Dreams Do Come True Jeff pre orders from the carNo menu searching/time wasting for this dinnerThe Catch is where we meet the potential surrogateYes, that is the name of the restaurant Jeff pickedAlexandra is blond and her...Read more
Jenni: Jeff Fanned the Flames
August 12, 2015 • 10:01 PM ET
Nastiness Does Not Solve Problems (I'm sure they teach this at Harvard Business School) I left his schedule on the deskWe are in the carMemo to Mr. Lewis Nastiness does not solve problemsLosing Stewie and the rushed moveEmotions are raw Gage's...Read more
Jenni on Joe's Dismissal
August 5, 2015 • 10:01 PM ET
Hooray for Hollywood…Off Sunset Jeff's house on GramercyHe said, she saidGate closedMoving to Hollywood Jeff is counting the days and nightsZoila will not miss the steps"50 up and 50 down" Jeff wants his appointments in the calendarThe word...Read more
Jenni: Jeff Wants George Eads to Be Happy
July 29, 2015 • 10:01 PM ET
What's Mud Go to Do With It? Jeff has plans for Finley's three bungalows1. The office 2. Jeff and Gage 3. Zoila Raznik who works with FranciscoRates top five for charactersMy previous pregnancy weight concerned JeffHe admits his teasing about it...Read more


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Bernardaud Naxos Serveware
Exemplifying a contemporary yet classic aesthetic, our Naxos dinnerware is simple and refined. Geometric motifs give depth and force to pure white porcelain, providing a clean canvas for your own culinary masterpieces. The porcelain ware is made by Bernardaud, the venerable family-run company established in Limoges in 1863. Dishwasher safe. Made in France.
Customer Reviews for
Bernardaud Naxos Serveware
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text:
Form Timeline
Click here to view Form Timeline for runners
Thu, 17th Jan, 2013
View Using Active Card View all races on one page
Formscan By Eamonn Murphy - Ruby Walsh has only ridden MIKAEL D'HAGUENET twice over fences – when coming down in the 2011 RSA Chase and when below par after that in the Powers Gold Cup. Basically it'll be a surprise if the French-bred has any problems in the jumping department over these obstacles, with Walsh up, and if he gets round he should beat these rivals. Anyone that read Aisling Crowe's piece in last weekend's Sunday Independent will be in no doubt about the magnitude of the achievement in having Western Leader back racing. He cheated death at Aintree in 2010 and it was something else to see him back in the number one berth at Fairyhouse on New Year's Day. He'll likely give Mikael D'Haguenet most to do. He'llberemembered is a decent horse in his own right but it'd be a surprise if he were to prove up to the task here. Tabhachtach needs luck to come out on top against this class of opponent.
Show Cards Help
A novice steeplechase for five yrs old and upwards which are E.B.F. eligible and have not won more than one steeplechase
Weights - A novice steeplechase for five yrs old and upwards which are E.B.F. eligible and have not won more than one steeplechase
Allowances - 5-y-o 10st 8lb; 6-y-o and up 11st 5lb; Mares allowed 7lb
Riders - Winner of a steeplechase 7lb extra;
Probable SP 11/8 Mikael D'Haguenet, 11/4 Western Leader, 7/1 He'llberemembered, 16/1 Tabhachtach.
Previous Years
2012 Quietly Fancied 8 11-0 Mr R P Quinlan 8/1 (Oliver McKiernan)


text: My Red Little Boots by Mon Petit Shoe £43.99$78.75€53.06$59.04 In stock This item is not available for delivery to United States. International delivery available sizesizeUK 4UK 4.5UK 5UK 7sizeUK 4UK 4.5UK 5UK 7sizeUK 4UK 4.5UK 5UK 7sizeUK 4UK 4.5UK 5UK 7 quantity by Mon Petit Shoe Location: London, GB read seller's profile see seller's complete range more items from this seller Striped Satin Baby Booties by Mon Petit Shoe £13.99$25.04€16.87$18.77 Damian Perforated Leather Shoes by Mon Petit Shoe £36$64.45€43.42$48.31 My Nursery Shoes by Mon Petit Shoe £36.99$66.22€44.62$49.64 Cuquito Out & About Shoe by Mon Petit Shoe £18.99$33.99€22.90$25.48 Velvet Mary Jane Shoes by Mon Petit Shoe £38.99$69.80€47.03$52.33 Little Marine Shoes by Mon Petit Shoe £22.50$40.28€27.14$30.19 view all you may also like PRODUCT DETAILS DELIVERY RETURNS ask seller a question save for later These lovely bordeux boots are a dream come true for little girls. They are warm, comfy, amazing quality, gorgeous design and water proof. These cute little boots are made by the luxury footwear brand Step2wo. The foundation of the Step2wo collection is its edgy, yet elegant approach to fashion, with each piece closely based on current adult trends. The collection is of the utmost quality and luxury and is designed in London and manufactured in Italy. They are great shoes for the winter and they will give yor little girl that extra touch of chicness when wearing. The bordeaux color is just stunning on these little shoes. made from: These toddler patent booties are available in bordeaux color and feature brogue detailing. They also feature a patent bow on the Velcro strap and have a Step2wo tag on the side of the shoe. They come up to the ankle wich is also great since it gives the child extra support while walking. dimensions: These boots come in the following sizes: UK 4/ EU 20 UK 4.5/ EU 21 UK 5/ EU 22 UK 6/ EU 23 UK 7/ EU 24 product code: 195773 This product is dispatched to you by Mon Petit Shoe. MAINLAND UK order now and receive this item by price Tracked Two Day Delivery Tuesday 2 August £2.95$5.28€3.55$3.95 Free Delivery Thursday 4 August FREEFREEFREEFREE NON-MAINLAND UK estimated delivery price Highlands & Islands of Scotland 2-5 working daysfrom dispatch FREEFREEFREEFREE Northern Ireland 2-5 working daysfrom dispatch FREEFREEFREEFREE Other UK Islands 2-5 working daysfrom dispatch FREEFREEFREEFREE INTERNATIONAL DELIVERY estimated delivery price Western Europe 2-5 working daysfrom dispatch £15.50$27.74€18.69$20.80 Rest of Europe 5-10 working daysfrom dispatch £15.50$27.74€18.69$20.80 You have 28 days, from receipt of cancellable goods, to notify the seller if you wish to cancel your order or exchange an item. Please note: goods that are personalised, bespoke or made-to-order to your specific requirements, perishable products and personal items sold with a hygiene seal (cosmetics, underwear) in instances where the seal is broken are non-refundable, unless faulty. Read more about our returns policy.Should you choose to return or exchange your order you will need to deliver the item(s) to the UK, where this seller is based. Mon Petit Shoe's return policy Please see notonthehighstreet.com's returns policy.


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In This Section
Journal Forum
A Meta-Analysis of High Resolution Audio Perceptual Evaluation - June 2016
2 comments
Synthetic Reverberator - January 1960
3 comments
Sound Board: High-Resolution Audio - October 2015
4 comments
AES E-Library
A Simplified-Hysteresis-Loop Model of the AC Biased Magnetic Recording Process
As a consequence of the well known complexity of the phenomena used for the magnetic recording of signals it seems to be very difficult, if not impossible to give one single theoretical picture which includes all the aspects of the recordng process. -Models- have been used instead, from which some factors are omitted in order to see the influence of other factors more clearly. It should be stressed that generally these models are not approximations in the mathematical sense of the word. Whereas in a mathematical approximation higher order effects are neglected, here the complex reality is replaced by a simplified scheme which can be handled. The final purpose of this procedure of -separating the variables- is to get the parts of the whole picture which can be put together to form an approximate quantitative description of a given recording configuration.
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Arsenal tops Cardiff, keeps Premier League lead
Updated: January 1, 2014, 3:01 PM ETAssociated Press
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Bermuda has a fantastic system of public transportation. You can purchase a daily pass or one for multiple days. Passes are good on the ferry which have schedules that go from St. George's, Hamilton and King's Wharf. From the ferry you can transfer to busses that take you all over the island. Some of the buses are referred to as sightseeing buses. #1 from the Grotto hotel is one. The driver was amazing, pointing out sights and plants. She stopped several times to point things out such as a rock outcropping that looks like George Washington. Drivers are very polite and friendly.
Own or manage this property? Claim your listing for free to respond to reviews, update your profile and much more.


text:
Hotel Duquesne Eiffel
No prices found through our partners. Please contact the business directly or check some of our recommended alternatives.
How has this hotel rated in the past?
- Airport Transportation
- Shuttle bus service
- Minbar in room
- Wireless internet connection in room (free)
- Breakfast Available
- Non-smoking hotel
Photos of Hotel Duquesne Eiffel
TravelPod Member ReviewsHotel Duquesne Eiffel Paris
Very nice hotel located in the 7th arrondissement. Our room was very comfortable with a view of the Eiffel Tower. Room and bathroom was very clean and nicely appointed. Desk staff was friendly and helpful.
Breakfast was delicious, typical Parisian, with great fresh breads. There is a lounge in the lobby to relax and have an evening nightcap.
All in all, a nice place to stay and a good location for touring. Pretty close to the Metro and very walkable sites nearby.
We would definitely stay again and would recommend it to friends.
This review is the subjective opinion of a TravelPod member and not of TravelPod.com.
Historical Traveler Reviews Hotel Duquesne Eiffel Paris
Small, but cozy
Visited hotel for Easter Holidays 2005, stayed in a superior room on 5th floor with my husband, with a wonderful view and only 10 minutes from the Torre Eiffel and 3 minutes from metro.
The staff was very helpful and the room was small, but very clean and with a small balcony.
Would suggest this hotel to all that would like to spend some days in beautifl Paris.
Would definitely stay here again...
Having originally planned to stay only Christmas eve and Christmas, we changed later reservations to this hotel for our whole trip. Excellent accomodations, newly remodeled (small, but aren't they all?) - view out our window of the Eiffel Tower was requested and fulfilled. Staff extremely attentive and polite. Felt comfortable here with limited knowledge of French. Went above and beyond, even assisting in getting a doctor when needed.
Near Eiffel Tower, Invalides, Metro Stop (Ecole Militaire), walking distance to Seine, museums, etc. The neighborhood is a bit sleepy, and there are hipper places to stay, but we cannot say enough good things about this hotel, and we'll be back again and again.
Not planned but great
We were scheduled for another hotel in the area for almost 10 months so when we arrived to find out our original hotel had a problem and would not be able to accomodate us I was dismayed. However they sent us to the Duquesne. We were pleasantly surprised. The rooms are small but very clean and the breakfast was good too. It is a couple of blocks from the nearest Metro station and the neighborhood is very nice. It is residential but there is alot of restuarants nearby and a grocery store. You are within walking distance of the rue cler and the bus lines run very close by. I am very glad we stayed with them, it was a very pleasant experience.
Loved the View!
We stayed at the Duquesne Eiffel for 3 wonderful nights. We had a "superior" room, which was in the corner and had windows on two walls, one of which looked out on the Eiffel Tower! The room was the perfect size, and very tastefully decorated. The breakfast, which is a bit expensive at 11 Euros, was WORTH EVERY PENNY! Fabulous. I found the staff to be very friendly and helpful. I can hardly wait to return to Paris, and I will definitely be staying here again!
Romantic . . .
I have stayed at the Duquesne Eiffel twice. It is a beautiful little hotel, very charming. I liked it so well the first time that I took my wife there on our honeymoon. I booked it well in advance, and I told the management that we would be on our honeymoon, so they gave us Room 55--in the corner on the top floor, with doors opening to the balcony on two sides, and a spectacular view of the Eiffel Tower. I have wonderful memories of the romance, watching the sunset behind the Eiffel Tower.We found the staff friendly and accommodating. The hotel is ideally situated close to the subway and major tourist attractions--without being "touristy" itself. The rooms are nicely decorated. They're not large, but who cares? It's Paris! I want to go back and do it all over again.
Clean and charming, but the tiniest room I've ever seen in Paris!
I travel to Paris a few times a year and have stayed in a number of different hotels. The Duquesne is ok. The room was well decorated and clean, but honestly, even by Parisian standards, it was small! There was no place to put the luggage, let alone walk from the side of the bed to the bathroom. The staff was helpful, but not especially friendly. The neighborhood is nice -- walking distance to the Eiffel Tower and the Invalides -- but not especially interesting. It is an upper class neighborhood, completely residential. So if you like to windowshop, forget it. There are some decent little restaurants, but they are filled with American tourists. If this is what you want, then try the Dusquesne. But please don't go there with great expectations.
Just sooooo sweet
My stay at the Duquesne Eiffel Hotel left me with a wonderful impression of Paris. This was my first trip out of the country and I was very nervous. My room was a little small, but still comfortable, and much larger than my friend's room at another hotel down the street.
The hotel staff was very helpful and made me feel that I was being given an insight into Paris that I could not have received elsewhere.
Thanks for a wonderful adventure!
Almost ruined my anniversary
We just came back from celebrating our 2nd Anniversary in Paris. We arrived early at the hotel and were able to be accommodated on the 2nd floor. Unfortunately, the glass panes are not double insulated so you hear the noise perfectly clear. My husband is 6-4 220lbs so when we opened the door . . . he asked me where I was going to sleep.
We went downstairs to see if anything can be done about the accommodations. The front desk receptionist said there was nothing she can do. It wasn't what she said as much as HOW she said it. She was blatantly rude and had no customer service skills at all. She said the higher floors were more expensive - as if we couldn't afford it - how ignorant of an assumption was that?!?!?!?! At the end of trying to find a solution, she said there's nothing she can do for us and suggested we'd try to find another hotel for our stay.
Luckily the Best Western up the street recommended a great and spacious hotel "Derby Garibaldi" with the customer service you'd expect when you're on vacation. Bon Voyage!
Loved this hotel
I was extremely pleased with this hotel. I booked an entire package through ... and was a bit wary as this was my first trip to Paris. They told me it was on a lovely tree lined street in a quiet neighborhood with a great view of the Eiffel Tower from the higher floors. The people who ran the hotel spoke English and were very helpful. Breakfast was included every day and the bus and metro stops were only at the end of the street. Lots of nice, inexpensive restaurants around the hotel where it seemed mostly French people were eating- not too many tourists. I would go back again and again to this hotel and also with France Vacations who were very knowledgeable.
Quiet & Quaint
This is a very quaint hotel about 10 minutes walking distance from the eiffel tower and I could see it from our small balcony. It was also centrally located two blocks from a subway station, which made it very easy to travel to other parts of the city. The rooms were very nice, but small. The bathroom was beautiful, with marble floors and walls. A traditional French breakfast was served every morning in the cellar. If you want an easy location to tour the city from, yet in a quiet setting, this is the place!
TripAdvisor Reviews Hotel Duquesne Eiffel Paris
Travel Blogs from Paris
Starting off in Paris
After a couple of easy flights, we arrived at Charles De Gaulle airport at 7:30am. It took a little while to get to and through passport control, but nothing terrible. They just seemed to be a little short staffed early in the morning.
We had decided to take the RER train instead of a shuttle service or taxi, but we won't do that next …
Paris on Two Wheels (Day Nine)
... avenue. This is one of the many streets that leads to a huge roundabout with the monumental Arc de Triomphe in the middle. To get to the centre, you have to go through an underground walkway, but I saw one family who crossed at street level. I was worried for them - but they somehow made it without getting run over! They found it very amusing after making it safely, but I don't recommend trying it. I decided to fork over 10 euros to go to the top of the ...


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Ready in 45 minutes
I LOVE Popeye's Biscuits, and if you don't have a Popeye's around you, they are very similar to KFC's, but sooo much better.....
548 people want to try | 626 have favorited
Preheat oven to 400
1. Mix together flour, sugar, salt, baking powder and baking soda in a medium bowl.
2. Slice cold butter into cubes and use a pastry blender to cut butter into dry mixture (a pastry blender is a GREAT thing to have, but if you don't have it, use two knives and cut the butter in) until no large chunks of butter remain.
3. Add buttermilk and milk and stir with a spoon until dough forms.
4. Roll out to 1/2 inch thick on a floured surface.
5. Cut biscuits with a 3 inch biscuit cutter or glass and arrange on a lightly greased baking sheet.
6. Bake for 22 to 24 minutes or until tops begin to turn a light brown.
7. Remove from oven and then brush melted butter on top
katieprovencher 2y ago
CupcakeGrenade 2y agoFantastic recipe, very yummy. I do wish they would have raised a little but higher though, I think next time I'll add half a teaspoon of cream of tartar. I added some freshly grated marble cheese to my recipe, tasted fantastic :)
Twistedmama 2y agoGreat biscuit recipe. Made a few modifications by using just milk, and they still turned out moist and delicious reminding us of Popeye's Biscuits.
lyvlaughlove7_13_2012 3y agoI tried this recipe. It worked great!!! Excellent for breakfast sandwiches!
katalia369 3y agoVery good
Sofia78 3y ago
Vdanielle30 3y ago
mogriff1 3y agoMine turned out flat and firm and looked more like cookies. They tasted nothing like Popeye's.
melinikki 3y agoLoved this recipe, biscuits came out so moist n delicious, will use this recipe for years to come!
dreid4 3y ago


text:
Analog Meter Megohmmeters Datasheets
from Duncan Instruments Canada, Ltd.
The MI-1050e general purposes megohmmeter is a truly portable equipment suitable to measure insulation resistances using test voltages up to 1 kV. It employs high-reliable, state-of-the-art technology for accurate measurements of ultra high insulation resistances up to 2.000.000 MO with four test... [See More]
- Display: AnalogMeter
- Resistance Range: 2.00E8 to 2.00E9
- DC Volts Out: 100 to 1000
- Testing Method: Semi-automatic
from RS Components, Ltd.
Type:Insulation tester; Maximum Test Voltage:5000V; Minimum Test Voltage:500V; Test Voltage Ranges:4; Display Type:Analog; Safety:IEC 1010-1, Cat. III EN61010 300 V; Timer PI and DAR:No [See More]
- Display: AnalogMeter
- DC Volts Out: 500 to 5000
from Weschler Instruments
The AEMC Model 6501 megohmmeter is a compact, self-contained, hand-cranked true megohmmeter. This practical and dependable instrument is designed for a broad range of plant and field service applications, such as acceptance testing and preventive maintenance of wiring, cables, switchgear and motors. [See More]
- Display: AnalogMeter; LEDIndicator
- Form Factor: Portable
- Resistance Range: 0.0 to 200000
- Safety Agency Rating: EN-61010-1, 600V CAT II, 300V CAT III
from All-Spec Industries
All points [See More]
- Display: AnalogMeter
- DC Current Out: 2.50E-4
- DC Volts Out: 1000
- Resistance Range: 1000 to 2.00E6
from Amprobe Test Tools
Tests insulation of wires, cables, transformers, motors and generators [See More]
- Display: AnalogMeter
- Testing Method: Manual
- Resistance Range: 0.0 to 100000
- Form Factor: Hand Held
from Chroma ATE, Inc.
High accuracy, hi and lo limit display, GO/NG judgment [See More]
- Display: AnalogMeter; DigitalMeter
- Resistance Range: 0.0 to 1.00E7
- DC Volts Out: 10 to 1000
- Power consumption: 90
from Seaward Group USA
A versatile portable test set providing simple straightforward operation and effective electrical safety testing of office equipment, power tools and appliances. The A252 electrical safety tester performs earth bond, flash and insulation tests. [See More]
- Display: AnalogMeter; LEDIndicator
- Resistance Range: 0.0 to 100000
- DC Volts Out: ? to 500
- Testing Method: Manual


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Freedom From Pants Ride
There's no better way to celebrate your independence than by dropping trou, hopping on your bike, and cruising around town feeling the breeze. That's exactly what's going to happen this July 4 holiday, during the annual Freedom From Pants Ride through downtown Minneapolis. The event starts promptly at 6 p.m. in the lot next to the Soap Factory. It begins as all good parties do: with some pre-ride socializing and disrobing. Riders will take off at 7 p.m., at which point pants-free participants will cruise through town with an occasional stop for drinks (BYOB; no bar stops) before returning to the Soap Factory for fireworks. Last year's event had over 200 participants of all ages, biking abilities, and underwear fashions, and the organizers are planning for an even bigger turnout this year. As for what's acceptable in terms of dress code, it's fairly open-ended. The only guidance provided is "flattering is good, funny is better, and flattering plus funny is a jackpot." Underwear, bikes, and freedom: If that doesn't scream "AMERICA!" what does? (Image by ThirteenofClubs via Flikr)
Wed., July 4, 6-11:30 p.m., 2012
Get the Things to Do Newsletter
Sign up for our weekly guide to events in Minneapolis & St. Paul, and never be bored again. With suggestions for every day of the week, our recommendations will keep you busy on any budget.
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514 2nd St. SE
Minneapolis, MN 55414
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text: I use the "Visual Subtitle" plugin which places a span tag in the title (http://wordpress.org/plugins/visual-subtitle/) . The span tag is being stripped out by this plugin. The previous version of Genesis Featured Widget Amplified did not do this, so I'm assuming this is something that changed with the upgrade. Can you have a look please?


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"What do you look for in a book of new poems? A voice like no other, incisive, musical? An imagination like no other trans-forming the world you thought you knew? A word-hoard deep enough for the demands of a big spender? Look and listen here." - Jon Stallworthy
"...the Howlin' Wolf of Hull poetry..." georgiasam blogCliff is a poet and painter. His latest collection Pilgrim Tongues (Wrecking Ball, 2015) travels from Hull to Vietnam and back, by way of Israel, Transylvania, California and Cambodia, continuing the voyages he embarked upon in Wake (Flarestack Poetry Pamphlet Prize 2009). Previous collections include: Vandemonian (Arc, 2013), which focuses on Van Diemen's land and its inhabitants – human and animal, newcomer and Aborigine – to piece together a fragmentary history of Tasmania Trans (The Collective Press, Wales, 2005) which culminates in a rewriting of Ovid's Metamorphoses. Hole, Cliff's satirical poem in which Philip Larkin leads us through a Dantean Hell (in search of Hull), is The Common online's March poetry feature: http://www.thecommononline.org/features/march-2016-poetry-feature NEWS: This website will be relaunched (at the same address) in the next week or so. There'll be more material, with many more and larger images of paintings, a slide-show facility, and more poems and translations from the books and ongoing projects. Do check back. Everything will be up, running, and also mobile-friendly by the end of July
"The high energy of Cliff Forshaw's poems makes me think particularly of John Donne and the other Metaphysicals: argument, wit, erudition and force of feeling all working to convey an authentic vision of the world we live in." Christopher Reid "As always, Cliff Forshaw's writing embodies a large intelligence. These are poems of voyage, exertion and discovery, enjoying the challenge of unpredictable and unusual locations, both geographical and psychological. At the same time, they demonstrate grounded, dependable craft. They never trick the reader, but, witty and exuberant, send us on our poetic journeys with new imaginative maps." Carol Rumens "Cliff Forshaw is a poet of rooted non-attachments, a nomad of the suburbs and a boulevardier of the wild places. As maps go, Pilgrim Tongues is the one that will get you lost, but you'll thank its author for it, later, or maybe even at the time." David Wheatley "The voice here is distinctive and mercurial - cool, intelligent yet engaged - the spirit of Larkin, perhaps, re-emerging, muscular and revitalised, in the 21st century." Flarestack Prize judges. " a dizzying, noisy world tour viewed through the prism of classical learning and told with dazzling accomplishment and technique. With a voice that is frank and inquisitive, the poet tackles complex themes and ideas with enviable accessibility and wit ... a rich, densely populated collection that speaks to the mind and the ear. It is multilingual and peripatetic in its concerns and always open to the strange and new. Above all this collection is characterised by an iridescent lyricism."Christopher James in Iota 95
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Hello everyone. I'm looking for a template that has a front page that shows only snippets of posts from a pre-defined set of categories.
For example:
Coda slider-type thing
snippet from | snippet from | snippet from |
latest post in | latest post in | latest post in |
category 1 | category 2 | category 3 |
If the snippets could display a specified image, that would be great too.
If it exists out there, I'd love to know about it, and I'm willing to pay if necessary. If it's NOT out there, can anyone direct me as how to do this?
I'm using wordpress 2.9.
Thank you so much!


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The printer is wireless and I haven't connected it via USB. I set the configurations for the Wi-Fi access point in the printer and it connected to my Wi-Fi. I have AT&T as the service provider and the 2Wire router. When I access the router in the browser I see that the printer is connected but when I run the software to detect the printer the computer can't find it. I have a Mac with Snow Leopard. I tried to detect the printer with another device and it can't find it. Is there a port that needs to be open for wireless printers? Because I tried putting the routers firewall settings for the device to its lowest and the computer still can't find it. Let me know. Thnx.
Have you tried inserting the IP address instead of using Bonjour to detect your printer (which I'm assuming you're doing)?
Check for the IP address in your router's configuration pages. Or it might show on your printer's screen?


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Andrew Young, Part I
NPR's Tavis Smiley talks with Andrew Young, a former U.S. ambassador to the United Nations, about the upcoming Leon H. Sullivan Summit, formerly the African-African American Summit. The week-long event will convene in Nigeria's capital on July 12. The late Rev. Leon H. Sullivan founded the summit movement as a tool to assist the human, economic and business development of Africa with special emphasis on Sub-Saharan Africa.
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