Elevated PD-1 expression and decreased telomerase activity in memory T cells of patients with symptomatic Herpes Zoster infection.

We investigated PD-1 levels on VZV-specific CD8+ T-cells of patients with zoster and the effect of PD-1 on the telomerase activity. CD3, CD8, CD137 and PD-1 expressions were analyzed on PBMCs from 9 symptomatic and 5 asymptomatic individuals. The effect of PD-1 blockade at the time of stimulation on the telomerase activity of non-senescent CD57-CD45RO+CD8+CD3+ memory T-cells was evaluated. PD-1 was elevated on CD8+ T-cells in patients. The frequency of PD-1+ and CD137- cells in total CD3+CD8+ T cells of patients was elevated compared to controls. Telomerase activity of non-senescent memory T-cells was lower than that of controls. Blockade of PD-1 at the time of stimulation increased telomerase activity of non-senescent memory T-cells, accompanied by increased CD137 expression. Low telomerase activity of the patients with reactivated zoster could be partially overcome by blocking PD-1 pathway.

Activation of H2AX and ATM in varicella-zoster virus (VZV)-infected cells is associated with expression of specific VZV genes.

Mammalian cells activate DNA damage response pathways in response to virus infections. Activation of these pathways can enhance replication of many viruses, including herpesviruses. Activation of cellular ATM results in phosphorylation of H2AX and recruits proteins to sites of DNA damage. We found that varicella-zoster (VZV) infected cells had elevated levels of phosphorylated H2AX and phosphorylated ATM and that these levels increased in cells infected with VZV deleted for ORF61 or ORF63, but not deleted for ORF67. Expression of VZV ORF61, ORF62, or ORF63 alone did not result in phosphorylation of H2AX. While BGLF4, the Epstein-Barr virus homolog of VZV ORF47 protein kinase, phosphorylates H2AX and ATM, neither VZV ORF47 nor ORF66 protein kinase phosphorylated H2AX or ATM. Cells lacking ATM had no reduction in VZV replication. Thus, VZV induces phosphorylation of H2AX and ATM and this effect is associated with the presence of specific VZV genes in virus-infected cells.

IFN-α production by human mononuclear cells infected with varicella-zoster virus through TLR9-dependent and -independent pathways.

Understanding the defense mechanisms of the host of an organism is important for infection control. In previous studies, we demonstrated that interferon-α (IFN-α), but not IL-12, was produced by human peripheral blood mononuclear cells infected with varicella-zoster virus (VZV). Here, we investigated what kind of cell(s) and which signal molecule(s) are involved in IFN-α production. Using cell isolation and ELISA, we found that plasmacytoid dendritic cells (pDCs) were responsible for IFN-α production during VZV infection. We also found that Toll-like receptor 9 (TLR9) was involved in VZV-induced IFN-α production because inhibitory CpG oligodeoxynucleotide inhibited IFN-α production. UV-inactivated VZV-induced IFN-α production was lower than that of active VZV, indicating another TLR9-independent pathway. Further studies demonstrated that double-stranded RNA-dependent protein kinase, but not DNA-dependent protein kinase was involved in VZV-induced IFN-α production. Together, these results suggest that pDCs play an important role in IFN-α production during VZV infection through TLR9-dependent and -independent pathways.

Histone deacetylases 1 and 2 are phosphorylated at novel sites during varicella-zoster virus infection.

ORF66p, a virion-associated varicella-zoster virus (VZV) protein, is a member of a conserved Alphaherpesvirinae kinase family with homology to herpes simplex virus US3 kinase. Expression of ORF66p in cells infected with VZV or an adenovirus expressing only ORF66p results in hyperphosphorylation of histone deacetylase 1 (HDAC1) and HDAC2. Mapping studies reveal that phosphorylation is at a unique conserved Ser residue in the C terminus of both HDACs. This modification requires an active kinase domain in ORF66p, as neither protein is phosphorylated in cells infected with VZV lacking kinase activity. However, hyperphosphorylation appears to occur indirectly, as within the context of in vitro kinase reactions, purified ORF66p phosphorylates a peptide derived from ORF62p, a known substrate, but does not phosphorylate HDAC. These results support a model where ORF66p is necessary but not sufficient to effect hyperphosphorylation of HDAC1 and HDAC2.

Expression of inducible nitric oxide synthase in skin lesions of acute herpes zoster.

Histopathologically, the skin lesions of acute herpes zoster (AHZ) are characterized by epidermal necrotic vesicles with inflammation. Nitric oxide (NO) is generated from L-arginine by nitric oxide synthase (NOS), and immune inflammation involves the activation of NOS in both effector cells and target cells. NO can cause apoptosis and necrosis of target cells such as keratinocytes. We proposed that a large burst of NO in AHZ may cause the epidermal necrosis. Skin biopsies were taken from 13 patients with AHZ. The expression of inducible-type NOS (iNOS) was examined by immunoperoxidase staining and reverse transcription-polymerase chain reaction (RT-PCR). In the skin specimen of AHZ, moderate-to-strong staining for iNOS was observed in inflammatory cells and necrotic keratinocytes, while weak staining was observed in non-necrotic peripheral keratinocytes. RT-PCR using skin specimen of AHZ corroborated the immunoperoxidase findings, yielding bright bands for iNOS. Normal control skin showed minimal or negative expression both by immunoperoxidase stains and RT-PCR. Increased expression of iNOS is consistent with the hypothesis that high level of NO induced by iNOS may be associated with the epidermal necrosis with inflammation seen in the skin lesions of AHZ.

The effect of sorivudine on dihydropyrimidine dehydrogenase activity in patients with acute herpes zoster.

OBJECTIVE: Bromovinyl-uracil (BVU) is the principal metabolite of sorivudine, a potent anti-zoster nucleoside. BVU binds to, and irreversibly inhibits, the enzyme dihydropyrimidine dehydrogenase (DPD). The objective of this study was to assess the time course of recovery of DPD activity after oral administration of sorivudine in patients with herpes zoster and to correlate restoration of DPD activity and levels of uracil with the elimination of sorivudine and its metabolite BVU from the circulation. METHODS: Sorivudine was given orally as 40 mg once-daily doses for 10 consecutive days to a total of 19 patients with herpes zoster. Serum sorivudine, BVU, and circulating uracil and DPD activity in peripheral blood mononuclear cells (PBMCs) were determined before, during, and after administration of sorivudine. RESULTS: BVU was eliminated from the circulation within 7 days after the last sorivudine dose. DPD activity in PBMCs, which was completely suppressed in 18 of the 19 subjects and markedly suppressed in the remaining subject during administration of sorivudine, recovered to baseline levels within 19 days after the last dose of sorivudine in all subjects and within 14 days in all but one of the subjects. The restoration of DPD activity was temporally associated with elimination of BVU from the circulation. The elevated uracil concentrations produced by inhibition of DPD activity fell rapidly after cessation of sorivudine administration and also were temporally associated with elimination of BVU from the circulation. The time course of recovery of DPD activity in three patients with renal impairment was similar to that of the other subjects. CONCLUSIONS: This study indicates that sorivudine therapy is associated with a profound depression of DPD activity. Recovery of DPD activity occurred within 4 weeks of the completion of sorivudine therapy, which indicates that fluorinated pyrimidines may be safely administered 4 weeks after completion of sorivudine therapy.

Alterations in mast cell proteinases and protease inhibitors in the progress of cutaneous herpes zoster infection.

The possible involvement of mast cell proteases in the cutaneous inflammation of herpes zoster was studied histochemically in ten patients. Mast cell tryptase and chymase bioactivities were demonstrated enzyme-histochemically. The localization of protease inhibitors as well as tryptase and chymase proteins in mast cells was established using a sequential double-staining method which first demonstrated bioactive tryptase or chymase, followed by immunohistochemical identification of these antigens. Biopsies were taken from involved vesicular and erythematous skin, as well as from normal healthy-looking skin. Tryptase-bioactive mast cells were significantly lower in number in the upper, but not in the deeper dermis of vesicular skin (68 +/- 37 cells/mm2, mean +/- SD) when compared with either healthy-looking (97 +/- 38) or erythematous skin (105 +/- 36) (t-test, P < 0.005). In contrast, chymase-bioactive mast cells were significantly reduced in number both in erythematous skin (44 +/- 20, P < 0.02) and even more so in vesicular skin (26 +/- 20, P < 0.0005) when compared with healthy-looking skin (64 +/- 27). The percentage of alpha 1-antitrypsin -immunoreactive and alpha 1-antichymotrypsin-immunoreactive mast cells in the upper dermis increased steadily from the values in healthy-looking skin (37.9 +/- 18.8 and 82.5 +/- 21.6 per cent) to those in erythematous (64.4 +/- 16.4 and 93.5 +/- 7.9 per cent) and vesicular skin (75.2 +/- 10.2 and 96.4 +/- 4 per cent). A novel finding was that cells showing tryptase immunoreactivity but no enzyme activity were found in two out of nine erythematous skin specimens and in four out of seven vesicular specimens. In healthy-looking skin, all cells with chymase immunoreactivity also displayed chymase bioactivity, but only 53.2 +/- 24.25 per cent of these mast cells in erythematous lesions and 44.4 +/- 15.9 per cent in vesicular lesions showed chymase bioactivity, suggesting inactivation of chymase by protease inhibitors. These results show prominent alterations in mast cell proteinases and protease inhibitors, indicating that these enzymes participate in the cutaneous inflammation due to herpes zoster.

[Creatine phosphokinase in the cerebrospinal fluid of patients with acute meningitis]. / Kreatinfosfokinaza v spinnomozgovoi zhidkosti u bol'nykh ostrymi meningitami.

A study is presented of the general activity and isoenzymatic spectrum of creatine phosphokinase (CPK) in the cerebrospinal fluid (CSF) in patients with acute meningitis of different etiology. The enzymatic activity proved to be increased due to the cerebral (CPK-BB) isoform. Liberation of CPK-BB from the brain tissue and its penetration into the intercellular fluid and then into the CSF was related to the functional-structural changes in the cellular membranes of the brain in meningitis.

Deoxythymidine kinases in varicella-zoster virus infected and biochemically transformed cells.

Deoxythymidine kinase (TK) activity induced in varicella-zoster virus (VZV)-infected human embryonic fibroblast (HEF) cells was immunologically distinguishable from that in non infected HEF cells and also from that in herpes simplex virus type 1 (HSV-1) infected HEF cells. The TKs in VZV-biochemically transformed cells were immunologically the same as that induced in VZV-infected human cells and immunologically different from that in Ltk- cells or in HSV-biochemically transformed cells. One peak of TK activity with an Rm value of 0.8-0.9, corresponding to mitochondrial TK, was observed on polyacrylamide gel electrophoresis of Ltk- cell extracts. VZV infected Ltk- cells had two peaks of TK activity with Rm values of 0.45-0.5 (peak I) and 0.8-0.9 (peak II). Peak I and II were concluded to be virus-specific TK and mitochondrial TK, respectively. VZV-biochemically transformed cells had a peak of activity with an Rm value of 0.4-0.5, corresponding to peak I in VZV-infected Ltk- cells; that is VZV-specific TK activity. The present study indicates that VZV has a gene coding for its own TK.