hMena: altered expression in psoriatic skin.

Psoriasis is a chronic inflammatory skin disease, characterized by an enhanced proliferation and a deregulated differentiation of keratinocytes. hMena is an actin regulatory protein involved in the control of cell motility and adhesion. hMena results up-modulated in several human tumors with respect to normal tissues and its expression has been positively correlated to proliferation rate, tumor size and aggressiveness in response to mitogenic stimuli, such as epidermal growth factor. The hyperproliferation of keratinocytes observed in psoriasis prompted us to evaluate hMena expression on biopsies collected from involved and uninvolved skin of 12 patients with active plaque-type psoriasis with respect to healthy skin. We analyzed the expression of hMena at transcript and protein levels by quantitative RT-PCR and immunohistochemistry. We correlated the expression of hMena to Ki67 proliferation index and to keratin 10 (K10) and keratin 16 (K16) used as markers of keratinocyte differentiation and activation. We demonstrated the expression of hMena in a hyperproliferative skin condition not related to neoplastic transformation. Interestingly, we observed that hMena is not expressed in healthy skin, but it becomes detectable in non-lesional areas and it is even more expressed in lesional psoriatic skin. In addition, we found that hMena expression is correlated to the rate of keratinocyte proliferation and activation. Hence, our observations indicate hMena as a new possible player, involved in the development and/or maintenance of the hyperproliferative state of psoriatic keratinocytes.

[Mechanisms underlying post-inflammatory hyperpigmentation: lessons from solar lentigo]. / Mécanismes de l'hyperpigmentation post-inflammatoire: ce que le lentigo actinique nous a appris.

Hyperpigmentation of the skin is a common dermatologic condition in all skin types but most prominent in brown-skinned population. In skin of color any inflammation or injury can be accompanied by alterations in pigmentation (hyper/hypo-pigmentation). Postinflammatory hyperpigmentation (PIH) can be observed in many skin conditions including acne, eczema, and contact dermatitis. In the control of skin pigmentation, parallel to the cross-talk between keratinocytes and melanocytes, increasing evidence has underlined the crucial role exerted by the interactions between mesenchymal and epithelial cells through the release of fibroblast-derived growth factors. Among these factors, the keratinocyte growth factor (KGF), alone or in combination with interleukin-1α, induces melanin deposition in vitro and hyperpigmented lesions in vivo. Furthermore, a moderate increase of KGF and a high induction of its receptor have been shown in solar lentigo lesions, suggesting the involvement of this growth factor in the onset of the hyperpigmented spots. Several studies highlight the possible contribution of the fibroblast-derived melanogenic growth factors to the hyperpigmentated lesions, in the context of the mesenchymal - epithelial interactions modulating melanocyte functions.

Angiokeratoma: decision-making aid for the diagnosis of Fabry disease.

Isolated angiokeratomas are common benign cutaneous lesions, generally deemed unworthy of further investigation. In contrast, diffuse angiokeratomas should alert the physician to a possible diagnosis of Fabry disease, a rare X-linked lysosomal storage disorder, characterized by α-galactosidase deficiency. Glycosphingolipids accumulate in cells throughout the body resulting in progressive multi-organ failure. Difficulties are encountered when trying to interpret the significance of angiokeratomas because they may also occur in other lysosomal storage disorders and rarely in an isolated manner in Fabry disease. We present an algorithm for the classification of angiokeratomas which might prove useful for the diagnosis and management of Fabry disease. Assessment of the clinical features and location of the lesions, personal and family history, skin biopsy, dermoscopy and electron microscopy imaging are sequential steps in the diagnostic process. Assessing the deficiency of α-galactosidase enzyme activity is essential to confirm the diagnosis in males, while mutation analysis is always needed in females. Potentially this algorithm can change the current approach to patients when Fabry disease is suspected, thus improving the diagnostic strategy and management of this disorder. It remains to be decided whether the use of an algorithm might reduce the number of genetic consultations. As evidence has shown the efficacy of enzyme replacement therapy in halting progression of the disease before the onset of irreversible organ damage, it is advisable to aim at an early diagnosis in order to achieve timely initiation of effective treatment with benefits for patients and appropriate use of medical resources.

Role of fibroblast-derived growth factors in regulating hyperpigmentation of solar lentigo.

BACKGROUND: Cutaneous pigmentation is regulated by a complex melanogenic network in which both keratinocytes and fibroblasts synthesize growth factors and cytokines. Solar lentigo (SL) is characterized by hyperpigmented lesions occurring on photodamaged skin areas. Despite the association of SL to ultraviolet (UV) exposure, the mechanisms underlying the development of these spots are not completely defined. OBJECTIVES: To analyse the involvement of the fibroblast-derived growth factors, hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and stem cell factor (SCF) in SL hyperpigmentation; to evaluate whether the photoageing process occurring in fibroblasts could be responsible for the altered expression of these cytokines; and to investigate a new possible role of KGF in regulating pigmentation through the specific induction of melanogenic cytokines by keratinocytes. METHODS: We performed immunohistochemical analysis of HGF, KGF and SCF on SL biopsies. We analysed the mRNA expression of these cytokines using an in vitro model of photoageing induced on fibroblasts. Finally, we evaluated the effects of KGF on the expression of melanogenic cytokines at the mRNA and protein levels on keratinocytes. RESULTS: We found positive staining for HGF, KGF and SCF in the upper dermis of SL lesions and a significant induction of the three cytokines in photoaged fibroblasts. We also demonstrated the contribution of KGF to pigmentation, showing its ability specifically to modulate the expression of SCF in keratinocytes. CONCLUSIONS: Fibroblasts may be persistently activated by UV exposure to release melanogenic growth factors; this inducible cytokine network acts both directly and indirectly through keratinocytes and may contribute to the hyperpigmentation of SL.

Co-localization of IgA and TG3 on healthy skin of coeliac patients.

BACKGROUND: Dermatitis herpetiformis (DH), the skin's expression of coeliac disease (CD), is induced by the presence of IgA antibodies and epidermal transglutaminase (TG3) as the main autoantigen, stored in the papillary dermis and on the vessel walls. AIMS: To evaluate the presence of IgA and TG3 deposits, considered to be the first step in inducing DH, in healthy skin of coeliac patients without cutaneous manifestations. METHODS: Punch biopsies were taken from 11 consecutive coeliac patients, two with DH and nine without cutaneous manifestations, three of whom were adhering to a gluten-free diet (GFD), and evaluated for the presence of deposits in the upper dermis and vessel walls by immunofluorescence and confocal microscopy. RESULTS: In coeliac patients affected by DH we found the presence of IgA and TG3 deposits mainly on the upper dermis, but also in vessel walls. In all coeliac patients without DH and also in those patients who were following a strict GFD, we found widely variable deposits of IgA and TG3 in both the papillary dermis and the vessel walls, although a lower intensity of the fluorescence signal was detected than with coeliac patients affected by DH. Double immunostaining with anti-IgA and anti-TG3 antibodies showed a strong co-localization in the upper dermis in patients with DH and a weaker co-localization in those without DH. CONCLUSIONS: We have demonstrated the presence of IgA and TG3 deposits in the healthy skin of coeliac patients, which are considered to play a central role in the pathogenesis of DH.

Involvement of hTERT in apoptosis induced by interference with Bcl-2 expression and function.

Here, we investigated the role of telomerase on Bcl-2-dependent apoptosis. To this end, the 4625 Bcl-2/Bcl-xL bispecific antisense oligonucleotide and the HA14-1 Bcl-2 inhibitor were used. We found that apoptosis induced by 4625 oligonucleotide was associated with decreased Bcl-2 protein expression and telomerase activity, while HA14-1 triggered apoptosis without affecting both Bcl-2 and telomerase levels. Interestingly, HA14-1 treatment resulted in a profound change from predominantly nuclear to a predominantly cytoplasmic localization of hTERT. Downregulation of endogenous hTERT protein by RNA interference markedly increased apoptosis induced by both 4625 and HA14-1, while overexpression of wild-type hTERT blocked Bcl-2-dependent apoptosis in a p53-independent manner. Catalytically and biologically inactive hTERT mutants showed a similar behavior as the wild-type form, indicating that hTERT inhibited the 4625 and HA14-1-induced apoptosis regardless of telomerase activity and its ability to lengthening telomeres. Finally, hTERT overexpression abrogated 4625 and HA14-1-induced mitochondrial dysfunction and nuclear translocation of hTERT. In conclusion, our results demonstrate that hTERT is involved in mitochondrial apoptosis induced by targeted inhibition of Bcl-2.

Immunohistochemical analysis of keratinocyte growth factor and fibroblast growth factor 10 expression in psoriasis.

The pathogenic mechanism underlying the hyperproliferation of keratinocytes in psoriasis is still not completely clarified. The production of cytokines released by activated T lymphocytes infiltrating the upper dermis probably has a crucial role. Even dermal fibroblasts can participate in the process through the secretion of growth factors, and some studies have reported an increased expression of the insulin-like growth factor 1. Few studies, however, have focused on the possible involvement of the keratinocyte growth factor (KGF/FGF-7) and the fibroblast growth factor 10 (FGF-10/KGF-2), which are secreted by fibroblasts and stimulate keratinocyte proliferation acting through a receptor specifically expressed by epithelial cells. The aim of this study was to investigate the expression of KGF and FGF-10 on the skin of patients with psoriasis by immunohistochemical analysis and to evaluate the correlation with the lymphocyte infiltrate and the epidermal proliferation. Immunostaining for KGF and FGF-10 showed that both the growth factors are upregulated in the upper dermis of psoriatic skin, and that the expression is correlated with the presence of T-cell infiltrate and with keratinocyte proliferation. Our data suggest that in psoriatic lesions activated lymphocytes can stimulate fibroblasts to produce KGF and FGF-10, which in turn contribute to sustain the hyperproliferative status of the keratinocytes.

Evidence of microevolution in a clinical case of recurrent Cryptococcus neoformans meningoencephalitis.

The aim of this study was to examine three serial isolates of Cryptococcus neoformans from a patient with AIDS for genotypical and phenotypical characteristics. The isolates were obtained during a first episode of cryptococcosis (simultaneous sampling of blood and cerebrospinal fluid) and after a relapse 3 years later (sampling of cerebrospinal fluid). Pulsed-field gel electrophoresis and random amplification of polymorphic DNA revealed that the blood isolate 1525 (first episode) was different from the two cerebrospinal fluid isolates (1526, first episode; 1782, relapse), yet the cerebrospinal fluid isolates were indistinguishable from each other regardless of the analysis performed. Phenotypical studies showed that isolate 1782 had significantly improved resistance to phagocytosis and killing by monocytes and polymorphonuclear cells and an altered efficacy in evoking cytokine response (augmentation of tumour necrosis factor-alpha, interleukin [IL]-1beta, IL-10, and interferon-gamma, decrease of IL-12). Interestingly, capsule size and antifungal drug resistance remained unchanged, while production of phospholipase and protease was consistently enhanced in the 1782 isolate with respect to the 1525 and 1526 isolates. In conclusion, in serial Cryptococcus neoformans isolates from a patient with AIDS, phenotypical changes but not molecular changes were documented, thus supporting the role of microevolution as a pathogenetic mechanism(s) for persistence/reactivation of fungal organisms.

Up-modulation of the expression of functional keratinocyte growth factor receptors induced by high cell density in the human keratinocyte HaCaT cell line.

Keratinocyte growth factor (KGF) is involved in the control of proliferation and differentiation of human keratinocytes. It binds to, and activates, the tyrosine kinase KGF receptor (KGFR), a splicing transcript variant of the fibroblast growth factor receptor 2. We have previously shown (C. Marchese et al., Cell Growth Differ., 8: 989-997, 1997) that differentiation of primary cultured keratinocytes triggered by high Ca2+ concentrations in the growing medium induced up-regulation of KGFR expression, which suggested that KGFR may play a crucial role in the control of the proliferative/differentiative program during transition from basal to suprabasal cells. Here we analyzed the process of modulation of the expression of KGFRs in the human keratinocyte cell line HaCaT, widely used as a model to study keratinocyte differentiation. Western blot and double immunofluorescence for KGFR and the K1 differentiation marker showed that cell differentiation and stratification induced by confluence and high cell density correlated with an increase in KGFR expression. KGFRs, present on suprabasal differentiated cells, appeared to be efficiently tyrosine-phosphorylated by KGF, which indicated that the receptors up-regulated by differentiation can be functionally activated by ligand binding. Bromodeoxyuridine incorporation assay revealed that a significant portion of suprabasal differentiated cells expressing KGFR seemed to be still able to synthesize DNA and to proliferate in response to KGF, which suggested that increased KGFR expression may be required for retention of the proliferative activity.

The BFRF1 gene of Epstein-Barr virus encodes a novel protein.

Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are approximately 100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. The BamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in the BamHI F region (BFRF1). Histidine and glutathione S-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitt's lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing the BFRF1 gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription of BFRF1, thus indicating that BFRF1 can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of the virus.

Intracellular transport and maturation pathway of human herpesvirus 6.

A peculiar characteristic of cells infected with human herpesvirus 6 (HHV6) is the absence of viral glycoproteins on the plasma membrane, which may reflect an atypical intracellular transport of the virions and/or the viral glycoproteins, different from that of the other members of the herpesvirus family. To investigate the maturation pathway of HHV-6 in the human T lymphoid cell line HSB-2, we used lectin cytochemistry and immunogold labeling combined with several electron microscopical techniques, such as ultrathin frozen sections, postembedding, and fracture-label. Immunolabeling with anti-gp116 and anti-gp82-gp105 monoclonal antibodies revealed that the viral glycoproteins are undetectable on nuclear membranes and that at the inner nuclear membrane nucleocapsids acquire a primary envelope lacking viral glycoproteins. After de-envelopment, cytoplasmic nucleocapsids acquire a thick tegument and a secondary envelope with viral glycoproteins at the level of neo-formed annulate lamellae or at the cis-side of the Golgi complex. Cytochemical labeling using helix pomatia lectin revealed that the newly acquired secondary viral envelopes contain intermediate forms of glycocomponents, suggesting a sequential glycosylation of the virions during their transit through the Golgi area before their final release into the extracellular space. Immunogold labeling also showed that the viral glycoproteins, which are not involved in the budding process, reach and accumulate in the endosomal/lysosomal compartment. Pulse-chase analysis indicated degradation of the gp116, consistent with its endosomal localization and with the absence of viral glycoproteins on the cell surface of the infected cells.

Viral glycoproteins accumulate in newly formed annulate lamellae following infection of lymphoid cells by human herpesvirus 6.

Ultrastructural analysis of HSB-2 T-lymphoid cells and human cord blood mononuclear cells infected with human herpesvirus 6 revealed the presence, in the cell cytoplasm, of annulate lamellae (AL), which were absent in uninfected cells. Time course analysis of the appearance of AL following viral infection showed that no AL were visible within the first 72 h postinfection and that their formation correlated with the expression of the late viral glycoprotein gp116. The requirement of active viral replication for AL neoformation was further confirmed by experiments using inactivated virus or performed in presence of the viral DNA polymerase inhibitor phosphonoacetic acid. Both conventional electron microscopic examination and immunogold fracture labeling with anti-endoplasmic reticulum antibodies indicated a close relationship of AL with the endoplasmic reticulum and nuclear membranes. However, when the freeze-fractured cells were immunogold labeled with an anti-gp116 monoclonal antibody, AL membranes were densely labeled, whereas nuclear membranes and endoplasmic reticulum cisternae appeared virtually unlabeled, showing that viral envelope glycoproteins selectively accumulate in AL. In addition, gold labeling with Helix pomatia lectin and wheat germ agglutinin indicated that AL cisternae, similar to cis-Golgi membranes, contain intermediate, but not terminal, forms of glycoconjugates. Taken together, these results suggest that in this cell-virus system, AL function as a viral glycoprotein storage compartment and as a putative site of O-glycosylation.

Prevalence of human papillomavirus, cytomegalovirus, and Epstein-Barr virus in the cervix of healthy women.

The prevalence of some sexually transmitted viruses, possibly involved in cervical carcinogenesis, was studied in the cervix of women with normal cytology. The presence of human papillomaviruses (HPV) type 16 and 18, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) genomes in cervical cells taken from 143 healthy Italian women was investigated using the polymerase chain reaction (PCR). The study population was divided into four groups with respect to age as follows: group I, 17 to 25 years, n = 48 women; group II, 26 to 35 years, n = 30; group III, 36 to 50 years, n = 32; and group IV, 51 to 70 years, n = 33. In the first age group prevalence rates of HPV 16, CMV and EBV infection of 23%, 21% and 19% were found respectively. The infection rates of HPV 16 and CMV were shown to decrease with age, with prevalences of HPV 16 at 10% in the second group, 6% in the third and 3% in the fourth and of CMV at 13% in the second and third and 6% in the fourth groups. The prevalence of EBV infection did not decrease with increasing age (19% in the first and third groups, 20% in the second and 18% in the fourth). The occurrence of HPV 18 genome was very low (0-3%) and independent of age. In the first age group a higher percentage of double infections (16.6%) was found than in the three other age groups (6% in the second and third and 3% in the fourth). The finding of multiple infections in younger women requires further study in order to clarify the implications of such viral infections in healthy women and their contribution to the development of genital tract malignancies.

[Changes in the bacterial flora of patients under antineoplastic treatment]. / Indagini sulla modificazione della flora batterica in pazienti sottoposti a trattamento antineoplastico.

The bacterial flora of the skin from different anatomical sites on cancer patients and a control group of medical personnel was examined. This was done to ascertain if antineoplastic therapy was able to change the pattern of microbial flora of patients and to provide a control for possible infectious complications. The results show that in the control group bacterial flora was normal and the antineoplastic treatment did not succeed in changing the bacterial pattern in the skin of patients deeply. Gram negative bacteria were isolated more frequently from the skin of leukemia patients than from either patients with malignant melanoma or other neoplastic diseases.

Antibodies to BK virus structural and tumor antigens in human sera from normal persons and from patients with various diseases, including neoplasia.

Sera from 1,279 patients with various diseases were examined for the presence of antibodies to BK virus (BKV) capsid antigens. The percentage of positive sera was comparable in all the diseases except rheumatoid arthritis and chronic nephropathies, where a slightly higher prevalence was found. Sera from 952 patients with tumors were examined for the presence of antibodies to BKV tumor and capsid antigens in comparison with a matched control group of 501 blood donors. Sera from 11 tumor patients (1.15%) and from 4 normal controls (0.80%) had antibodies to BKV tumor antigen. No higher prevalence of antibodies to BKV capsid antigens was found in any cancer type except in carcinomas of the urinary bladder, where the percentage of positive sera and of sera with high titers was higher than in other groups. BKV infection is discussed in relation to its possible connection with human non-neoplastic diseases as well as with human tumors and to its activation under conditions of immunosuppressive therapy.