Establishment of Human Pluripotent Stem Cell-Derived Skin Organoids Enabled Pathophysiological Model of SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19) patients with impact on skin and hair loss are reported. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is detected in the skin of some patients; however, the detailed pathological features of skin tissues from patients infected with SARS-CoV-2 at a molecular level are limited. Especially, the ability of SARS-CoV-2 to infect skin cells and impact their function is not well understood. A proteome map of COVID-19 skin is established here and the susceptibility of human-induced pluripotent stem cell (hiPSC)-derived skin organoids with hair follicles and nervous system is investigated, to SARS-CoV-2 infection. It is shown that KRT17+ hair follicles can be infected by SARS-CoV-2 and are associated with the impaired development of hair follicles and epidermis. Different types of nervous system cells are also found to be infected, which can lead to neuron death. Findings from the present work provide evidence for the association between COVID-19 and hair loss. hiPSC-derived skin organoids are also presented as an experimental model which can be used to investigate the susceptibility of skin cells to SARS-CoV-2 infection and can help identify various pathological mechanisms and drug screening strategies.

Follicle-sinus complexes in muzzle skin of domestic and wild animals as diagnostic material for detection of rabies.

We previously reported a novel diagnostic method using follicle-sinus complexes (FSCs) in the muzzle skin for postmortem diagnosis of rabies in dogs. However, whether this method works in other animal species remains unclear. Here, FSCs were collected from a wolf, a red fox, 2 bats, and a cat, and examined for the presence of viral antigen, viral mRNA, and viral particles. Viral antigen and viral mRNA were confirmed in Merkel cells (MCs) in FSCs of all species. Electron microscopy performed using only samples from wolf and cat confirmed viral particles in MCs of FSCs. These results suggested that this novel diagnostic method using FSCs might be useful for detection of rabies not only in domestic but also wild animals.

Assessment of Prolonged Dengue Virus Infection in Dermal Fibroblasts and Hair-Follicle Dermal Papilla Cells.

Dengue virus (DENV)-mediated hair loss is one of the post-dengue fatigue syndromes and its pathophysiology remains unknown. Whether long-term or persistent infection with DENV in the scalp results in hair loss is unclear. In this study, we cultured human dermal fibroblasts (WS1 cells) and primary human hair-follicle dermal papilla cells (HFDPCs) in the long term with DENV-2 infection. The production of virion, the expression of inflammatory and anti-virus genes, and their signaling transduction activity in the infected cells were analyzed. DENV-2 NS3 protein and DENV-2 5' UTR RNA were detected in fibroblasts and HFDPCs that were subjected to long-term infection with DENV-2 for 33 days. A significant amount of DENV-2 virion was produced by both WS1 cells and HFDPCs in the first two days of acute infection. The virion was also detected in WS1 cells that were infected in the long term, but HFDPCs failed to produce DENV-2 after long-term culture. Type I and type III interferons, and inflammatory cytokines were highly expressed in the acute phase of DENV infection in HFPDC and WS1 cells. However, in the long-term cultured cells, modest levels of anti-viral protein genes were expressed and we observed reduced signaling activity, which was correlated with the level of virus production changes. Long-term infection of DENV-2 downregulated the expression of hair growth regulatory factors, such as Rip1, Wnt1, and Wnt4. This in vitro study shows that the long-term infection with DENV-2 in dermal fibroblasts and dermal papilla cells may be involved with the prolonged-DENV-infection-mediated hair loss of post-dengue fatigue syndrome. However, direct evidence for viral replication in the human hair of a dengue victim or animal infection model is required.

Statistical analysis of the usefulness of follicle-sinus complexes as a novel diagnostic material for canine rabies.

In the present study, follicle-sinus complexes (FSCs) were harvested from the muzzle skin of 123 dogs with suspected canine rabies, and the sensitivity and specificity of FSC analysis were compared with those of brain tissue immunohistochemistry analysis. In the FSCs, viral antigen was detected from Merkel cells. Sensitivity was 97.3%, specificity was 100%, and the coefficient κ was 0.88. These results reconfirm that FSCs are very useful for the postmortem diagnosis of canine rabies, and suggest that 5 FSCs can yield results that are almost equivalent to those derived from brain tissue analysis in rabid dogs.

Dengue Virus Infects Primary Human Hair Follicle Dermal Papilla Cells.

During the epidemic of the dengue virus (DENV) infection in Taiwan in 2014 and 2015, we observed an abnormally high frequency of increased scalp hair shedding in infected individuals that could not be explained by telogen effluvium. In this study, the mechanism of hair loss caused by DENV was explored. Human hair follicle dermal papilla cells (HFDPCs) are essential for hair follicle morphogenesis and cycling. Thus, we established an in vitro DENV infection model in HFDPCs. On immunofluorescence analysis, HFDPCs that were susceptible to DENV infection responded to type I interferon (IFN) treatment, and the cells showed antibody-dependent enhancement (ADE) effect. The expression of the pro-inflammatory cytokines, interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-α), revealed an inflammatory response in DENV-infected HFDPCs. In particular, DENV infection impaired cell viability, and it activated caspase-associated cell death signaling in HFDPCs. In conclusion, our data indicate that direct infection with DENV causes inflammation and cell death in HFDPCs, which is involved in the mechanisms of hair loss after DENV infection. The knowledge of DENV infection in an immune-privileged tissue, such as hair follicles, may suggest their use for further studies on post-dengue fatigue syndrome (PDFS).

Detection of novel Betapapillomaviruses and Gammapapillomaviruses in eyebrow hair follicles using a single-tube 'hanging droplet' PCR assay with modified pan-PV CODEHOP primers.

A modified pan-PV consensus-degenerate hybrid oligonucleotide primer (CODEHOP) PCR was developed for generic and sensitive detection of a broad-spectrum of human papillomaviruses (HPVs) infecting the cutaneous epithelium. To test the analytical sensitivity of the assay we examined 149 eyebrow hair follicle specimens from immunocompetent male patients. HPV DNA was detected in 60 % (89/149) of analysed eyebrow samples with a total of 48 different HPV sequences, representing 21 previously described HPVs and 27 putative novel HPV types. Evidence for ten novel HPV subtypes and seven viral variants, clustering to three out of five genera containing cutaneous HPVs, was also obtained. Thus, we have shown that the modified pan-PV CODEHOP PCR assay is able to identify multiple HPV types, even from different genera, in the same clinical sample. Overall, these results demonstrate that the pan-PV CODEHOP PCR is an excellent tool for screening and identification of novel cutaneous HPVs, even in samples with low viral loads.

Possible chromosomal and germline integration of human herpesvirus 7.

Human herpesvirus 7 (HHV-7) is a betaherpesvirus, and is phylogenetically related to both HHV-6A and HHV-6B. The presence of telomeric repeat sequences at both ends of its genome should make it equally likely to integrate into the human telomere as HHV-6. However, numerous studies have failed to detect germline integration of HHV-7, suggesting an important difference between the HHV-6A/-6B and HHV-7 genomes. In search of possible germline integrated HHV-7, we developed a sensitive and quantitative real-time PCR assay and discovered that primers designed against some parts of the HHV-7 genome can frequently miss HHV-7 positive clinical samples even though they work efficiently in cell-culture-derived HHV-7 positive materials. Using a primer pair against the U90 ORF of HHV-7, we identified a possible case of germline integration of HHV-7 with one copy of viral genome per cell in both peripheral blood cells and hair follicles. Chromosomal integration of HHV-7 in these individuals was confirmed by fluorescence in situ hybridization analysis. Germline integration of HHV-7 was further confirmed by detection of ~2.6 copies of HHV-7 in the hair follicles of one of the parents. Our results shed light on the complex nature of the HHV-7 genome in human-derived materials in comparison to cell-culture-derived materials and show the need for stringent criteria in the selection of primers for epidemiological HHV-7 studies.

Localization of the rabies virus antigen in Merkel cells in the follicle-sinus complexes of muzzle skins of rabid dogs.

The direct fluorescent antibody test (dFAT) on fresh brain tissues is the gold standard for rabies virus antigen detection in dogs. However, this method is laborious and holds a high risk of virus exposure for the experimenter. Skin biopsies are useful for the diagnosis of humans and animals. In mammals, the tactile hair, known as the follicle-sinus complex (FSC), is a specialized touch organ that is abundant in the muzzle skin. Each tactile hair is equipped with more than 2,000 sensory nerve endings. Therefore, this organ is expected to serve as an alternative postmortem diagnostic material. However, the target cells and localization of rabies virus antigen in the FSCs remain to be defined. In the present study, muzzle skins were obtained from 60 rabid dogs diagnosed with rabies by dFAT at the Research Institute of Tropical Medicine in the Philippines. In all dogs, virus antigen was clearly detected in a part of the outer root sheath at the level of the ring sinus of the FSCs, and the majority of cells were positive for the Merkel cell (MC) markers cytokeratin 20 and CAM5.2. Our results suggest that MCs in the FSCs of the muzzle skin are a target for virus replication and could serve as a useful alternative specimen source for diagnosis of rabies.

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.

Trichodysplasia spinulosa: a polyomavirus infection specifically targeting follicular keratinocytes in immunocompromised patients.

Trichodysplasia spinulosa (TS) is a rare skin disease, caused by a specific polyomavirus, occurring in immunocompromised patients. The pathophysiological mechanisms of TS are not yet fully understood. By using polymerase chain reaction and skin biopsy immunostaining we report evidence, in a paediatric case, of follicular keratinocytes being the primary target of trichodysplasia spinulosa-associated polyomavirus.

Spiny Papules in an Immunosuppressed Child.

Trichodysplasia spinulosa is a rare folliculocentric polyomavirus infection observed in the setting of immunosuppression. We report a 7-year-old boy with pre-B-cell lymphoblastic leukemia who presented with folliculocentric spiny papules on the face. Histologic evaluation revealed hypertrophic bulbs, an expanded inner root sheath, and numerous brightly eosinophilic trichohyalin granules. We present this case to raise awareness of this rare but recognizable entity and to highlight the availability of appropriate diagnostic and therapeutic modalities.

Invasion of Herpes Simplex Virus Type 1 into Murine Epidermis: An Ex Vivo Infection Study.

Herpes simplex virus type 1 (HSV-1) invades its human host via the skin or mucosa. We aim to understand how HSV-1 overcomes the barrier function of the host epithelia, and for this reason, we established an ex vivo infection assay initially with murine skin samples. Here, we report how tissue has to be prepared to be susceptible to HSV-1 infection. Most efficient infection of the epidermis was achieved by removing the dermis. HSV-1 initially invaded the basal epidermal layer, and from there, spreading to the suprabasal layers was observed. Strikingly, in resting stage hair follicles, only the hair germ was infected, whereas the quiescent bulge stem cells (SCs) were resistant to infection. However, during the growth phase, infected cells were also detected in the activated bulge SCs. We demonstrated that cell proliferation was not a precondition for HSV-1 invasion, but SC activation was required as shown by infection of aberrantly activated bulge SCs in integrin-linked kinase (ILK)-deficient hair follicles. These results suggest that the status of the bulge SCs determines whether HSV-1 can reach its receptors, whereas the receptors on basal keratinocytes are accessible irrespective of their proliferation status.

Cidofovir gel as treatment of follicular spicules in multiple myeloma.

IMPORTANCE: The cause of follicular spicules in multiple myeloma (MM) is not known. OBSERVATIONS: We present a case of follicular spicules in a patient with MM, which is very reminiscent of trichodysplasia spinulosa caused by a polyomavirus. No trichodysplasia spinulosa-associated polyomavirus could be isolated from the skin lesions; however, the spicules were positive for Merkel cell carcinoma virus, which is also a polyomavirus. CONCLUSIONS AND RELEVANCE: Follicular spicules in MM are probably not caused by the trichodysplasia spinulosa-associated virus. Merkel cell polyomavirus could contribute to the origin of this dermatosis.

Human papillomavirus load in eyebrow hair follicles and risk of cutaneous squamous cell carcinoma.

BACKGROUND: Beta-human papillomavirus (betaPV) may play a role in the development of cutaneous squamous cell carcinoma (SCC). However betaPV is highly prevalent, and it may only be people with a higher viral load who have increased risk of SCCs. We therefore examined the association between betaPV load and SCCs. METHODS: We recruited 448 immunocompetent cases with SCCs and 464 controls from Italy and Australia and 497 immunosuppressed organ transplant recipients (OTR; 179 cases and 318 controls) from Europe. We used reverse hybridization to genotype 25 betaPV types in eyebrow hair follicles and determined the viral load for eight selected types using quantitative PCR. We used logistic regression to assess associations between type-specific and cumulative viral load and SCCs. RESULTS: Australian and OTR participants in the highest cumulative load tertile were at significantly higher risk of SCCs than those in the lowest tertile. Those with more than four betaPV types in the high load tertile were at approximately three-fold increased risk of SCCs. In Australia, HPV23 and 36 loads were significantly associated with SCCs, with borderline associations for HPV5 and 38. In OTR, HPV8 and 38 loads were significantly associated and HPV20 and 36 were borderline. We found little evidence for an association between load and SCCs in Italy. CONCLUSIONS: High viral load may be associated with risk of cutaneous SCCs, with total load seemingly more important than the load of any specific type. IMPACT: Our findings lend weight to the hypothesis that HPV plays a role in skin carcinogenesis.

[Human papillomavirus in the pubic hair follicles of men and cervical lesions in their female sexual partners].

OBJECTIVE: To investigate the association of human papillomavirus (HPV) in the pubic hair follicles of males with HPV infection in their female sexual partners. METHODS: We included in this study 21 female patients with HPV infection, including 8 cases of cervical cancer, 5 cases of atypical cervical hyperplasia, 5 cases of cervical condyloma, and 3 cases with unidentified causes. We also enlisted 52 men without visible condyloma acuminatum in the external genitalia as healthy controls. We detected HPV in the pubic hair follicles of the female patients' male sexual partners and the healthy male controls by PCR and reverse hybridization in situ. RESULTS: HPV positive was found in 6 (28.6%) of the 21 women's male partners, in whom the HPV types were correspondent situ. to those of the female patients. CONCLUSION: HPV in the pubic hair follicles of men might be one of the causes of HPV-related cervical lesions in their female sexual partners.