Serge Debrus

Comparative immunohistochemical study of herpes-simplex and varicella-zoster infections

Herpes simplex (HSV) and varicella-zoster (VZV) skin infections share so many histological similarities that distinguishing between them may prove to be impossible. We developed and characterized a new monoclonal antibody, VL8, IgG kappa isotype, directed to the VZV envelope glycoprotein gpI. Immunohistochemistry with VL8 appeared highly sensitive and specific on formalin-fixed paraffin-embedded biopsies and a clear-cut distinction between HSV and VZV infections was possible. The pattern of VL8 immunolabelling in VZV infections was strikingly different from that found in HSV infections studied with polyclonal antibodies to HSV I and II. Double immunolabelling revealed the VL8 positivity of sebaceous cells, endothelial cells, Mac 387- and CD68-positive monocyte-macrophages, and factor XIIIa-positive perivascular, perineural and interstitial dendrocytes. Intracytoplasmic VL8 labelling of endothelial cells and perivascular dendrocytes was found at the site of leukocytoclastic vasculitis.

Varicella-zoster virus latency in the adult rat is a useful model for human latent infection.

A model of latent infection by varicella-zoster virus (VZV) was obtained in the adult rat. Inoculation of VZV-infected cells in the skin led to infection of the peripheral nervous system. Latency was characterized by a long-lasting presence of the viral genome, of selected viral gene transcripts, and of at least one viral protein in the dorsal root ganglia. Reactivation has not been obtained in vivo, but has occurred ex vivo after repeated stresses. Many similarities with VZV latency in humans were found, making this model useful for vaccine and antiviral studies. NEUROLOGY 1995;45(Suppl 8): S18-S20

Distribution of varicella zoster virus and herpes simplex virus in disseminated fatal infections.

AIMS: To study the cutaneous and visceral distribution of herpes simplex virus (HSV) and varicella zoster virus (VZV) in fatal infections. METHODS: Standard histology, immunohistochemistry (monoclonal antibodies VL8 and VL2 and polyclonal antibody IE63 directed against VZV; monoclonal antibodies IBD4 and HH2 and polyclonal antibodies directed against HSVI and HSVII) and in situ hybridisation (anti-HSV and anti-VZV probes) were applied to formalin fixed, paraffin wax sections. RESULTS: On histological examination, Herpesviridae infection was evident in various organs including the lungs, liver and skin. In addition, immunohistochemistry and in situ hybridisation revealed the presence of HSV and VZV antigens and nucleic acids in several cell types and tissues showing no cytopathological alterations suggestive of Herpesviridae infection. The organs with histological evidence of infection also contained VZV or HSV antigens and their genes. CONCLUSIONS: These findings suggest that organ failure in disseminated VZV and HSV infections is primarily caused by HSV or VZV induced cell damage and lysis. They also indicate that immunohistochemistry and in situ hybridisation can provide an accurate, type-specific diagnosis on formalin fixed, paraffin wax embedded tissue even when classic histological and cytological characteristics are lacking.

Viral glycoproteins in herpesviridae granulomas

Granulomatous reactions after varicella zoster virus (VZV) and herpes simplex virus (HSV) infections are rare, and their pathogenesis remains unclear. We studied by immunohistochemistry and in situ hybridization early granulomatous reactions after VZV and HSV infections. In the five cases studied, the VZV glycoproteins gp I and gp II were present in cells abutted to altered vessels, but the corresponding genome sequences were disclosed in similar locations in only one of these cases. In an immunocompromised patient with diffuse HSV eruption, HSV 1 antigens were present in cells of the reticular dermis, while viral nucleic acids were not evident. Immunophe-notyping of the granulomas showed strong Mac 387 and CD68 positive labelings of macrophages/monocytes, without any involvement of Factor XIlla-positive cells. These findings suggest that the major viral envelope glycoproteins, rather than complete viral particles could trigger granuloma formation following HSV and VZV skin infections.

Localization of varicella-zoster virus nucleic acids and proteins in human skin

The pathogenic mechanisms involved in varicella-zoster virus (VZV) infections remain elusive. The pattern of cutaneous distribution of the IE63 protein and of the gpI (gE) and gpII glycoproteins with their corresponding genome sequences during VZV infections was studied by immunohistochemistry and in situ hybridization. Skin biopsy specimens were obtained from immunocompetent and immunocompromised patients with varicella, herpes zoster, or atypical VZV lesions. The first evidence for VZV infection consisted of the presence of IE63 in keratinocytes. In the vesicles and pustules, the viral transcripts gpI, gpII, and IE63 and the corresponding nucleic acids for gpI and gpII were identified in keratinocytes, sebocytes, Langerhans cells, dermal dendrocytes, monocytes/macrophages, and endothelial cells. The gpI and gpII glycoproteins were essentially located on the cellular membranes while IE63 expression was generally restricted to the nuclei. In three biopsies of early herpes zoster, viral proteins were disclosed in dermal nerves and in perineurial type I dendrocytes. This was never encountered in varicella. Vasculitic changes and endothelial cell involvement were more prominent in varicella than in herpes zoster. It is concluded that the secondary viremia in varicella that affects the dermal endothelial cells is followed by a cell-to-cell spread to keratinocytes. In herpes zoster, the viral progression through cutaneous nerves primarily extends to the pilosebaceous units with a secondary involvement of epidermal keratinocytes, followed by a further spread to dermal cells. NEUROLOGY 1995;45(Suppl 8): S47-S49

Intracellular distribution of the ORF4 gene product of varicella-zoster virus is influenced by the IE62 protein.

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for genes of heterologous viruses. The major regulatory immediate-early protein of VZV (IE62) is a transactivator of VZV gene expression. In transfection assays, IE4 has been shown to enhance activation induced by IE62. To investigate the functional interactions underlying this observation, indirect immunofluorescence studies were undertaken to determine whether IE62 could influence IE4 intracellular localization in transfected cells. In single transfections, IE4 was predominantly found in cytoplasm. In cotransfection with IE62, the IE4 localization pattern was altered, with nuclear staining predominating over cytoplasmic staining. This effect was specific to the IE62 protein since the gene products of ORF63 and ORF61, which are also regulatory proteins, did not influence IE4 distribution. The use of IE62 mutants indicated that IE62 influence is independent of its transactivation function and that the integrity of regions 3 and 4 is required. IE62 remained nuclear whether IE4 was present or not. These observations underline differences in the regulation of gene expression between VZV proteins and their herpes simplex virus type 1 homologues. In infected cells, IE4 was only sometimes found to colocalize with IE62 in nuclei. This observation suggests that when all VZV proteins are present, complex interactions probably occur which could diminish the influence of IE62.

Comparative in vitro and in vivo cytotoxic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and its arabinosyl derivative, (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), against tumor cells expressing either the Varicella zoster or the Herpes simplex virus thymidine kinase

The inhibitory effects of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) and its arabinosyl derivative (E)-5-(2-bromovinyl)-1-β-D-arabinofuranosyluracil (BVaraU) on the growth of both MDA-MB-435 human breast carcinoma and 9L rat gliosarcoma cells expressing the thymidine kinase (tk)-encoding gene of the Varicella zoster virus (VZV) or the Herpes simplex virus (HSV) were evaluated. In vitro, BVDU and BVaraU effectively killed both cell types expressing VZVtk, with 50% inhibitory concentration values ranging from 0.06 to 0.4 μM, whereas ganciclovir (GCV) lacked activity. On HSVtk+ cells, BVDU had high cytotoxic activity, with 50% inhibitory concentration values that were similar to those of GCV, whereas BVaraU was inactive. In vivo, BVDU applied intraperitoneally caused a 50% tumor growth inhibition in nude mice inoculated subcutaneously with VZVtk+ as well as HSVtk+ mammary tumor cells. In mice and at variance with the in vitro results, BVaraU had very little activity against the VZVtk+ mammary cells; GCV had the highest activity on the HSVtk+ cells, resulting in a 50% eradication of the tumors. With the 9L rat gliosarcoma model, the VZVtk/BVDU system completely failed to inhibit the development of VZVtk+ glioma tumors induced subcutaneously in syngeneic rats, although BVDU had a similar 45-minute half-life in both rats and mice. Factors other than degradation of the prodrug and related to the mode of action of these analogs are possibly involved in the observed discrepancies between the in vitro and in vivo results.

Detection of Cytokines in Human Sural Nerve Biopsies: An Immunohistochemical and Molecular Study

Abstract. In vitro and in vivo models have implicated numerous cytokines as major modulators of inflammation, destruction and repair in the peripheral nervous system (PNS). The in situ production of cytokines in human peripheral nerve disorders is still poorly documented. We studied the expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10, IL-4, IL-3 and nerve growth factor (NGF) in 35 human sural nerve biopsies using immunohistochemistry; additional reverse transcription-polymerase chain reaction and mRNA in situ hybridization were performed for IL-4 and NGF. Expression of IL-1β and TNF-α was shown in both morphologically normal nerves and various neuropathies, and macrophages appeared as their predominant source. Levels of IL-1β and TNF-α expression were significantly correlated (P<0.01) with each other and with expression of NGF. Multiple endoneurial sources were suggested for IL-6 and IL-10 with low immunoreactivity in the vast majority of cases. Conversely, IL-4 and IL-3 expression were found in neuropathies of various etiologies and Schwann cells appeared to be a predominant source of IL-4 in double-labeling immunofluorescence studies. IL-3 immunoreactivity correlated with IL-1β, TNF-α and IL-6. In this retrospective study, no specific cytokine profile of expression could be assigned to a precise subgroup of neuropathies. This is the first report of IL-4 and IL-3 expression in human neuropathies, and it may be important given the potential role of these cytokines in modulating macrophage activity in the PNS.

Lessons to be learned from varicella-zoster virus

Varicella-zoster virus (VZV) is an alphaherpesvirus responsible for two human diseases: chicken pox and shingles. The virus has a respiratory port of entry. After two successive viremias, it reaches the skin where it causes typical lesions. There, it penetrates the peripheral nervous system and it remains latent in dorsal root ganglia. It is still debatable whether VZV persists in neurons or in satellite cells. During latency, VZV expresses a limited set of transcripts of its immediate early (IE) and early (E) genes but no protein has been detected. Mechanisms of reactivation from ganglia have not been identified. However, dysfunction of the cellular immune system appears to be involved in this process. The cell-associated nature of VZV has made it difficult to identify a temporal order of gene expression, but there appears to be a cascade mechanism as for HSV-1. The lack of high titre cell-free virions or recombination mutants has hindered so far the understanding of VZV gene functions. Five genes, ORFs 4, 10, 61, 62, and 63 that encode regulatory proteins could be involved in VZV latency. ORF4p activates gene promoters with basal activities. ORF10p seems to activate the ORF 62 promoter. ORF61p has trans-activating and trans-repressing activities. The major IE protein ORF62p, a virion component, has DNA-binding and regulatory functions, transactivates many VZV promoters and even regulates its own expression. ORF63p is a nuclear IE protein of yet unclear regulatory functions, abundantly expressed very early in infection. We have established an animal model of VZV latency in the rat nervous system, enabling us to study the expression of viral mRNA and protein expression during latency, and yielding results similar to those found in humans. This model is beginning to shed light on the molecular events in VZV persistent infection and on the regulatory mechanisms that maintain the virus in a latent stage in nerve cells.

Mice transgenic for a soluble form of murine cytotoxic T lymphocyte antigen 4 are refractory to murine acquired immune deficiency sydrome development.

Interactions between B and CD4+ T cells are central to the pathogenesis of retrovirus‐induced murine acquired immune deficiency virus (MAIDS). Prompted by previous work showing that treatment with cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) partly inhibited the disease, we studied the course of infection in mice deficient for CD28–B7 interactions (mCTLA4‐Hγ1 transgenic mice). Despite a relative viral load identical to that of non‐transgenic mice, the transgenic mice did not develop any of the major MAIDS symptoms (i.e. lymphoproliferation and immune anergy). The mCTLA4‐Hγ1 did not however, completely inhibit B‐cell activation as indicated by a slight hypergammaglobulinaemia and microscopic blastic transformation. Absence of MAIDS in transgenic mice was associated with much lower levels of both interleukin‐4 and interferon‐γ transcripts following viral infection. These results support the theory that the CD28/B7 costimulatory pathway is a critical determinant to MAIDS development.