Vertical mother-to-infant transmission of herpes simplex virus 2 is correlated with tropism due to mutations in viral UL13.

Although neonates are commonly exposed to vaginal herpes simplex virus (HSV)-2, neonatal herpes is rare. Therefore, we analyzed paired infant and maternal HSV-2 isolates from two cases of mother-to-infant transmission to identify viral factors contributing to vertical transmission. Sixteen infant isolates with neonatal herpes and 27 genital isolates in their third trimester were included. The infant isolates were significantly more temperature-independent than the maternal isolates. Sequence comparison revealed viral UL13 protein kinase (UL13-PK) mutation in the infant isolates in both cases. In the expanded cohort, infant isolates (5/18) had significantly more UL13-PK mutations than genital isolates (1/29). Isolates within 8 days post-birth (3/4) had a significantly higher frequency of UL13-PK mutation than those after 9 days (2/14), suggesting a close association between UL13-PK mutations and vertical transmission. Elongation factor 1-delta was identified as a target of UL13-PK by proteomic analysis of UL13-PK-positive and -negative HepG2 cells. The mixed infant isolates with the intact and mutated UL13-PK conferred altered cell tropism, temperature independence adapting to fetal temperature, and better growth properties in Vero and hepatoblastoma HepG2 cells than in HSV-2 with intact and mutated UL13-PK alone, indicating that viral UL13-PK mutation is essential for vertical HSV-2 transmission.

The wound/burn guidelines - 4: Guidelines for the management of skin ulcers associated with connective tissue disease/vasculitis.

The Japanese Dermatological Association prepared guidelines focused on the treatment of skin ulcers associated with connective tissue disease/vasculitis practical in clinical settings of dermatological care. Skin ulcers associated with connective tissue diseases or vasculitis occur on the background of a wide variety of diseases including, typically, systemic sclerosis but also systemic lupus erythematosus (SLE), dermatomyositis, rheumatoid arthritis (RA), various vasculitides and antiphospholipid antibody syndrome (APS). Therefore, in preparing the present guidelines, we considered diagnostic/therapeutic approaches appropriate for each of these disorders to be necessary and developed algorithms and clinical questions for systemic sclerosis, SLE, dermatomyositis, RA, vasculitis and APS.

The wound/burn guidelines - 5: Guidelines for the management of lower leg ulcers/varicose veins.

Varicose veins are treated at multiple clinical departments, but as patients often visit the dermatology clinic first due to leg ulcers, the present Guidelines for the Management of Lower Leg Ulcers/Varicose Veins were prepared in consideration of the importance of the dermatologist's role. Also, the disease concept of chronic venous insufficiency or chronic venous disorders and the CEAP classification of these disorders are presented. The objective of the present guidelines is to properly guide the diagnosis and treatment of lower leg ulcers/varicose veins by systematically presenting evidence-based recommendations that support clinical decisions.

The wound/burn guidelines - 6: Guidelines for the management of burns.

Burns are a common type of skin injury encountered at all levels of medical facilities from private clinics to core hospitals. Minor burns heal by topical treatment alone, but moderate to severe burns require systemic management, and skin grafting is often necessary also for topical treatment. Inappropriate initial treatment or delay of initial treatment may exert adverse effects on the subsequent treatment and course. Therefore, accurate evaluation of the severity and initiation of appropriate treatment are necessary. The Guidelines for the Management of Burn Injuries were issued in March 2009 from the Japanese Society for Burn Injuries as guidelines concerning burns, but they were focused on the treatment for extensive and severe burns in the acute period. Therefore, we prepared guidelines intended to support the appropriate diagnosis and initial treatment for patients with burns that are commonly encountered including minor as well as moderate and severe cases. Because of this intention of the present guidelines, there is no recommendation of individual surgical procedures.

Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma.

To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal in any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.

Detection of Merkel cell polyomavirus in Merkel cell carcinoma and Kaposi's sarcoma.

Merkel cell carcinoma is a rare malignancy that sometimes occurs in the skin of elderly people. Recently, a new human polyomavirus, Merkel cell polyomavirus (MCPyV) was identified in Merkel cell carcinoma. In the present study, MCPyV-DNA was detected in 6 of 11 (55%) cases of Merkel cell carcinoma by nested PCR and real-time PCR. Histologically, MCPyV-positive cases showed round and vesicular nuclei with a fine granular chromatin and small nucleoli, whereas MCPyV-negative cases showed polygonal nuclei with diffusely distributed chromatin. Real-time PCR analysis to detect the MCPyV gene revealed that viral copy numbers ranged 0.04-0.43 per cell in cases of Merkel cell carcinoma. MCPyV was also detected in 3 of 49 (6.1%) cases of Kaposi's sarcoma (KS), but not in 192 DNA samples of other diseases including 142 autopsy samples from 20 immunodeficient patients. The MCPyV copy number in KS was lower than that in Merkel cell carcinoma. PCR successfully amplified a full-length MCPyV genome from a case of KS. Sequence analysis revealed that the MCPyV isolated from KS had 98% homology to the previously reported MCPyV genomes. These data suggest that the prevalence of MCPyV is low in Japan, and is at least partly associated with the pathogenesis of Merkel cell carcinoma.

Loop-mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16, and 18.

A new method was developed for detection of human papillomavirus (HPV) by loop-mediated isothermal amplification (LAMP), which was compared with the polymerase chain reaction (PCR), and real-time PCR for specificity and sensitivity. All initial validation studies with the control DNA proved to be type-specific. In order to evaluate the reliability of HPV type-specific LAMP detecting HPV DNA from clinical samples, tissue specimens were obtained from 27 patients with external genital polypoid lesions. The histologic diagnoses included condyloma acuminatum (n = 21), bowenoid papulosis (n = 2), seborrheic keratosis (n = 2), epidermolytic acanthoma (n = 1), and hairy nymphae (n = 1). HPV-6 DNA and HPV-11 DNA were detected in 18 and 3 of 21 condylomata acuminata, respectively, and there was no simultaneous infection. HPV-16 DNA was detected in one of two bowenoid papuloses. HPV DNA was not detected in the seborrheic keratoses, epidermolytic acanthoma, and hairy nymphae. These results correlated perfectly with those from real-time PCR analysis. Most positive samples contained high copy numbers of HPV DNA. HPV-11 DNA was detected in one case that could not be detected by PCR. The average reaction time was about 59 min. There was a linear correlation between the genome quantity and reaction time to reach the threshold. The LAMP method has an additional advantage as a quantitative method, and is superior in terms of sensitivity, specificity, rapidity, and simplicity, and can potentially be a valuable tool for the detection of HPV DNA.

Role of neutralizing antibody in the pathogenesis of zoster and the correlation of severity with anti-gE: gI antibody response.

Antibody response to varicella-zoster virus glycoproteins and neutralizing antibodies were examined to correlate them with the severity of the zoster. Antibody rise to whole viral antigen was observed in 17 of 19 patients, that to gE: gI was not increased in 7 patients, and antibody to gE: gI in severe cases was significantly lower compared with mild cases in the convalescent phase than in the acute phase (p<0.05 Fisher's exact test). Antibody rise to gE: gI was well correlated with limited development of zoster lesions and, in contrast, antibody titer without rise was associated with increased severity and wider development of zoster lesions. As gE is the most abundant glycoprotein in the infected cells, the antibodies may be consumed in severe and widespread lesions, resulting in an antibody response to gE: gI without a rise. When the ratio of neutralizing antibody to anti-gE: gI antibody titers in the acute phase was compared with that in the recovery phase, the former was significantly lower in the severe cases than the latter (p=0.0082 Student t-test). This suggested that neutralizing antibody was specifically consumed in the acute phase of zoster among the anti-VZV antibodies. The role of humoral immunity in the pathogenesis of zoster might have been reflected in the antibody response to the neutralizing antibody in the acute phase and antibody to gE: gI in the severe cases.