Multiple exposures during a norovirus outbreak on a river-cruise sailing through Europe, 2006.

In the summer of 2006, several cruise-related viral gastroenteritis outbreaks were reported in Europe. One report came from a river-cruise, belonging to a ship-owner who had two other ships with outbreaks. This situation warranted onsite investigation in order to identify a potential common source of infection. A retrospective cohort study was performed among 137 people on board. Epidemiological questionnaire data were analysed using logistic regression. Stool, food, water and surface samples were collected for norovirus detection. Norovirus GGII.4-2006b was responsible for 48 gastroenteritis cases on this ship as confirmed in six patients. Identical norovirus sequences were detected in stool samples, on surfaces and in tap water. Epidemiological and microbiological data indicated multiple exposures contributing to the outbreak. Microbiological results demonstrated person-to-person transmission to be clearly present. Epidemiological results indicated that consuming tap water was a risk factor; however, this could not be concluded definitively on the basis of the available data. A common source for all cruise-related outbreaks was unlikely. The ongoing outbreaks on this ship demonstrated that evidence based guidelines on effective disinfection strategies are needed.

Differential regulation of plasminogen activation in normal keratinocytes and SCC-4 cells by fibroblasts.

The plasminogen activator (PA)/plasmin system is thought to be involved in processes such as tumor invasion and wound healing, during which epithelial and mesenchymal cells come close together. However, information on regulation of the PA/plasmin system during epithelial-mesenchymal interactions is scarce. Therefore, we examined the in vitro modulation of the production and activity of the components of the PA/plasmin system in squamous carcinoma cells (SCC-4) and normal human keratinocytes in relation to cell density and the presence or absence of fibroblasts (3T3 cells). There was an inverse relation between cell density and mRNA expression for urokinase-type plasminogen activator (u-PA) and u-PA receptor in both SCC-4 cells and keratinocytes. In addition, such a relation was found for plasminogen activator inhibitor types 1 (PAI-1) and 2 (PAI-2) in SCC-4 monocultures, but not in keratinocyte monocultures. In contrast to monocultures, variation of cell density did not affect the mRNA expression of the components of the PA/plasmin system in cocultures of SCC-4 cells or keratinocytes with 3T3 cells. However, the relative expression of mRNAs in co-cultures was clearly different from that in monocultures, especially at low cell density. For most of the components of the PA/plasmin system, a decrease in mRNA expression and u-PA receptor protein was observed at most cell densities, whereas for PAI-1 only in keratinocytes a marked increase was documented. Zymography of supernatants revealed that the levels of both free u-PA and PA-PAI were increased in SCC-4/3T3 co-cultures, whereas in keratinocytes/3T3 co-cultures, only levels of the PA-PAI complex were increased, while the amount of free u-PA activity decreased. This occurred despite the increase u-PA immunoreactivity and was probably caused by the markedly elevated levels of immunoreactive PAI-1. The results of the present study reveal that the production and synthesis of various components of the PA/plasmin system in keratinocytes and SCC-4 cells depend on the density of epithelial cells and are modulated by fibroblasts, probably through a direct cell-cell or cell-matrix contact. Fibroblast-induced modulations are similar in keratinocytes and SCC-4 cells except for the regulation of PAI-1, which is markedly enhanced only in keratinocytes. This suggests that the modulation of PA activity in the direct microenvironment may be different under physiologic and pathologic conditions.

Association between epidermodysplasia verruciformis-associated human papillomavirus DNA in plucked eyebrow hair and solar keratoses.

Epidermodysplasia-verruciformis-associated human papillomavirus DNA has been demonstrated in squamous cell carcinomas and plucked hair from immunocompetent patients and renal transplant recipients. This study investigated the association between infection with epidermodysplasia-verruciformis-associated human papillomavirus, identified by the detection of viral DNA in plucked eyebrow hairs, and solar keratoses. These lesions are strongly predictive of squamous cell carcinoma. In a cross-sectional study 518 individuals were enrolled from a randomly selected sample of a subtropical Australian community. Epidermodysplasia-verruciformis-associated human papillomavirus DNA in eyebrow hair was detected using a nested polymerase chain reaction specific for epidermodysplasia-verruciformis-associated human papillomavirus types. Epidermo dysplasia-verruciformis-associated human papillomavirus DNA was present in 121 (49%) of 245 men and 116 (44%) of 262 women. There was a strongly significant increase in epidermodysplasia-verruciformis-associated human papillomavirus infection with age (p < 0.00001), with prevalences of 29% in the 25-39 y age group, 42% at 40-59 y and 65% in the 60-79 y age group. Among men there was a strong association between epidermodysplasia-verruciformis-associated human papillomavirus and solar keratoses with an odds ratio, adjusted for age, skin color, and occupational sun exposure, of 3.40 (95% confidence interval, 1.77-6.53). No such association was found among women [odds ratio 1.03 (95% confidence interval 0.59-1.77, after adjustment for the same factors)]. Differences in occupational sun exposure and smoking histories could not explain these apparently different associations between epidermodysplasia-verruciformis-associated human papillomavirus infection and solar keratoses in men and women. In conclusion, epidermodysplasia-verruciformis-associated human papillomavirus infection is associated with solar keratoses in men suggesting that epidermodysplasia-verruciformis-associated human papillomavirus infection, in conjunction with sex specific factors (like androgens), may be involved in neoplastic changes of keratinocytes.

Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers.

We have previously detected a group of human papillomaviruses originally found in skin lesions of epidermodysplasia verruciformis (EV) patients in skin cancers from renal transplant recipients and from non-immunosuppressed patients. The reservoir of EV-HPVs is still unknown. In the current study we investigated whether EV-HPV DNA can be detected in plucked hairs from renal transplant recipients and healthy volunteers. Hairs were plucked from eyebrows, scalp, arms, and/or legs and DNA was subsequently isolated. To detect EV-HPV, we used nested PCR with degenerate primers located in the HPV L1 open reading frame. HPV DNA was detected in hairs from one or more sites in all 26 renal transplant recipients tested. Forty-five of 49 samples (92%) from these 26 patients were positive. The HPV type was successfully determined by sequencing in 38 samples, and all types belonged to the EV-HPVs. In ten of 22 healthy volunteers (45%), EV-HPV DNA was also detected in hairs from one or more sites. Twenty of 38 samples (53%) were positive, of which 17 samples were typed as EV-HPV types. These findings indicate that EV-HPV is subclinically present in the skin of the general population. Immunosuppression may lead to activation of the virus, explaining the finding that the apparent prevalence of EV-HPV in plucked hairs from renal transplant patients is higher than in those from the volunteers. If a dose-response situation exists for the carcinogenic potential of HPV infection, this finding may be relevant to the increased risk of skin cancer in this group of patients.

Transduction of the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papillomaviruses alters human keratinocyte growth and differentiation in organotypic cultures.

Epidermodysplasia-verruciformis-associated human papilloma virus DNA has been detected in skin cancers, in premalignant and benign skin lesions, and in plucked hairs from immunocompetent and immunosuppressed patients. The role of epidermodysplasia-verruciformis-associated human papilloma virus in the pathogenesis of nonmelanoma skin cancer is still enigmatic. In organotypic cultures we investigated the effects of retroviral transduction of the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papilloma virus types 5, 12, 15, 17, 20, and 38 on the growth and differentiation of human keratinocytes. Differentiation was disturbed to different degrees as revealed by histology and by the expression patterns of differentiation markers keratin 10 and small proline rich protein 2. Conversely, proliferating cell nuclear antigen was induced in some of the suprabasal, differentiated cells to varying extent. No unscheduled DNA synthesis was detected in these cells, however, as probed by 5'-bromo-2'-deoxyuridine incorporation. Most intriguingly, when the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papilloma virus types 15 and 17 were transduced, a broadening layer of basal cells and an accelerated differentiation were observed. In addition, "papilla-like structures" comprising basal-like keratinocytes arose from the basal layer into the differentiated layers. These cells did not express the differentiation markers keratin 10 and small proline rich protein 2, but did actively replicate DNA. These observations warrant further research by using this system to elucidate the replication strategy of epidermodysplasia-verruciformis-associated human papilloma virus types in keratinocytes and to shed light on the role of these human papilloma virus types in the pathogenesis of skin cancer.

Parathyroid hormone related protein and interleukin-6 mRNA expression in larynx and renal cell carcinomas from normocalcaemic and hypercalcaemic patients.

AIMS: To determine the expression of parathyroid hormone related protein (PTHrP) and interleukin-6 (IL-6) mRNAs and their possible relation in malignant tumours, derived from patients with and without hypercalcaemia, commonly associated with humoral hypercalcaemia of malignancy. METHODS: PTHrP and IL-6 mRNA expression was studied by northern blot analysis in tumour specimens from 13 consecutive patients. Six patients (two with hypercalcaemia) had squamous cell carcinomas of the larynx and seven (one with hypercalcaemia) had renal cell carcinomas. RESULTS: There was no relation between the histological features of the tumours and the expression of either PTHrP or IL-6 mRNAs. PTHrP mRNA was detected in all squamous cell carcinomas, expression being highest in the two patients with hypercalcaemia. In the renal cell carcinomas PTHrP mRNA was expressed only in the patient with hypercalcaemia. IL-6 mRNA was detected in nearly all tumours studied but there was no apparent relation between its expression and that of PTHrP mRNA or serum calcium concentrations. CONCLUSIONS: PTHrP mRNA expression is increased in patients with hypercalcaemia but is not related to IL-6 mRNA expression. The results suggest a quantitative relation between PTHrP gene expression and hypercalcaemia, and imply that different mechanisms account for this expression in squamous and renal cell carcinomas.

Role of fibroblasts in the regulation of proinflammatory interleukin IL-1, IL-6 and IL-8 levels induced by keratinocyte-derived IL-1.

In the epidermis, the keratinocytes are the first cells to be encountered by external stimuli and they are able to promote the inflammatory response by increased production and release of various cytokines. In their turn, these cytokines may directly affect the production of proinflammatory cytokines in human dermal fibroblasts. In addition, in both epithelial and mesenchymal cells cytokine production may be modulated by their mutual interaction, and thereby regulate the inflammatory response. The present study aimed to examine the role of fibroblasts in the regulation of proinflammatory IL-1, IL-6 and IL-8 levels induced by keratinocyte-derived IL-1. The data show that in fibroblasts exposed to conditioned media derived from cultures of normal human keratinocytes or squamous carcinoma cells (SCC-4), both the IL-8 and IL-6 mRNA expression as well as protein production were elevated. In addition, it was shown that these effects were induced by IL-1 alpha. The IL-1 alpha-induced increase in IL-8 and IL-6 production, both on the protein level as well as on the mRNA level, were concentration dependent and occurred almost simultaneously. While the induction of IL-6 and IL-8 occurred simultaneously, the IL-6 mRNA remained elevated for longer. In contrast to increased IL-6 and IL-8 production the IL-1 alpha levels markedly decreased upon culturing of fibroblasts in keratinocyte-derived conditioned medium. From internalization experiments it could be concluded that binding of IL-1 to IL-1 receptors, and its subsequent internalization and intracellular degradation is the most likely mechanism involved in the reduction of IL-1 levels by fibroblasts. Comparing the rate of IL-1 reduction in the presence of various cell types indicated that the rate of IL-1 reduction is directly related to the number of IL-1 receptors found on these cell types. In conclusion, these results indicate that the release of IL-1 alpha by activated keratinocytes may act as an inducer of IL-8 and IL-6 production in neighbouring fibroblasts. This may be an important pathway for the amplification of the inflammatory response. The amounts of both cytokines produced by fibroblasts were at least two to three orders of magnitude higher than those produced by keratinocytes, suggesting an important role of fibroblasts in the general inflammatory response. Furthermore, fibroblasts might be involved in turning off this inflammatory response by reducing IL-1 levels, most likely via IL-1 receptor-mediated uptake.

HPV-DNA is not detectable in outgrowing cells from explant cultures of skin lesions established at the air-liquid-interface.

Keratinocyte cultures established from HPV containing skin cancers were described earlier to lose their HPV DNA after passaging in vitro. A different approach was therefore used in this study. Explant cultures were generated by depositing small pieces of various benign and (pre)malignant skin specimens of renal transplant recipients and non-immunosuppressed patients on fibroblast-populated collagen lattices or on de-epidermized dermis. Subsequently, the cultures were maintained at the air-liquid interface. At various time points, samples were collected for both HPV analysis, using a nested PCR approach, and morphology. The outgrowing keratinocytes developed into multilayered epithelial structures showing terminal differentiation. No histological differences were observed between cultures established from HPV positive and negative lesions. Eighteen biopsy specimens were tested for their HPV content before and after culture. Before culture 11 out of these skin specimens contained DNA of the Epidermodysplasia Verruciformis-related HPV types (EV-HPV). Comparison of the HPV types detected in two different parts of the same skin specimen before culture was strongly suggestive for a non-homogeneous distribution of EV-HPV in the lesions. From the explant cultures derived from the 11 HPV-positive biopsies, 31 samples from the originally explanted pieces of tissue and 38 samples from the outgrowing multilayered epithelial sections were collected. HPV DNA was detected in 10 of the 31 and in 3 of the 38 samples (Chi-square test, P = 0.01), respectively. These results indicate that EV-HPV positive keratinocytes do not efficiently proliferate or lose their HPV DNA in this culture system or EV-HPV DNA is present in only a few basal cells, making it improbable that these cells are located at the outgrowing margins.

Case-control study in a subtropical Australian population to assess the relation between non-melanoma skin cancer and epidermodysplasia verruciformis human papillomavirus DNA in plucked eyebrow hairs. The Nambour Skin Cancer Prevention Study Group.

Epidermodysplasia verruciformis human papillomavirus (EV-HPV) DNA has been demonstrated in malignant and benign skin lesions and in hairs plucked from renal transplant recipients and immunocompetent patients. We investigated the association between EV-HPV DNA in hairs plucked from eyebrows and the occurrence of non-melanoma skin cancer (NMSC) in a community-based study. Within a cohort of residents of a Queensland township (Nambour), nested case-control studies of recently developed NMSC (64 cases), basal-cell carcinoma (BCC) (51 cases) and squamous-cell carcinoma (SCC) (25 cases) were conducted. EV-HPV DNA in hair and a small number of available tumour samples was detected using a nested PCR specific for EV-HPV types. EV-HPV DNA was detected in hairs from 94 of 143 individuals (66%), and 36 (39%) of the samples contained 2 or more different EV-HPV types. Only known or putatively new EV-HPV types were detectable after sequencing 93 samples. EV-HPV status agreed for 12 of 20 subjects who had both hair and skin tumour samples available. In 4 of 5 pairs of positive samples, the same EV-HPV type was found. There were non-significant negative associations between EV-HPV and NMSC (OR 0.77, 95% CI 0.34-1.8) and BCC (OR 0.58, 95% CI 0.23-1.5) but a non-significant positive association with SCC (OR 2.00, 95% CI 0.50-8.0).

Human papillomavirus type 5 is commonly present in immunosuppressed and immunocompetent individuals.

DNA of a wide spectrum of epidermodysplasia verruciformis-associated human papillomavirus (HPV) types (EV-HPV) has been detected in skin lesions and plucked hairs from both immunosuppressed and a considerable proportion of non-immunosuppressed persons. Recently, the skin of psoriatic patients was claimed to be an important reservoir for a particular EV-HPV type, HPV 5, which is considered as a high-risk HPV type for skin carcinomas. In the present study, we analysed plucked hairs from immunosuppressed renal transplant patients and immunocompetent individuals, utilizing an HPV 5-specific nested polymerase chain reaction. HPV 5 was detected in hairs derived from 14 of 31 (45%) immunosuppressed patients and 21 of 135 (16%) immunocompetent individuals. Both the immunosuppressed and the immunocompetent groups consisted of individuals with and without non-melanoma skin cancer. HPV 5 DNA was detected in similar proportions of hair samples plucked from individuals with and without skin cancer in either group. Our results indicate that HPV 5 is commonly present in the population. The role of HPV 5 in the pathogenesis of skin carcinomas and psoriasis remains to be established.

Detection of human papillomavirus types 6 and 11 in pubic and perianal hair from patients with genital warts.

Genital human papillomavirus (HPV) types 6 and 11 are of clinical importance due to their role in the development of anogenital warts. A pilot study was performed to investigate whether DNAs from HPV types 6 and 11 are present in hairs plucked from the pubic and perianal regions and eyebrows of patients with genital warts at present and patients with a recent history of genital warts. Genital HPV DNA was detected in 9 of 25 (36%) pubic hair samples and in 11 of 22 (50%) perianal hair samples by the CPI/CPIIg PCR. After sequencing of 17 of 20 samples, HPV type 6 or 11 was detected in 6 of 25 (24%) hair samples from the pubis and 8 of 22 (36%) hair samples from the perianal region. These types were not detected in plucked eyebrow hairs. In contrast, the HPV types associated with epidermodysplasia verruciformis were detected in similar proportions (62%) in both samples of pubic and eyebrow hairs. Moreover, HPV type 6 and 11 DNAs were detected in pubic hairs plucked from two patients who had been successfully treated and who did not show any lesion at the time of hair collection; this finding is an argument that HPV DNA may persist in this region. The presence of genital HPV types in plucked pubic and perianal hair suggests that there is an endogenous reservoir for HPV which may play a role in the recurrences of genital warts.

Proteomic analysis of skin irritation reveals the induction of HSP27 by sodium lauryl sulphate in human skin.

BACKGROUND: There is an increasing need for screening of mild irritants in vitro to reduce animal testing. OBJECTIVES: Proteomics were used to search for new markers of which the expression changes after mild irritation. METHODS: Sodium lauryl sulphate (SLS) was applied topically on excised human skin. Epidermal proteins were isolated from SLS-treated skin specimens that showed hardly any morphological changes. The proteins were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and proteins that significantly increased or decreased after SLS treatment in a dose-dependent way were characterized by mass spectrometry. Subsequently, immunohistochemistry was performed on skin samples treated with SLS in vivo and nonanoic acid (NAA) or benzalkonium chloride (BC) in vitro to evaluate one of the identified proteins for its predictive value. RESULTS: We identified seven proteins as potentially new epidermal markers for skin irritation. Among these seven proteins, the 27 kDa heat shock protein (HSP27) was identified as the most prominently upregulated protein. A strong nuclear HSP27 staining was seen in the SLS-treated skin, whereas in the vehicle controls only cytoplasmic staining was observed. Moreover, nuclear staining was also observed after topical application of SLS in vivo and after exposure to NAA and BC in vitro. CONCLUSIONS: Our findings suggest that HSP27 may serve as a sensitive marker of skin irritation and eventually as a novel tool in clinics for testing the sensitivity of the patient for a panel of irritants.