CD34+ cells in the peripheral blood transport herpes simplex virus DNA fragments to the skin of patients with erythema multiforme (HAEM)

Herpes simplex virus (HSV)-associated erythema multiforme (HAEM) is a recurrent disease characterized by the presence and expression of HSV DNA fragments in lesional skin. Our studies examined the mechanism of viral DNA transport to the skin of HAEM patients. CD34+ cells were isolated from the blood of normal subjects and HSV and HAEM patients during acute lesions and at quiescence. They were cultured with cytokines that favor their differentiation into Langerhans cells (LC) precursors (CD1a+/CD14-) and examined for HSV replication, HSV-induced cellular alterations, viral DNA fragmentation, and clearance. CD34+ cells from all study groups were non-permissive for HSV replication but infection favored their differentiation into CD1a+/CD14- LC precursors and upregulated E-cadherin expression, thereby assisting LC targeting to the skin. Only HAEM patients had CD34+ cells that retained viral DNA fragments, notably polymerase DNA, for at least 7 d of in vitro culture. The percentages of circulating CD34+ (and CD34+/CLA+) cells were significantly higher in HAEM patients at the time of acute lesions. A similar increase was not seen for HSV patients. The data are the first report implicating CD34+ cells in HAEM pathogenesis, likely by transporting HSV DNA fragments to lesional skin.     

Erythema multiforme lesions are associated with expression of a herpes simplex virus (HSV) gene and qualitative alterations in the HSV-specific T-cell response

A common form of erythema multiforme, herpes-associated erythema multiforme (HAEM), occurs following infection with herpes simplex virus (HSV). Here we report that HSV gene expression and the qualitative nature of the virus-specific T-cell responses are related to HAEM lesion development. Skin from HAEM lesions and 1–3 months healed HAEM lesional skin were positive for the viral DNA polymerase gene (Pol) by polymerase chain reaction. However, gene expression as determined by immunohistochemistry with Pol-specific antibody was seen only in HAEM lesions, suggesting that lesion development is associated with Pol gene expression. Similar HSV-specific T-cell lymphoproliferative responses were seen in peripheral blood mononuclear cells (PBMCs) from patients with acute or healed HAEM lesions or HSV lesions and from HSV-seropositive patients with unrelated inflammatory diseases. However, the T-cell receptor variable (Vβ ) chain repertoire of HSV-stimulated PBMCs obtained from HAEM lesions was altered; the prevalence of some families of variable chain (namely Vβ16 and Vβ19) was reduced, whereas the prevalence of others was increased (namely Vβ2 and Vβ7). Vβ2 cells were found in HAEM lesional skin positive for Pol antigen, suggesting that these cells home to viral antigen-positive skin.     

Detection of herpes simplex virus DNA in cutaneous lesions of erythema multiforme

The association between erythema multiforme (EM) and herpes simplex virus (HSV) infection has long been appreciated, although the exact role which HSV may play in the pathogenesis of this herpes-associated EM (HAEM), is unknown. Previous studies have suggested, but not definitively demonstrated, the presence of HSV in lesions of HAEM. The presence of HSV would support the hypothesis that an immune-mediated response directed against HSV-specific antigens in the skin is central to lesion development in HAEM. The purpose of this study was to examine lesions of EM for the presence of HSV DNA by using the polymerase chain reaction (PCR). In addition, in situ hybridization using an HSV-specific RNA probe was performed to further localize the HSV nucleic acids within the skin. DNA was extracted from formalin-fixed, paraffin-embedded specimens of cutaneous lesions of HAEM and also from EM for which no precipitating factor could be documented, otherwise known as idiopathic EM (IPEM). DNA from lesions of bullous pemphigoid served as a negative control. Using PCR to specifically amplify HSV sequences which might be present, and then performing Southern analysis, we demonstrated HSV DNA in 9/13 HAEM and 6/9 IPEM biopsies. No HSV was detected in six lesions of bullous pemphigoid. In situ hybridization of three cutaneous HAEM lesions using an 35S-labeled HSV-specific RNA probe localized the HSV nucleic acids predominantly to the epidermis. Three biopsies of chronic dermatitis, used as negative controls, did not demonstrate this specific hybridization. These findings confirm the presence of HSV in lesions of HAEM and are consistent with the concept of an HSV-specific immune-mediated pathogenesis for this disease. In addition, most cases of IPEM appear to be herpes associated despite the absence of clinically apparent HSV infection.     

Longitudinal study of a patient with herpes-simplex-virus-associated erythema multiforme: viral gene expression and T cell repertoire usage.

BACKGROUND: Erythema multiforme is a polymorphous self-limited, often recurrent eruption that can follow herpes simplex virus (HSV) infection, hereby designated HAEM. Studies of relatively large groups of patients during one recurrent episode indicated that HAEM pathogenesis is associated with HSV gene expression, Vbeta2 T cell infiltration of lesional skin and altered T cell receptor (TCR) repertoire usage by HSV-stimulated peripheral blood mononuclear cells (PBMC). However, HAEM recurrences are not always preceded by overt HSV eruptions and virus cannot be isolated from HAEM lesional skin. Therefore, it is unknown whether all HAEM recurrences experienced by a given patient are HSV related. OBJECTIVE: The studies described in this report were designed to examine whether all HAEM recurrences experienced by a given patient are HSV related. METHODS: We describe one patient who was studied longitudinally during 6 HAEM recurrences and in the intervening lesion-free periods. Lesional skin from all HAEM episodes was studied for HSV gene expression and infiltration by Vbeta2 and Vbeta3 T cells. PBMC obtained at these times were assayed for TCR repertoire usage upon HSV stimulation. RESULTS: Lesional skin from all HAEM episodes was positive for HSV gene expression (RNA and protein) as well as Vbeta2 T cell infiltration. HSV-stimulated PBMC obtained at these times had an altered TCR repertoire characterized by a predominance of Vbeta2 cells. The duration of viral gene expression, Vbeta2 cell infiltration and altered TCR repertoire usage correlated with the duration of clinical symptoms. CONCLUSION: The data suggest that HSV and a virus-specific immunopathology component are involved in the causation of all HAEM episodes experienced by the patient.     

Herpes simplex virus (HSV)-associated erythema multiforme (HAEM): a viral disease with an autoimmune component

Erythema multiforme (EM) is a clinical conundrum the name of which reflects the broad morphological spectrum of the lesions. Molecular and immunologic evidence that herpes simplex virus (HSV) causes a subset of EM lesions [herpes-associated EM (HAEM)] is reviewed, and new data are presented which suggest that autoreactive T-cells triggered by virus infection play an important role in HAEM pathogenesis. Disease development begins with viral DNA fragmentation and the transport of the DNA fragments to distant skin sites by peripheral blood mononuclear cells (PBMCs). HSV genes within DNA fragments deposited on the skin [notably DNA polymerase (Pol)] are expressed, leading to recruitment of HSV-specific CD4+ Th1 cells that respond to viral antigens with production of interferon-gamma (IFN-gamma). This step initiates an inflammatory cascade that includes expression of IFN-gamma induced genes, increased sequestration of circulating leukocytes, monocytes and natural killer (NK) cells, and the recruitment of autoreactive T-cells generated by molecular mimicry or the release of cellular antigens from lysed cells. The PBMCs that pick up the HSV DNA [viz. macrophages or CD34+ Langerhans cells (LC) precursors], their ability to process it, the viral proteins expressed in the skin and the presence of epitopes shared with cellular proteins may determine whether a specific HSV episode is followed by HAEM development. Drug-associated EM (DIEM) is a mechanistically distinct EM subset that involves expression of tumor necrosis factor alpha (TNF-alpha) in lesional skin. It is our thesis that the polymerase chain reaction (PCR) assay for HSV DNA detection in lesional skin and staining with antibodies to IFN-gamma and TNF-alpha, are important criteria for the diagnosis of skin eruptions and improved patient management.     

Detection of herpes simplex virus DNA in the peripheral blood during acute recurrent herpes labialis.

BACKGROUND: Although herpes simplex virus (HSV) has been detected in the peripheral blood of immunocompromised patients and in neonates with disseminated disease, the extent to which this virus may be present in the blood during a localized infection in otherwise healthy adults is unknown. OBJECTIVE: The purpose of this study was to determine whether HSV may be detected in the peripheral blood during acute recurrent herpes labialis. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from otherwise healthy adults with recurrent herpes labialis, both during an acute episode and several weeks after the lesions had healed. The PBMCs were examined for the presence of HSV with the polymerase chain reaction (PCR) and viral culture. RESULTS: By PCR, HSV DNA was detected in 7 of 34 specimens from an acute episode but in none of 24 specimens in the convalescent stage (p less than 0.004). PBMCs from seven donors, who were seronegative for HSV, were also negative for HSV by PCR. Viral cultures of 22 PBMC specimens were negative (including four specimens that were positive by PCR). CONCLUSION: The presence of HSV DNA in the blood is a transient phenomenon limited to the period of active infection in a minority of patients with herpes labialis, although it may be important in the development of disseminated disease as well as in the pathogenesis of herpes-associated cutaneous processes such as erythema multiforme.     

Detection of herpes simplex virus genomic DNA in various subsets of Erythema multiforme by polymerase chain reaction

BACKGROUND: The wide variation in the detection of herpes simplex virus (HSV) DNA (36-75%) by polymerase chain reaction (PCR) in erythema multiforme (EM) may be partly attributed to differences in case selection in terms of subsets of EM studied. OBJECTIVE: To determine the frequencies of detection of HSV DNA in specific subsets of EM. METHODS: Nested PCR was used to detect HSV DNA in skin biopsies with histologically proven EM. RESULTS: PCR was performed on skin biopsies from 63 patients with EM. HSV DNA was detected in 3/11 (27.2%) of single-episode HSV-associated EM (HAEM), 6/10 (60%) of recurrent HAEM, 1/4 (25%) of single-episode idiopathic EM and 6/12 (50%) of recurrent idiopathic EM. HSV DNA was not detected in atypical EM (0/11), suspected drug-induced EM (0/9) or Stevens-Johnson syndrome (0/6). CONCLUSION: The overall PCR positive rates of HAEM (42.9%) and idiopathic EM (43.8%) were comparable suggesting that idiopathic EM is likely to be related to a subclinical HSV infection.     

Identification of herpes simplex virus DNA in lesions of erythema multiforme by the polymerase chain reaction.

An association between erythema multiforme and herpes simplex virus infection has been supported by clinical studies and by the detection by immunofluorescence of herpes viral antigen in sera and skin biopsy specimens of patients with erythema multiforme. In rare cases, the virus has also been isolated in cultures of skin biopsy specimens of erythema multiforme. To investigate further the association between erythema multiforme and herpes simplex virus, we used the polymerase chain reaction for herpes simplex virus to examine skin lesions from patients with erythema multiforme. In this study herpes simplex virus DNA was detected in 11 of 31 biopsy specimens of erythema multiforme; six additional cases showed equivocal amplification results, which is suggestive of low amounts of viral DNA. Seven skin and mucosal biopsy specimens with the histologic changes of herpes virus infection served as positive controls: all were positive for herpes simplex virus DNA. Viral DNA was not detected in control biopsy specimens from skin excised for unrelated conditions. These studies support the association of herpes simplex virus in the pathogenesis of some cases of erythema multiforme. The polymerase chain reaction provides a quick and effective method of detecting herpes simplex virus in lesions of herpes-associated erythema multiforme. Furthermore, the polymerase chain reaction may delineate those cases of erythema multiforme that are etiologically related to herpes virus infection and therefore might be treated with acyclovir to prevent recurrence.     

HSV‐associated erythema multiforme in a patient after hematopoietic stem cell transplantation

Herpes simplex virus (HSV)–associated erythema multiforme (HAEM) is an acute and self‐limiting mucocutaneous hypersensitivity reaction triggered by herpes virus infections. We reported a patient with HAEM after hematopoietic stem cell transplantation (HSCT). A 55‐year‐old man received HSCT 7 months ago. He suffered from chronic graft versus host disease 4 months after HSCT and was treated with prednisone and tacrolimus. One week ago, he developed generalized macules with leukopenia. Dermatological examination revealed multiple iris‐like erythemas on his trunk and extremities. The skin lesions and leukopenia resolved upon anti‐HSV treatment.     

Viral DNA detection and RAS mutations in actinic keratosis and nonmelanoma skin cancers

Background Actinic keratosis (AK) is a well‐established precancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). Basal cell carcinoma (BCC) is a locally aggressive slowly growing tumour that rarely metastasizes. A number of viruses have been proposed to play a role in the development of nonmelanoma skin cancers (NMSC), but the most plausible evidence to date suggests that cutaneous human papillomavirus (HPV) is the key instigating factor. Objectives To evaluate the prevalence of HPV, cytomegalovirus (CMV), herpes simplex virus (HSV) and Epstein–Barr virus (EBV) and investigate their relationship with the presence of RAS gene mutations in cutaneous lesions obtained from nonimmunosuppressed patients. Methods HPV, CMV, HSV and EBV detection was performed using polymerase chain reaction (PCR) in skin biopsies (26 AK, 12 SCC and 15 BCC samples) that were collected from immunocompetent patients. The RAS mutation incidence was also investigated in all cutaneous lesions by use of PCR/restriction fragment length polymorphism and direct DNA sequencing. Results Seventeen out of 53 (32%) skin lesions were found to be positive for HPV DNA. The highest incidences of HPV infection were five of 15 (33%) in BCC and four of 12 (33%) in SCC specimens. The HPV incidence was eight of 26 (31%) in AK and eight of 53 (15%) in normal skin tissue. Twelve out of 53 (23%) skin lesions were CMV‐positive. The highest incidence of CMV infection was six of 15 (40%), observed in BCC specimens. The CMV incidence was two of 26 (8%) in AK and four of 12 (33%) in SCC. No normal skin biopsy was found to be positive for CMV. All cutaneous samples were negative for HSV and EBV DNA, as assessed by our PCR‐based assays. Only three samples, one AK (4%), one BCC (6%) and one SCC (8%), were found to carry a G>T transversion at the second position of HRAS codon 12. Both HRAS mutant SCC and BCC biopsies were HPV‐ and CMV‐positive, as well. Conclusions HPV DNA is detected in NMSC, AK and normal skin biopsies. Our results also indicate that CMV is involved in NMSC at higher levels than in premalignant lesions, whereas the virus was not detected in normal skin biopsies. HSV and EBV do not appear to be involved in the pathogenesis of cutaneous lesions. Moreover, we suggest that the HRAS codon 12 mutation is not a very common event in AK or NMSC. Finally, both viral infection and HRAS activation appear to represent independent factors in the aetiology of NMSC, samples of which were obtained from immunocompetent patients.     

寻常型银屑病与单纯疱疹病毒1型相关性的研究

目的 探讨寻常型银屑病与单纯疱疹病毒1型(HSV-1)的可能相关性。方法 应用PCR法检测患者皮损、外周血单一核细胞(PBMCs)和咽拭子中HSV-1DNA,ELISA法检测患者血清中抗HSV-1的IgM、IgG抗体,并与正常人对照做比较。结果 患者皮损、PBMCs和咽拭子中HSV-1DNA检出率分别为37.5%、18.6%和18.8%,血清中抗HSV-1的IgM、IgG抗体检出率分别为37.2%和53.5%.经χ²检验,患者皮损、PBMCs中HSV-1DNA和血清中IgM抗体检出率显着高于正常人对照,点滴状患者的皮损、PBMCs和咽拭子中HSV-1DNA以及血清中抗HSV-1IgM抗体检出率显着高于斑块状患者。结论 寻常型银屑病尤其是皮损呈点滴状者与HSV-1显着相关,患者可能存在HSV-1的近期感染。     

Merkel cell polyomavirus DNA detection in lesional and nonlesional skin from patients with Merkel cell carcinoma or other skin diseases

Summary Background A novel polyomavirus, the Merkel cell polyomavirus (MCPyV), has recently been identified in Merkel cell carcinoma (MCC). Objectives To investigate the specificity of this association through the detection, quantification and analysis of MCPyV DNA in lesional and nonlesional skin biopsies from patients with MCC or with other cutaneous diseases, as well as in normal skin from clinically healthy individuals. Methods DNA was extracted from lesional and nonlesional skin samples of patients with MCC or with other cutaneous diseases and from normal‐appearing skin of clinically healthy subjects. MCPyV DNA was detected by polymerase chain reaction (PCR) and quantified by real‐time PCR. Additionally, the T antigen coding region was sequenced in eight samples from seven patients. Results MCPyV DNA was detected in 14 of 18 (78%) patients with MCC, five of 18 (28%) patients with other skin diseases (P = 0·007) and one of six (17%) clinically healthy subjects. In patients with MCC, viral DNA was detected in nine of 11 (82%) tumours and in 10 of 14 (71%) nontumoral skin samples (P = 0·66). MCPyV DNA levels were higher in MCC tumours than in nontumoral skin from patients with MCC, and than in lesional or nonlesional skin from patients with other cutaneous disorders. Signature mutations in the T antigen gene were not identified in the two MCC tumour specimens analysed. Conclusions High prevalence and higher levels of MCPyV DNA in MCC supports a role for MCPyV in tumorigenesis. However, the high prevalence of MCPyV in the nontumoral skin and in subjects without MCC suggests that MCPyV is a ubiquitous virus.     

Investigation of herpes simplex virus DNA in pityriasis rosea by polymerase chain reaction

BACKGROUND: Pityriasis rosea (PR) is an acute, inflammatory disease of unknown cause. Clinical and experimental findings indicate an infectious etiology of PR. Our purpose is to examine the skin lesions and blood samples of PR patients by polymerase chain reaction (PCR) for the presence of HSV type 1 and 2 DNA. METHODS: The lesional skin biopsies from 10 patients and blood samples from two randomized patients with clinically and histologically confirmed pityriasis rosea were examined by PCR. RESULTS: No HSV 1 and HSV 2 DNA was detected in the lesional biopsy and blood samples. CONCLUSIONS: We could not identify a relationship between HSV 1, HSV 2 and PR.     

The herpes-specific immune response of individuals with herpes-associated erythema multiforme compared with that of individuals with recurrent herpes labialis

Infection with herpes simplex virus (HSV) is the most common precipitating factor in the development of erythema multiforme (EM). It is not known why only a few of the many individuals who experience recurrent HSV infection also develop herpes-associated EM (HAEM), although a difference in the HSV-specific immune response has been postulated. The purpose of this study was to compare the HSV-specific immune response of individuals with HSV infection alone with that of individuals with HAEM. There were 21 patients in each of the two groups. Four parameters of the HSV-specific immune response were examined: (1) anti-HSV IgG titers were measured by ELISA; (2) antibody neutralization was assessed using a plaque assay; and (3) antibody-dependent complement-mediated cytotoxicity, and (4) antibody-dependent cellular cytotoxicity were investigated using a previously described in vitro HSV-specific cytotoxicity assay. No statistically significant differences were detected between the two patient groups. Thus, a difference in these HSV-specific immune mechanisms does not explain the development of HAEM in some individuals with recurrent HSV infection.Grant support: AI 16637 from the National Institutes of Allergy and Infectious Disease     

Detection of HSV-specific DNA in biopsy tissue of patients with erythema multiforme by polymerase chain reaction

Formalin-fixed paraffin-embedded skin biopsies of lesions of erythema multiforme (EM) from 32 patients and 13 controls were examined for the presence of herpes simplex virus (HSV) by polymerase chain reaction (PCR) and for histological findings by direct immunofluorescence and staining with haematoxylin and eosin. HSV-specific DNA was detected in 23 (72%) patients. A history of recurrent skin rash was present in 59% of the PCR-positive cases, while 55% had had suspected HSV infections. Only two PCR-positive specimens were found in patients without a history of recurrent rash and/or previous oral lesions. One biopsy was positive for HSV by conventional cell cultures. There was no significant difference in histology between HSV-related and HSV-negative cases of EM. In the 13 control specimens [bullous pemphigoid (3), dermatitis herpetiformis (2), lichen planus (1), aphthous ulcer (1), fixed-drug eruption (1), varicella-zoster (1), hypereosinophilic syndrome (1), photocontact dermatitis (1), contact dermatitis (1), and cellulitis (1)], no HSV-DNA was detected.     

Detection of spirochaetal DNA simultaneously in skin biopsies, peripheral blood and urine from patients with erythema migrans

Lyme borreliosis is an emerging zoonosis transmitted by infected hard-bodied ticks. The disease is multisystemic. In the initial stage its typical manifestation is the erythema migrans, a cutaneous lesion that occurs in up to 90% of patients. In order to investigate the presence of the specific agent, Borrelia burgdorferi, in the early stages of the disease, DNA from skin biopsies, urine and peripheral blood of 30 patients with clinically documented erythema migrans and without apparent systemic involvement was analysed by polymerase chain reaction. Borrelia DNA in both blood and skin biopsies was detected in 23 patients, while in 9 patients it was discovered in urine and skin biopsies. These results demonstrate that Borrelia DNA is detectable systemically also in patients with early Lyme borreliosis and strongly suggest a possible dissemination of the causative agents even when only a local infection is assumed.     

Immunohistochemical and virological study of skin in the papular-purpuric gloves and socks syndrome

Summary The pathogenesis of viral exanthems remains unclear. We have undertaken an immunohistochemical study of lesional skin biopsies in patients with the papular-purpuric gloves and socks syndrome (PPGSS) secondary to parvovirus B19 infection. Intracytoplasmic staining of the dermal endothelial cells, keratinocytes, and sweat glands was shown with an antibody to parvovirus B19. There were perivascular dermal infiltrates with T cells, sometimes with exocytosis. By polymerase chain reaction, virus DNA was detected in all skin biopsies and in one serum sample. The cutaneous manifestations of parvovirus infection seem secondary to infection of the endothelium and epidermis.     

Erythema multiforme: Differences between HSV‐1 and HSV‐2 and management of the disease—A case report and mini review

Erythema multiforme (EM) is an immune‐mediated reaction characterized by target lesions and with possible mucosal involvement. Its most frequent cause is HSV, with HSV‐1 more common than ?2. It is usually self‐limited but it can show recurrences. We report a peculiar case of recurrent herpes‐associated erythema multiforme (HAEM) in a 35‐year‐old man. The patient was affected by both herpes labialis and genitalis, but the typical target lesions were only associated with recurrent herpes labialis. Here, we hypothesize about the pathogenic differences between HSV‐1 and HSV‐2, and discuss the therapeutic management of HAEM.     

A case of erythema multiforme associated with primary Epstein-Barr virus infection

We present a case of primary Epstein-Barr virus (EBV) infection with erythema multiforme. A 1-year-old Japanese boy presented with skin eruptions, including typical target lesions and a low-grade fever. Just before the skin biopsy, 95 copies/μg DNA of EBV genome was detected in peripheral blood mononuclear cells, which subsequently increased to 6,834 copies/μg DNA. Skin tissue collected from the skin lesion showed the typical pathologic findings of erythema multiforme. EBV-encoded small nuclear RNA signals were not detected in the skin tissue by in situ hybridization.