T cell reactivity during infectious mononucleosis and persistent gammaherpesvirus infection in mice

Intranasal infection of mice with murine gammaherpesvirus 68 causes a dramatic increase in numbers of activated CD8(+) T cells in the blood, analogous in many respects to EBV-induced infectious mononucleosis in humans. In the mouse model, this lymphocytosis has two distinct components: an early, conventional virus-specific CD8(+) T cell response, and a later response characterized by a dramatic increase among CD8(+) T cells that bear Vbeta4(+) TCRs. We previously demonstrated that Vbeta4(+)CD8(+) T cells recognize an uncharacterized ligand expressed on latently infected B cells in an MHC-independent manner. The frequency of Vbeta4(+)CD8(+) T cells increases dramatically following the peak of viral latency in the spleen. In the current studies, we show that elevated Vbeta4(+)CD8(+) T cell levels are sustained long-term in persistently infected mice, apparently a consequence of continued ligand expression. In addition, we show that Vbeta4(+)CD8(+) T cells can acquire effector functions, including cytotoxicity and the capacity to secrete IFN-gamma, although they have an atypical activation profile compared with well-characterized CD8(+) T cells specific for conventional viral epitopes. The characteristics of Vbeta4(+)CD8(+) T cells (potential effector function, stimulation by latently infected B cells, and kinetics of expansion) suggested that this dominant T cell response plays a key role in the immune control of latent virus. However, Ab depletion and adoptive transfer studies show that Vbeta4(+)CD8(+) T cells are not essential for this function. This murine model of infection may provide insight into the role of unusual populations of activated T cells associated with persistent viral infections.     

Analysis of virus-specific CD4(+) t cells during long-term gammaherpesvirus infection.

Major histocompatibility complex class II-mediated antigen presentation after intranasal infection with murine gammaherpesvirus 68 differs in mediastinal lymph nodes and spleen. Evidence that virus-specific CD4(+) T cells were being stimulated was found as late as 6 to 8 months after infection, and cells specific for the viral gp150(67-83) and ORF11(168-180) peptides were maintained as a fairly stable proportion of the total response.     

Type I interferon inhibition and dendritic cell activation during gammaherpesvirus respiratory infection

The respiratory tract is a major mucosal site for microorganism entry into the body, and type I interferon (IFN) and dendritic cells constitute a first line of defense against viral infections. We have analyzed the interaction between a model DNA virus, plasmacytoid dendritic cells, and type I IFN during lung infection of mice. Our data show that murine gammaherpesvirus 68 (gammaHV68) inhibits type I IFN secretion by dendritic cells and that plasmacytoid dendritic cells are necessary for conventional dendritic cell maturation in response to gammaHV68. Following gammaHV68 intranasal inoculation, the local and systemic IFN-alpha/beta response is below detectable levels, and plasmacytoid dendritic cells are activated and recruited into the lung with a tissue distribution that differs from that of conventional dendritic cells. Our results suggest that plasmacytoid dendritic cells and type I IFN have important but independent roles during the early response to a respiratory gammaHV68 infection. gammaHV68 infection inhibits type I IFN production by dendritic cells and is a poor inducer of IFN-alpha/beta in vivo, which may serve as an immune evasion strategy.     

Differential type I interferon induction by respiratory syncytial virus and influenza a virus in vivo

Type I interferon (IFN) induction is an immediate response to virus infection, and very high levels of these cytokines are produced when the Toll-like receptors (TLRs) expressed at high levels by plasmacytoid dendritic cells (pDCs) are triggered by viral nucleic acids. Unlike many RNA viruses, respiratory syncytial virus (RSV) does not appear to activate pDCs through their TLRs and it is not clear how this difference affects IFN-alpha/beta induction in vivo. In this study, we investigated type I IFN production triggered by RSV or influenza A virus infection of BALB/c mice and found that while both viruses induced IFN-alpha/beta production by pDCs in vitro, only influenza virus infection could stimulate type I IFN synthesis by pDCs in vivo. In situ hybridization studies demonstrated that the infected respiratory epithelium was a major source of IFN-alpha/beta in response to either infection, but in pDC-depleted animals only type I IFN induction by influenza virus was impaired.     

Development of a bioassay from isolated digestive gland cells of the cuttlefish Sepia officinalis L. (Mollusca Cephalopoda): effect of Cu, Zn and Ag on enzyme activities and cell viability

The purpose of this study was to establish a bioassay from isolated digestive gland cells of the cuttlefish Sepia officinalis in order to observe the effect of heavy metals on digestive enzyme activities. Digestive cells were isolated using a pronase enzyme that was removed by several washings of the cell suspension. Cell viability was tested by the MTT assay (3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium) and microscopic analysis. The results showed that isolated digestive cells could be maintained 24 h with preservation of whole digestive functionality, measured in terms of MTT test. In fact, the viability was maintained at a high level during 24 h and the intra- and extracellular digestive enzyme activities became stabilised rapidly. Furthermore, suspension cells responded to calcium ionophore and 8-Bromo-cAMP by an unspecific secretion of extracellular digestive enzyme, trypsin, which demonstrated that isolated digestive cells were functional. Using the bioassay, ecotoxicological studies showed that heavy metals could have effects on digestive enzyme activities after 24 h of an incubation time of the metal with the cells. In fact, zinc and silver affected trypsin and/or cathepsins specific activity of the cells. On the contrary, copper had no effect on digestive enzyme activities. Zinc, which is a trace element in all living animals, generated two different responses of cathepsins and cell viability. At a low concentration (0.02 μM), it increased viability and cathepsins specific activity, whereas at a high concentration (0.02 mM), zinc inhibited the cathepsins specific activity with an inhibition of cathepsins. For silver, whatever the tested concentration (0.02 mM or 0.02 μM), it has no impact on digestive gland isolated cell viability. Nevertheless, heavy metal induced high disturbance of enzymatic systems.     

Intranasal Administration of dsRNA Analog Poly(I:C) Induces Interferon-α Receptor-Dependent Accumulation of Antigen Experienced T Cells in the Airways

Polyriboinosinic-polyribocytoidylic acid (pIC), a synthetic dsRNA, acts as an adjuvant that boosts immune responses and protection. Intranasal (IN) administration of pIC has recently been used to adjuvant influenza virus vaccines; however, the effects of IN pIC administration on pulmonary T cell responses remain unclear. Here we show that a single IN administered dose of dsRNA into mice induced local Th1 chemokine production in the lungs and airways, and generated a biphasic and sustained migration of T lymphocytes to the airways. Furthermore, IN pIC-induced chemokine production and T cell recruitment to the airways were interferon-α receptor (IFNAR) signaling dependent. The effect of dsRNA on T cell recruitment to the airways was also dependent on the presence of high molecular weight (HMW) pIC, as a low molecular weight (LMW) pIC preparation known to only interact with TLR3 did not elicit the same effect on T cell migration to the airways, suggesting that the observed effects were dependent upon dsRNA recognition by multiple pattern recognition receptors (PPRs). IN pIC was additionally capable of stimulating low levels of T cell proliferation in the draining lymph nodes approximately 4–6 days after treatment that preceded a small population of de-novo T cells found in the airways by day 10. Taken together, these results demonstrate that the adjuvant effect of IN pIC that results in enhanced T cell proliferation and sustained T cell recruitment to the airways requires multiple PRRs and IFNAR signaling.     

Antibody-mediated control of persistent gamma-herpesvirus infection

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, establish life-long latency and can reactivate in immunocompromised individuals. T cells play an important role in controlling persistent EBV infection, whereas a role for humoral immunity is less clear. The murine gamma-herpesvirus-68 has biological and structural similarities to the human gamma-herpesviruses, and provides an important in vivo experimental model for dissecting mechanisms of immune control. In the current studies, CD28(-/-) mice were used to address the role of Abs in control of persistent murine gamma-herpesvirus-68 infection. Lytic infection was controlled in the lungs of CD28(-/-) mice, and latency was maintained in B cells at normal frequencies. Although class-switched virus-specific Abs were initially generated in the absence of germinal centers, titers and viral neutralizing activity rapidly waned. T cell depletion in CD28(-/-) mice with compromised Ab responses, but not in control mice with intact Ab responses, resulted in significant recrudescence from latency, both in the spleen and the lung. Recrudescence could be prevented by passive transfer of immune serum. These data directly demonstrate an important contribution of humoral immunity to control of gamma-herpesvirus latency, and have significant implications for clinical intervention.     

A Long-Term Response of Chlorophyll Fluorescence Induction to One-Shot Application of Cyanazine on Barley Plants and Its Relation to Crop Yield

Field-grown plants of spring barley (Hordeum vulgare L. cv. Akcent) in the growth phase 30 DC (beginning of stem extension) were exposed to a one-shot application of a commercial product containing cyanazine (Bladex 50 SC) in two doses, C₃₀ and C₆₀ (30 and 60 mg m⁻²). The reaction of the plant photosynthetic system was followed non-destructively using chlorophyll fluorescence induction (the O-J-I-P transient) within three weeks after the application in the fifth developed leaf and three further gradually appearing leaves. An immediate response of plants to the application of cyanazine and a regeneration of plants from cyanazine action were detected. The biological (plant dry mass) and crop yield production (the number and mass of grains in a spike) were analyzed in time of full ripeness. The crop yield was lowered by the herbicide effect to the same level for the two doses used. This revised version was published online in August 2006 with corrections to the Cover Date.