Molluscum contagiosum virus MC80 sabotages MHC-I antigen presentation by targeting tapasin for ER-associated degradation.

The human specific poxvirus molluscum contagiosum virus (MCV) produces skin lesions that can persist with minimal inflammation, suggesting that the virus has developed robust immune evasion strategies. However, investigations into the underlying mechanisms of MCV pathogenesis have been hindered by the lack of a model system to propagate the virus. Herein we demonstrate that MCV-encoded MC80 can disrupt MHC-I antigen presentation in human and mouse cells. MC80 shares moderate sequence-similarity with MHC-I and we find that it associates with components of the peptide-loading complex. Expression of MC80 results in ER-retention of host MHC-I and thereby reduced cell surface presentation. MC80 accomplishes this by engaging tapasin via its luminal domain, targeting it for ubiquitination and ER-associated degradation in a process dependent on the MC80 transmembrane region and cytoplasmic tail. Tapasin degradation is accompanied by a loss of TAP, which limits MHC-I access to cytosolic peptides. Our findings reveal a unique mechanism by which MCV undermines adaptive immune surveillance.

Primitive follicular induction in molluscum contagiosum.

BACKGROUND: Molluscum contagiosum (MC) is the commonest human poxvirus infection. Follicular induction has rarely been observed in the epidermis surrounding lesions of MC. A virus-induced localized proliferation of germinative/stem cells of the folliculosebaceous-apocrine unit has been suggested as the underlying cause, however few reports of this peculiar phenomenon exist in the literature and the mechanisms involved in this proliferation require further study. METHODS: We prospectively collected MC cases showing multifocal areas of primitive follicular induction involving the adjacent undersurface epidermis. Immunohistochemical expression of BerEP4, PHLDA1 and cytokeratin 20 (CK20) was evaluated in the basaloid germs surrounding the lesions. For PHLDA1, we used epidermal melanocytes as a positive internal control. For BerEP4, we employed a basal cell carcinoma (BCC) and for CK20, colon as positive external controls. An incubation without the primary antibody functioned as an external negative control. RESULTS: All the cases studied showed an intense positive staining of the basaloid buds with BerEP4 and weaker stain for PHLDA1. CK20 showed the presence of scattered Merkel cells within the induced epidermal basaloid proliferations favoring their reactive origin. DISCUSSION: The pathogenetic mechanisms behind the development of these microscopic features and the link between follicular induction and poxvirus infection are explored. Awareness of this unusual phenomenon by dermatopathologists will be helpful in avoiding a misdiagnosis of a superficial BCC in such cases. CONCLUSIONS: BerEP4 and PHLDA1 were consistently expressed in the areas of primitive follicular induction surrounding lesions of MC. CK 20 stained the Merkel cells present in the basaloid buds. All these findings support the reactive origin of this phenomenon, which we believe is most probably viral-induced.

Immune escape phenomenon in molluscum contagiosum and the induction of apoptosis.

Molluscum contagiosum (MC) may persist for many weeks, evading host immunity. We studied the mechanism of immune escape phenomenon in MC, and the possible inducer of apoptosis. Using tissue samples of MC, we examined the numbers of epidermal Langerhans cells (LC), the expression levels of macrophage inflammatory protein-3α (MIP-3α) and thymic stromal lymphopoietin (TSLP), and the apoptotic signals. After molluscum contagiosum virus (MCV) genotyping, we studied the expression of MCV-encoded MC148 mRNA and MC159 mRNA which correspond to viral antagonist for CCR8 and viral Fas-linked interleukin (IL)-1ß converting enzyme (FLICE)-like inhibitor protein (vFLIP), respectively. The nutlin-3-induced apoptosis in MC was observed ex vivo. The numbers of CD1a(+) or Langerin(+) epidermal LC and the expression levels of MIP-3α were markedly decreased in MC. The expression of TSLP was enhanced in the lesional epidermis of atopic dermatitis and human papillomavirus-induced warts, whereas the expression was observed locally in MC. All 14 MC samples examined harbored MCV type 1. The MC148 mRNA was detected in all 14 samples and the MC159 mRNA was detected in 13 samples. Apoptotic cells were absent or at a background level in the living layers of MC, but their numbers were increased in the molluscum bodies by overnight incubation with 5 µmol/L nutlin-3 in culture medium. In conclusion, molluscum bodies are protected from host immune responses and apoptotic signals by being surrounded by LC-depleted epidermal walls and viral immunosuppressive molecules, but could be eradicated by reagents inducing p53-dependent apoptosis.

Immunolocalization of toll-like receptor 2 in viral warts and molluscum contagiosum.

OBJECTIVE: To explore toll-like receptor 2 (TLR2) immunolocalization and its possible role in the innate immune response against viral warts and molluscum contagiosum (MC). STUDY DESIGN: Using standard immunohistochemical techniques, we examined 50 cases (25 cases with viral warts and 25 cases with MC) together with normal skin biopsies of 25 age and sex-matched subjects who comprised the control group. RESULTS: TLR2 was expressed in the epidermis of all controls and in 94% of study cases. Staining patterns were cytoplasmic, membranous, nuclear and combined. There was a significant difference between cases and controls regarding the intensity (p = 0.0001) and pattern (p = 0.001) of TLR2 expression, where intense expression was in favor of cases and membranous and nuclear pattern of staining was seen only in cases. The intensity of TLR2 expression was significantly associated with patients of young age (p = 0.04), short disease duration (p = 0.04), facial location (p = 0.009), and MC category (p = 0.02). CONCLUSION: Our study suggests that upregulation of TLR2 is involved in the induction of defense mechanism against human papillomavirus and MC virus. Its nuclear localization may be related to virus pathogenesis, virus-TLR interaction, or to other unknown molecular events.

The MC159 protein from the molluscum contagiosum poxvirus inhibits NF-κB activation by interacting with the IκB kinase complex.

Molluscum contagiosum virus (MCV) causes persistent neoplasms in healthy and immunocompromised people. Its ability to persist likely is due to its arsenal of viral immunoevasion proteins. For example, the MCV MC159 protein inhibits TNF-R1-induced NF-κB activation and apoptosis. The MC159 protein is a viral FLIP and, as such, possesses two tandem death effector domains (DEDs). We show in this article that, in human embryonic kidney 293 T cells, the expression of wild-type MC159 or a mutant MC159 protein containing the first DED (MC159 A) inhibited TNF-induced NF-κB, or NF-κB activated by PMA or MyD88 overexpression, whereas a mutant protein lacking the first DED (MC159 B) did not. We hypothesized that the MC159 protein targeted the IκB kinase (IKK) complex to inhibit these diverse signaling events. Indeed, the MC159 protein, but not MC159 B, coimmunoprecipitated with IKKγ. MC159 coimmunoprecipitated with IKKγ when using mouse embryonic fibroblasts that lack either IKKα or IKKß, suggesting that the MC159 protein interacted directly with IKKγ. MC159-IKKγ coimmunoprecipitations were detected during infection of cells with either MCV isolated from human lesions or with a recombinant MC159-expressing vaccinia virus. MC159 also interacts with TRAF2, a signaling molecule involved in NF-κB activation. However, mutational analysis of MC159 failed to reveal a correlation between MC159-TRAF2 interactions and MC159's inhibitory function. We propose that MC159-IKK interactions, but not MC159-TRAF2 interactions, are responsible for inhibiting NF-κB activation.

Kaposi sarcoma in association with molluscum contagiosum: an uncommon diagnosis in a single biopsy and potential diagnostic pitfall.

Molluscum contagiosum is a cutaneous poxviral infection that is rarely associated with other skin diseases, such as cutaneous neoplasms. Such associations are likely to be coincidental, except in immunocompromised patients. Kaposi sarcoma, an angioproliferative neoplasm derived from lymphatic endothelium, is mediated by human herpes virus-8 infection and occurs with increased frequency in immunocompromised individuals. We report an unusual case of molluscum contagiosum with underlying cutaneous Kaposi sarcoma diagnosed in a single skin biopsy of a human immunodeficiency virus-positive patient. Our case highlights the importance of adequate sampling to avoid missing secondary diagnoses in histopathologic sections and alerts pathologists and dermatologists to the possibility of coinfection in high-risk patients by 2 virally-mediated skin conditions.

Soluble adenylyl cyclase defines a nuclear cAMP microdomain in keratinocyte hyperproliferative skin diseases.

Cyclic adenosine monophosphate (cAMP) is a nearly ubiquitous signaling molecule important for numerous signaling pathways in human skin. We studied a novel class of mammalian adenylyl cyclase, the soluble adenylyl cyclase (sAC). We examined sAC localization in normal human skin and found it to be present in keratinocytes, melanocytes, mononuclear cells, eccrine ducts, and nerves. In normal skin, sAC keratinocyte staining was evenly distributed throughout the cell. However, in certain hyperproliferative disorders of the skin, including psoriasis, verruca vulgaris, and SCCIS on sun-damaged skin, sAC keratinocyte staining was predominantly nuclear. In contrast, in other hyperproliferative disorders, such as basal cell carcinoma, sAC staining was similar to normal human skin. Using a model of epithelial differentiation, we established that sAC migrates into the nucleus when differentiated cells are induced to reenter the cell cycle. Previous work had determined that nuclear sAC activates the cAMP-response-element-binding (CREB) transcription factor, and we found that in psoriasis lesions, nuclear sAC occurs concomitantly with activation of CREB. Hence, sAC may play a role in the pathogenesis of certain hyperproliferative skin disorders via modulation of gene expression.

Increased expression of human beta-defensin 3 in mollusca contagiosum.

The human beta defensins (hBDs)-2 and -3 are inducible antimicrobial peptides present in human skin. Besides an important role in fighting bacteria, they also have an antiviral function. Molluscum contagiosum (MC) is a cutaneous viral disease caused by the MC virus. Lesions show a tendency towards spontaneous regression, which might be caused by antiviral proteins such as defensins. We report a marked increase in hBD-3 immunoreactivity in MC lesions in contrast to hBD-2, which was only marginally increased. We suggest a role for the hBD-3 peptide in MC pathogenesis.

Molluscum contagiosum: immunomorphological aspects of keratinocytes markers of differentiation and adhesion.

BACKGROUND: Molluscum contagiosum (MC) is a Molluscipox virus infection of keratinocytes with hyperplasia and intracytoplasmic inclusions - the molluscum bodies (MBs). Few papers address cytokeratins (K) profile in MC, mainly focusing terminal keratinization process. METHODS: Forty-one MC lesions were subjected to immunohistochemical technique to verify K1, K10, K14, K16, involucrin, filaggrin, E-cadherin and p63 expression. MC immunolabeling pattern was compared to adjacent normal appearing epidermis (ANAE). RESULTS: In MC and ANAE, K1/K10 were expressed in suprabasal layers, K14 was expressed in basal and suprabasal layers and K16 was expressed through all spinous layer. Involucrin and filaggrin were observed in granular, spinous and in basal layer of ANAE and MC. E-cadherin was present up to the first layers of MC while ANAE exhibited E-cadherin labeling at basal and spinous layers. Basal and spinous layers keratinocytes nuclei, in both MC and ANAE, express p63. CONCLUSION: Infection by Molluscipox virus alters keratinocyte differentiation status. The presence of K14 and p63 in spinous layer, as well as early expression of involucrin and filaggrin, associated to a hyperproliferative state disclosed by K16 expression, may be a result of disruption in keratinocytes maturation process. The changes observed at ANAE may represent early events in keratinization disturbance.

Expression of Toll-like receptors in verruca and molluscum contagiosum.

Recent studies indicate that several Toll-like receptors (TLRs) are implicated in recognizing viral structures and instigating immune responses against viral infections. The aim of this study is to examine the expression of TLRs and proinflammatory cytokines in viral skin diseases such as verruca vulgaris (VV) and molluscum contagiosum (MC). Reverse transcription-polymerase chain reaction and immunostaining of skin samples were performed to determine the expression of specific antiviral and proinflammatory cytokines as well as 5 TLRs (TLR2, 3, 4, 7, and 9). In normal human skin, TLR2, 4, and 7 mRNA was constitutively expressed, whereas little TLR3 and 9 mRNA was detected. Compared to normal skin (NS), TLR3 and 9 mRNA was clearly expressed in VV and MC specimens. Likewise, immunohistochemistry indicated that keratinocytes in NS constitutively expressed TLR2, 4, and 7; however, TLR3 was rarely detected and TLR9 was only weakly expressed, whereas 5 TLRs were all strongly expressed on the epidermal keratinocytes of VV and MC lesions. In addition, the mRNA expression of IFN-beta and TNF-alpha was upregulated in the VV and MC samples. Immunohistochemistry indicated that IFN-beta and TNF-alpha were predominantly localized in the granular layer in the VV lesions and adjacent to the MC bodies. Our results indicated that VV and MC skin lesions expressed TLR3 and 9 in addition to IFN-beta and TNF-alpha. These viral-induced proinflammatory cytokines may play a pivotal role in cutaneous innate immune responses.

Prominent eosinophilic intranuclear inclusions in melanocytes of a melanocytic nevus: the aftermath of an infection with molluscum contagiosum? A case report.

A 65-year-old Latino man presented to his dermatologist for the removal of two melanocytic nevi from the back. The first nevus was removed from the right scapula and contained melanocytes with prominent eosinophilic nuclear inclusion bodies. The second nevus was removed from the paravertebral region, without evidence of inclusion bodies. Ultrastructurally, the inclusions in the first nevus contained dispersed finely granular, homogenous bodies without a limiting membrane. Immunohistochemistry characterized them as ubiquitin-positive material. Reverse transcriptase in situ polymerase chain reaction analysis was positive for molluscum-specific primers, suggesting that the inclusions encountered in the first nevus were secondary to a remote, local molluscum viral infection of melanocytes.

Expression of Toll-Like Receptors in Verruca and Molluscum Contagiosum

Recent studies indicate that several Toll-like receptors (TLRs) are implicated in recognizing viral structures and instigating immune responses against viral infections. The aim of this study is to examine the expression of TLRs and proinflammatory cytokines in viral skin diseases such as verruca vulgaris (VV) and molluscum contagiosum (MC). Reverse transcription-polymerase chain reaction and immunostaining of skin samples were performed to determine the expression of specific antiviral and proinflammatory cytokines as well as 5 TLRs (TLR2, 3, 4, 7, and 9). In normal human skin, TLR2, 4, and 7 mRNA was constitutively expressed, whereas little TLR3 and 9 mRNA was detected. Compared to normal skin (NS), TLR3 and 9 mRNA was clearly expressed in VV and MC specimens. Likewise, immunohistochemistry indicated that keratinocytes in NS constitutively expressed TLR2, 4, and 7; however, TLR3 was rarely detected and TLR9 was only weakly expressed, whereas 5 TLRs were all strongly expressed on the epidermal keratinocytes of VV and MC lesions. In addition, the mRNA expression of IFN-beta and TNF-alpha was upregulated in the VV and MC samples. Immunohistochemistry indicated that IFN-beta and TNF-alpha were predominately localized in the granular layer in the VV lesions and adjacent to the MC bodies. Our results indicated that VV and MC skin lesions expressed TLR3 and 9 in addition to IFN-beta and TNF-alpha. These viral-induced proinflammatory cytokines may play a pivotal role in cutaneous innate immune responses.

Treatment of molluscum contagiosum with 585 nm collagen remodeling pulsed dye laser.

The inevitable regression of molluscum contagiosum (MC) has been the major argument in favor of leaving the lesions to spontaneous involution. But the infection is often widespread and recurrent. Conventional therapies are frequently ineffective and require multiple visits. Flashlamp-pumped pulsed dye laser is now recommended in the therapy of MC in case reports. There is no evaluation of a pulsed dye laser collagen remodeling (wavelength of 585 nm) as a possible therapeutic alternative. We treated 76 patients with cutaneous MC with 1 to 176 MC (mean 27 MC) in a prospective study from April 2002 to September 2002 (over a period of six months). The female/male sex ratio was of 1.2:1 (42 girls, and 34 boys). Patients were aged from 1 to 15 years, with a mean of 4.9 years. We used 585 nm collagen remodeling, double flashlamp excited pumped dye laser ED2000 (manufactured by Deka(c) MELA Calenzano, Italy), spot size 5 mm, energy density (fluence J/cm(2)) from 2 to 4 J/cm(2), emission modality (repetition rate) at 0.5 Hz, with a short pulse duration of 250 microsec in all cases. The therapy was well tolerated. No scars or pigment anomalies were observed. 96.3% of the lesions healed after the first treatment, the remaining 3.7% after the second (two weeks later). Laser photocoagulation causes selective damage to abnormal vessels and surrounding connective tissue. The heating effect in these skin layers triggers the release of various growth factors that stimulate collagen remodeling and tightening. It appears to be a cell-mediated reaction, which brings about an elevation in the T lymphocytes, capable of affecting pox viridae. Dye laser photocoagulation however, cannot protect against relapse. Hyperpigmentation may occur at nearly all sites, however this fades after 1 to 6 months. The pulsed dye laser for collagen remodeling is an effective, bloodless, quick, and easy therapeutic alternative for MC. The advantage of using a collagen remodeling pumped dye laser is the absence of pain, because of the short pulse duration (half that of a normal pulsed dye laser), and the use of low fluence (less than 4 J/cm(2)). It enables the treatment of young patients with a large number of lesions, which is impossible with a normal pulsed dye laser. There are no side effects.

An immunohistochemical study of abnormal keratinocyte proliferation in molluscum contagiosum.

BACKGROUND: Molluscum contagiosum is a common cutaneous tumour that is characterized by usually spontaneous involution and self-limited spreading in immunocompetent individuals. OBJECTIVE: We aimed to investigate the apoptosis and the expression of cell-cycle proteins in molluscum contagiosum lesions. METHODS: The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL)-based apoptotic index and the expression of the cell-cycle proteins Ki-67, p53, p21WAF and Bcl-2 were investigated in molluscum contagiosum lesions obtained from the trunk of 20 immunocompetent patients and in normal skin samples from the trunk of six healthy volunteers. RESULTS: Whereas molluscum contagiosum lesions displayed a TUNEL-based apoptotic index similar to that of normal skin, they exhibited an increased Ki-67 index, which was confined to the basal and first suprabasal layers (P < 0.001). Compared with normal non-sun-exposed skin, molluscum contagiosum lesions also exhibited increased p53 staining in basal cells (P < 0.01), increased p21WAF in suprabasal cells (P < 0.001) and loss of Bcl-2 expression. CONCLUSIONS: These results indicate that molluscum contagiosum lesions exhibit an increased proliferation rate of keratinocytes, which is likely to be partially counteracted by accumulation of p53.

Counts and areas of S-100-positive epidermal dendritic cells in atypical molluscum contagiosum affecting HIV+ patients.

Molluscum contagiosum is a common and self-limiting viral infection, that in HIV+ patients courses as an opportunist affection with atypical clinical features. Impaired cell-mediated immune response could be involved in such atypical growth. We evaluated the density and area of Langerhans cells (LC) using S-100 immunohistochemistry in seven atypical molluscum contagiosum. LC density was quantified by three different methods using computer-assisted morphometry as well as estimating the relative area of LC with respect to epidermal area. Results were compared with two control groups (normal skin specimens and molluscum contagiosum affecting non-AIDS healthy patients). We found a virtual absence of LC in areas of molluscum lesions affecting both HIV+ and non-AIDS patients. Likewise we observed an evident decrease in LC density in perilesional epidermis of atypical molluscum with respect to both control groups. Upon comparing the counts and areas, we observed that this reduction in LC count was statistically significant only when considering LC related to length of basement membrane in atypical molluscum with respect to normal skin specimens. Our finding of a reduced number of LC in the perilesional epidermis of HIV+ patients with atypical molluscum could explain the high frequency and clinical challenge of molluscum contagiosum in immunocompromised people. In spite of these results, further studies of LC kinetics and functions are required to precisely elucidate their role in the course of molluscum contagiosum in HIV+ patients.

An immunohistochemical study of abnormal keratinocyte differentiation in molluscum contagiosum.

To investigate abnormalities in the keratinization process in lesional epidermis of molluscum contagiosum, production of filaggrin, loricrin, Ted-H-1 antigen, involucrin, cystatin A and CD95 ligand (CD95L) was investigated using specific antibodies. Anti-filaggrin monoclonal antibody (MoAb) did not react with keratohyalin granules (KHG), but with the substance around virus particles in the stratum corneum. KHG reacted with anti-loricrin polyclonal antibody (PoAb) and anti-Ted-H-1 MoAb. Anti-involucrin PoAb and anti-cystatin A PoAb reacted with materials in the cytoplasm of the middle stratum spinosum to the stratum granulosum. CD95L was expressed in the cell membrane region of the living cell layers in lesional epidermis. These observations suggest that the keratinization process may be altered in molluscum contagiosum.