Evidence obtained from both animal models and humans suggests that T cells specific for HSV-1 and HSV-2 glycoprotein D (gD) contribute to protective immunity against herpes infection. However, knowledge of gD-specific human T cell responses is limited to CD4+ T cell epitopes, with no CD8+ T cell epitopes identified to date. In this study, we screened the HSV-1 gD amino acid sequence for HLA-A*0201-restricted epitopes using several predictive computational algorithms and identified 10 high probability CD8+ T cell epitopes. Synthetic peptides corresponding to four of these epitopes, each nine to 10 amino acids in length, exhibited high-affinity binding in vitro to purified human HLA-A*0201 molecules. Three of these four peptide epitopes, gD53–61, gD70–78, and gD278–286, significantly stabilized HLA-A*0201 molecules on T2 cell lines and are highly conserved among and between HSV-1 and HSV-2 strains. Consistent with this, in 33 sequentially studied HLA-A*0201-positive, HSV-1-seropositive, and/or HSV-2-seropositive healthy individuals, the most frequent and robust CD8+ T cell responses, assessed by IFN-γ ELISPOT, CD107a/b cytotoxic degranulation, and tetramer assays, were directed mainly against gD53–61, gD70–78, and gD278–286 epitopes. In addition, CD8+ T cell lines generated by gD53–61, gD70–78, and gD278–286 peptides recognized infected target cells expressing native gD. Lastly, CD8+ T cell responses specific to gD53–61, gD70–78, and gD278–286 epitopes were induced in HLA-A*0201 transgenic mice following ocular or genital infection with either HSV-1 or HSV-2. The functional gD CD8+ T cell epitopes described herein are potentially important components of clinical immunotherapeutic and immunoprophylactic herpes vaccines.

In this report, rat hypothalamic nitric oxide synthase (NOS) activity is shown to be partially inhibited by physiological concentrations of the pineal hormone melatonin. In vitro studies demonstrate that 1 nM melatonin, which approximates the physiological concentration of the hormone at night, significantly inhibited NOS activity. In vivo studies show that administering melatonin or collecting the hypothalamus from animals at night, when endogenous melatonin levels are elevated, results in a significant decrease of NOS activity. Results also show that calmodulin may be involved in this process since its presence in the incubation medium prevents the inhibitory effect of melatonin on NOS activity.

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The muscarinic-gated atrial potassium (I KACh ) channel contributes to the heart rate decrease triggered by the parasympathetic nervous system. I KACh is a heteromultimeric complex formed by Kir3.1 and Kir3.4 subunits, although Kir3.4 homomultimers have also been proposed to contribute to this conductance. While Kir3.4 homomultimers evince many properties of I KACh , the contribution of Kir3.1 to I KACh is less well understood. Here, we explored the significance of Kir3.1 using knock-out mice. Kir3.1 knock-out mice were viable and appeared normal. The loss of Kir3.1 did not affect the level of atrial Kir3.4 protein but was correlated with a loss of carbachol-induced current in atrial myocytes. Low level channel activity resembling recombinant Kir3.4 homomultimers was observed in 40% of the cellattached patches from Kir3.1 knock-out myocytes. Channel activity typically ran down quickly, however, and was not recovered in the inside-out configuration despite the addition of GTP and ATP to the bath. Both Kir3.1 knock-out and Kir3.4 knock-out mice exhibited mild resting tachycardias and blunted responses to pharmacological manipulation intended to activate I KACh . We conclude that Kir3.1 confers properties to I KACh that enhance channel activity and that Kir3.4 homomultimers do not contribute significantly to the muscarinic-gated potassium current.

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