Antiviral effect of an oligo(nucleoside methylphosphonate) complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5.

Selective inhibition of regulatory immediate early (IE) genes of herpes simplex virus type 1 (HSV-1) should inhibit virus growth. Treatment of HSV-1-infected cells with the oligo(nucleoside methylphosphonate) d(TpCCTCCTG) (deoxynucleoside methylphosphonate residues in italic), which is complementary to the acceptor splice junction of HSV-1 IE pre-mRNA 4 and 5, before (1-24 hr) or at the time of infection caused a dose-dependent inhibition in virus replication. Virus titers were decreased 50% and 90% in cells treated with 25 microM and 75 microM oligomer, respectively; at 300 microM, a 99% reduction in virus production was observed. Viral DNA synthesis was reduced 70-75% and there was a 90% reduction in synthesis of viral proteins, including other IE species and viral functional (130-kDa major DNA-binding) and structural (glycoprotein gB) proteins. In the same concentration range, d(TpCCTCCTG) caused a minimal reduction (0-30%) in protein synthesis and growth rates (less than 40%) of uninfected cells. The data suggest that oligo(nucleoside methylphosphonate)s may be effective in antiviral chemotherapy.     

Regulation of expression of herpes simplex virus (HSV) glycoprotein D in vaccinia recombinants affects their ability to protect from cutaneous HSV-2 disease

The effect of regulation of herpes simplex virus (HSV) type 1 glycoprotein D (gD-1) gene expression on HSV-specific immune response and protection from cutaneous HSV-2 disease was studied using vaccinia virus recombinants containing gD-1 under the control of early (VP176) or late (VP254) vaccinia virus promoters. Expression of gD-1 in VP176-infected cells was first observed at 2 h after infection. It did not depend on viral DNA replication. In VP254-infected cells, gD-1 was first observed at 24 h after infection and its expression depended on DNA replication. Immunized guinea pigs had similar titers of HSV-specific neutralizing antibody. However, HSV-specific T cell responses were significantly higher in VP176- than in VP254-immunized animals as determined by lymphoproliferation (P less than .005) and delayed type hypersensitivity (P less than .01). The reduced T cell responses of VP254-immunized guinea pigs correlated with poor gD-1 expression in VP254-infected antigen presenting cells (splenic adherent and epidermal cells). Immunization with VP176, but not with VP254, protected guinea pigs from primary (P less than .0005) and recurrent (P less than .0005) cutaneous HSV-2 lesions.     

Tumorigenic transformation induced by a specific fragment of DNA from herpes simplex virus type 2.

Transfection of Syrian hamster embryo cells with limit digests of Bgl II-, Hpa I-, or Bgl II/Hpa I-cleaved DNA from herpes simplex virus type 2 (strain S-1) but not with salmon sperm DNA resulted in the appearance of refractile, morphologically altered cells at a frequency of 10(-5)/0.005 microgram of viral DNA within two to four passages. Transformed lines manifested reduced serum requirement and anchorage-independent growth and were tumorigenic in newborn hamsters. They expressed ICP10, a viral protein immunologically identical to the cervical-tumor-associated AG-4 antigen. Transforming activity was localized in the 16.5 x 10(6)-dalton Bgl II/Hpa I double-digest fragment CDs-1, which exhibited sequence homology to the Bgl II/Hpa I fragment CD of DNA from herpes simplex virus type 2 strain 333, mapping between coordinates 0.43 and 0.58 on the physical map of strain 333 DNA. This fragment, CD333, was also shown to induce neoplastic transformation.     

Phase 2 of collaborative action around the implementation of virtual hearing aid care: Evaluation of a clinical practice guideline

Rationale

Following the onset of the COVID-19 pandemic, a clinical practice guideline (CPG) around virtual hearing aid practices was developed to fill a knowledge gap within the field of audiology. Details outlining the development and mobilization of this draft guideline were outlined as Phase 1 (described in a paired paper).

Aims and Objectives

This study describes Phase 2 of this project as part of the Knowledge-to-Action Framework, including an evaluation of the methodological quality of the guideline and the resulting tailored version of the document (v2.0).

Method

The Appraisal of Guidelines for Research and Evaluation II instrument was used to assess methodological quality and to guide revisions. Twenty-two clinicians, from a variety of clinical backgrounds, participated in the evaluation.

Results and Conclusion

Findings reported across six domains suggest high mean scores, ranging from 78% to 81%, in order of scope and purpose (highest rated), stakeholder involvement, rigour of development, applicability, clarity of presentation, and editorial independence. Specific recommendations made by in international co-creation team during the evaluation informed the final version of the CPG. Future development and evaluation efforts should aim to include greater representation from nontraditional practice contexts to strengthen global applicability.     

Immortalization and neoplastic transformation of normal diploid cells by defined cloned DNA fragments of herpes simplex virus type 2.

Diploid Syrian hamster embryo (SHE) cells were passaged after transfection with recombinant plasmids containing herpes simplex virus type 2 (HSV-2) DNA inserts Bgl II focus-forming fragment N, Bgl II transforming fragment C, and EcoRI/HindIII fragment AE. Cultures transfected with salmon DNA or with 0.1-5.0 micrograms of Bgl II fragment N reached crisis and senesced. Those transfected with 0.1-0.5 micrograms of Bgl II fragment C or its left-hand 64% subclone EcoRI/HindIII fragment AE escaped senescence and formed continuous lines. At early passages, these lines as well as isolated clones grew in 2% serum but formed small (less than or equal to 0.1 mm) colonies in 0.3% agarose and were nontumorigenic. Serial passaging of Bgl II fragment C-induced cultures and isolated clones resulted in the appearance of large (greater than 0.25 mm) colonies in agarose followed by tumorigenicity. This behavior was not exhibited by the EcoRI/HindIII fragment AE-induced cultures that remained nontumorigenic after 53 passages. DNA from normal SHE cells exhibited homology to Bgl II fragment C but, under relatively stringent conditions, DNAs from transformed and tumor-derived lines exhibited discrete hybridizing bands comigrating with authentic viral fragments. These results indicate that neoplastic transformation of normal diploid SHE cells by HSV-2 DNA fragments involves at least two distinct steps--i.e., immortalization and conversion to tumorigenicity. EcoRI/HindIII fragment AE representing the left 64% of Bgl II fragment C is sufficient to induce immortalization. However, DNA sequences from both left-hand 64% and right-hand 36% subfragments of Bgl II fragment C are required for tumorigenic transformation.     

Synthesis and cytotoxicity against tumor cells of pincer N-heterocyclic ligands and their transition metal complexes

The complexes: [CoL₂](ClO₄)₂ (1), [FeL₂](ClO₄)₂ (2), [NiL₂](ClO₄)₂ (3) and [MnLCl₂] (4), with L = diethyl-1,1′-(pyridine-2,6-diyl)bis(5-methyl-1H-pyrazole-3-carboxylate), were synthesized and fully characterized. Structural analysis revealed two distinct patterns influenced by the counter ions where L acts as a tridentate chelating ligand. The in vitro antitumor activity of L and L′ (diethyl 2,2′-(pyridine-2,6-diylbis(5-methyl-1H-pyrazole-3,1-diyl)) diacetate) as well as their metal complexes, was tested by the measurement of their cytostatic and cytotoxic properties towards the blood cancer mastocytoma cell line P815. We have also investigated their interactions with the antioxidant enzyme system. As a result, [MnL′Cl₂] (1′) exhibited the strongest activity compared to reference cis-platin with no cytotoxicity towards normal cells PBMCs (Peripheral Blood Mononuclear Cells). On the other hand, the antioxidant enzyme activity showed that the efficiency of metal complex 1′ against P815 tumor cells was via the rise in the SOD activity and inhibition of CAT enzyme activity. This proof of concept study allows disclosure of a new class of molecules in cancer therapeutics.

The complexes: [CoL₂](ClO₄)₂ (1), [FeL₂](ClO₄)₂ (2), [NiL₂](ClO₄)₂ (3) and [MnLCl₂] (4), with L = diethyl-1,1′-(pyridine-2,6-diyl)bis(5-methyl-1H-pyrazole-3-carboxylate), were synthesized and fully characterized.     

Intracellular localization and serological identification of a HSV-2 protein in cervical cancer

Exfoliated atypical cells from US and Italian patients with cervical intraepithelial neoplasia or invasive cancer stained in indirect immunofluorescence with a monoclonal antibody to a high MW HSV-2 protein (ICP10). The proportion of staining cells increased as a function of the severity of the cervical anaplasia. In the majority of cases with mild or moderate dysplasia staining was restricted to the cytoplasm while atypical cells from 6 of 7 cases with marked dysplasia and 7 of 7 cases with invasive cancer evidenced also nuclear staining. Normal exfoliated cells did not stain even when obtained from tissue adjacent to the tumor mass. Patients with ICP10 positive atypical cells had antibody to ICP10 as determined by the Western blot assay. Serologic analyses indicated that there was a good correlation between: (i) the Western blot and the CF assay previously used to detect antibody to AG-4 (Aurelian, et al, Cancer, 48, 455, 1981) and (ii) cervical anaplasia and ICP10 seropositivity. A similar correlation with cervical anaplasia was not observed with respect to antibody to two other viral proteins (ICP12/14 and 68K).     

Expression of herpes simplex virus type 2 protein ICP10 PK rescues neurons from apoptosis due to serum deprivation or genetic defects

Previous studies have shown that the herpes simplex virus type 2 protein kinase ICP10 PK activates the Ras/MEK/MAPK pathway in nonneuronal cells. Here we report that ectopically expressed ICP10 PK has anti-apoptotic activity in various paradigms of neuronal cell death. Neuronally differentiated PC12 cells and primary murine hippocampal cultures transfected with an expression vector for ICP10 PK were protected from cell death resulting from growth factor withdrawal. Protection from apoptosis was also seen in ICP10 PK-transfected hippocampal neurons from the trisomy 16 mouse, a naturally occurring genetic abnormality the human analog of which is Down syndrome. Cells transfected with an expression vector for a mutant that lacks kinase activity were not protected, although it was expressed as well as ICP10 PK. The data indicate that ICP10 PK has a broad anti-apoptotic activity in neuronal cells which depends on a functional PK.