A preliminary examination of the role of NFAT 3 in human skin, cultured keratocytes and dermal fibroblasts.

BACKGROUND: Ciclosporin A (CsA) is widely utilized for the treatment of inflammatory skin diseases such as psoriasis.The therapeutic effects of CsA are thought to be mediated via its immunosuppressive action on infiltrating lymphocytes in skin lesions. CsA and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor Nuclear Factor of Activated T cells (NFAT). METHODS: RT-PCR and Western Analysis were used to investigate the presence of NFAT-3 mRNA and protein in human keratocytes. Tissue culture of human keratocytes and immunostaining of cells on coverslips and confocal microscopy were used to assess the degree of nuclear localisation of NFAT-3 in cultured cells. Keratome biopsies were taken from patients with psoriasis (lesional and non-lesional skin) and normal skin and immunohistochemistry was used to assess the NFAT-3 localisation in these biopsies using a well characterized anti-NFAT-3 antibody. RESULTS: The NFAT-3 mRNA and protein expression was demonstrated using RT-PCR and Western blotting. The expression of NFAT-3 in human keratocytes and response to different agonists provides perhaps a unique opportunity to examine the regulation, subcellular localization and kinetics of translocation of different NFATs in primary cultured human cells. As with NFAT 1, NFAT 2 and recently NFAT 5, differentiation-promoting agents that increase intracellular calcium concentration induced nuclear translocation of NFAT-3 in cultured keratocytes but with different kinetics. CONCLUSION: These data provide the first evidence of that NFAT-3 is expressed in normal skin, psoriasis and that NFAT-3 functionally active in human keratocytes and that nuclear translocation of NFAT-3 in human skin cells has different kinetics than NFAT 1 suggesting that NFAT-3 may play an important role in regulation of keratocytes proliferation and differentiation at a different stage. Inhibition of this pathway in human epidermal keratocytes many account, in part for the therapeutic effects of CsA and tacrolimus in skin disorders such as psoriasis.

Epithelial sheath neuroma: be aware of benign perineural invasion!

Perineural involvement by epithelial cells is usually considered as a sign of malignancy and is seen in a variety of malignant skin neoplasms. However, there are other benign conditions characterized by the presence of perineural involvement by epithelial cells. We present a case of epithelial sheath neuroma in a 43-year-old male. The clinicopathological features of this newly described entity are discussed together with the differential diagnosis and the different hypotheses of pathogenesis. Both pathologists and dermatologists should be aware of this entity to avoid misdiagnosis of malignancy.

Gallbladder sarcoma: a clinicopathological study of seven cases from the UK and Austria with emphasis on morphological subtypes.

BACKGROUND: Primary sarcoma of the gallbladder (PGBS) is rare, with only 40 cases reported in the literature. Most of these have been diagnosed as leiomyosarcoma. We aimed to evaluate the histological features of a case series of this rare tumor and correlate these with clinical features. DESIGN: Cases recorded as "gallbladder sarcoma" from different institutes were reviewed and the clinicopathological features of these cases were recorded. Only primary gallbladder wall mesenchymal tumors were included. Epithelial tumors, mixed tumors (carcinosarcoma or sarcomatoid carcinoma), and tumors extending into the gallbladder from the abdomen or sarcoma with other known primaries were specifically excluded. RESULT: PGBS occurred in one male and six females with a median age of 70 (range 64-82) years. Patients presented with acute or chronic cholecystitis, abdominal pain, weight loss, and pruritus. They were generally found to have elevated alkaline phosphatase and bilirubin, and leukocytosis. Tumors ranged from 1.1 to 4 cm with a median size of 3 cm. Most PGBS arose in the body but one arose in the fundus. All tumors were associated with ulcerated mucosa. Based on morphological and immunohistochemical features of the PGBS, there were three myxofibrosarcomas (malignant fibrous histiocytoma, MFH, storiform pleomorphic), one leiomyosarcoma (LMS), one angiosarcoma (AS), and two liposarcomas (LS). All patients received cholecystectomy and three received adjuvant chemotherapy. Follow-up revealed that six patients died of the disease 6 weeks to 2 years after diagnosis and one died of unrelated causes. CONCLUSION: PGBS are rare and mainly occur in the gallbladder body in middle-aged females. They generally present with acute cholecystitis and have a very poor prognosis. A variety of sarcoma types are found with MFH being the predominant variant.

Superficial acral fibromyxoma: a clinicopathological analysis of 32 tumors including 4 in the heel.

AIMS: To examine a series of superficial acral fibromyxomas (SAFs) and discuss our experience with this new entity and its differential diagnosis in the past 5 years. METHODS AND RESULTS: Thirty-two new cases of SAF were studied between 2001 and 2006. The patients included 22 males and 10 females with an age range between 23 and 82 years (mean 51, median 53) presenting with a solitary mass or nodule with an average size of 2 cm. The sites were the toes (n = 15) and fingers (n = 13) with 66.6% of tumors close to or involving the nail bed. Four tumors occurred in the heel where SAF has not been previously observed. Local recurrences developed in 3 of 14 patients (22%). Histologically, all tumors presented with spindle cells with a vague storiform and fascicular pattern embedded in a myxoid/fibromyxoid/collagenous stroma. A characteristic immunophenotype included CD34+, CD99+/- and EMA+ focally. One case showed moderate cytological atypia with 1 mitosis per 10 HPF, but a 4-year follow up showed no evidence of recurrence. CONCLUSION: Thirty-two new cases of SAF confirm this tumor as a reproducible entity. Occurrence in the heel, a new site for this tumor, was reported in four cases. Recurrence rate of this tumor may exceed 20%.

Granular perineurioma: the first report of a rare distinctive subtype of perineurioma.

Perineurioma represents a relatively recently described neoplasm in the spectrum of benign peripheral nerve sheath tumors composed of perineurial cells staining immunohistochemically positive for epithelial membrane antigen. Although intraneural, extraneural and sclerosing perineurioma, rare variants of perineurioma, do occur, and knowledge of them is important in the differential diagnosis of mesenchymal tumors of different lines of differentiation and more importantly if their clinical course differs from that of other perineuriomas. We report herein the first case in the world literature of granular perineurioma arising in the dermal and subcutaneous tissues of the trunk of a 28-year-old female. The diagnosis was confirmed morphologically and immunohistochemically. More interestingly, 3 years later the patient complained of right lower extremity pain, for which magnetic resonance imaging studies showed an intraneural perineurioma confined to the sciatic nerve. The latter finding was confirmed both histopathologically and immunohistochemically to have exactly the same appearances of the original dermal and subcutaneous mass. Neoplastic cells stained positively for epithelial membrane antigen and for the newly described antibodies claudin-1 and glut-1. Interestingly, the granular component of this large tumor (4.5 cm in maximum diameter) was negative for S100, but positive for NKI-C3. The morphology, immunohistochemistry, and the clinical behavior for this tumor and the differential diagnoses are discussed.

Localization of calcineurin/NFAT in human skin and psoriasis and inhibition of calcineurin/NFAT activation in human keratinocytes by cyclosporin A.

Systemic cyclosporin A and tacrolimus are effective treatments for psoriasis. Cyclosporin A and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor nuclear factor of activated T cells (NFAT). Inhibition of T cell activation is thought to account for their therapeutic action in psoriasis. We investigated whether nonimmune cells in human skin express calcineurin and NFAT1 and whether cyclosporin A and tacrolimus block activation of calcineurin/NFAT in epidermal keratinocytes. The expression patterns of the principal components of calcineurin/NFAT signaling pathway in normal human skin and psoriasis were determined by immunohistochemistry. We assessed calcineurin/NFAT activation in cultured keratinocytes by measuring the degree of nuclear localization of calcineurin and NFAT1 using immunofluorescence/confocal microscopy and assessed if cyclosporin A and tacrolimus blocked nuclear translocation of these proteins. A variety of cell types in normal and psoriatic skin expressed calcineurin and NFAT1, but expression was particularly prominent in keratinocytes. The principal cyclosporin A and tacrolimus binding proteins cyclophilin A and FKBP12 were also expressed by keratinocytes and nonimmune cells in skin. NFAT1 was predominantly nuclear in normal basal epidermal keratinocytes. Increased nuclear localization of NFAT1 was observed in suprabasal keratinocytes within lesional and to a lesser extent nonlesional psoriatic epidermis compared to normal skin (p = 0.001 and p = 0.03, respectively), suggesting increased activation of calcineurin in psoriatic epidermal keratinocytes. Agonists that induce keratinocyte differentiation, specifically 12-0-tetradecanoyl-phorbol-13-acetate (TPA) plus ionomycin, TPA, and raised extracellular calcium, induced nuclear translocation of NFAT1 and calcineurin in keratinocytes that was inhibited by pretreatment with cyclosporin A or tacrolimus. In contrast in human dermal fibroblasts, TPA plus ionomycin or TPA did not significantly alter the proportion of nuclear-associated NFAT1. These data provide the first evidence that calcineurin is functionally active in human keratinocytes inducing nuclear translocation of NFAT1 and also indicate that regulation of NFAT1 nuclear translocation in skin is cell type specific. Inhibition of this pathway in epidermal keratinocytes may account, in part, for the therapeutic effect of cyclosporin A and tacrolimus in skin diseases such as psoriasis.

Infection and cervical neoplasia: facts and fiction.

Whilst there is strong evidence that human papillomavirus (HPV) is the principal aetiological agent in cervical neoplasia, some other sexually transmitted agents may either contribute or protect against cervical carcinogenesis, such as the herpes virus family (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV) or Chlamydia trachomatis (CT). Epidemiological studies suggest that HSV may have a role in cervical neoplasia, but there is no clear supportive experimental evidence. Serological studies have also failed to reveal a difference in the prevalence of antibodies to CMV and EBV between patients with cervical cancer and controls. However, longitudinal seroepidemiological studies have provided evidence that CT is an independent risk factor for the development of cervical squamous carcinoma and this association is serotype specific. The increased risk of cervical neoplasia in patients infected with HIV has been recognised for over a decade and HIV may interact with HPV either by alternating HPV gene transcription or by immunosuppression. Finally extensive experimental and limited epidemiological evidence suggests that adeno-associated viruses (AAV) may have antioncogenic activity in man and may protect against the development of cervical cancer. At present the mechanism of this action is unclear but may relate to AAV-induced regulation of HPV gene expression and the HPV life cycle. In this review we summarize the current literature relating to the associations and mechanisms of cervical carcinogenesis by each of these infectious microorganisms.

Disseminated coccidioidomycosis: a case report from the United kingdom.

We reported the first case of disseminated coccidioidomycosis from the UK successfully treated with amphotericin B, where skin biopsy was the initial clue for the correct diagnosis.

Modulation of NFAT-5, an outlying member of the NFAT family, in human keratinocytes and skin.

BACKGROUND: Cyclosporin A (CsA) and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor Nuclear Factor of Activated T cells (NFAT). NFAT compose a family of transcription factors that are turned on during T cell activation. AIMS: To study the expression of NFAT-5 mRNA and protein in normal human keratinocytes and to investigate the cellular and subcellular pattern of expression of NFAT-5 in normal human skin and psoriasis, and analyze effects of different agonists and ultraviolet radiation on NFAT-5 in normal human skin. METHODS: Tissue cultures, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), Western analysis, immunostaining, confocal microscopy. RESULTS: Sequencing of RT-PCR products confirmed the identity of the product that showed 100 % homology with the predicted NFAT-5 sequence. anti-NFAT-5 mainly detected a single band in cultured keratinocytes and dermal fibroblasts using Western analysis. Immunohistochemistry showed that epidermal keratinocytes and dermal fibroblasts in normal human and psoriatic skin express NFAT-5. NFAT-5 showed predominantly nuclear localization in epidermal keratinocytes and dermal fibroblasts within five normal adult skin biopsies. Our data also suggest that UV irradiation reduces NFAT-5 nuclear localization within the epidermis. Unlike NFAT 1-4, NFAT-5/TonEBP was localized to both nucleus and cytoplasm of cultured keratinocytes. Cyclosporin A induces nuclear membrane translocation of NFAT-5 in cultured keratinocytes and raffinose (a hypertonicity inducing agent) induces more nuclear localization of NFAT-5 compared to untreated cells. In addition, differentiation-promoting agonists that induce sustained rise in intracellular calcium did not result in changes in NFAT-5 localization in cultured keratinocytes. CONCLUSION: These studies provide the first observation of expression of NFAT-5/TonEBP mRNA protein in cultured keratinocytes and dermal fibroblasts and possible functional regulation in cultured keratinocytes. CsA and raffinose effects on NFAT-5/TonEBP in cultured keratinocytes suggest diverse intracellular signaling pathways for NFAT-5/TonEBP in these cells, and that NFAT-5/TonEBP might function to translate different extracellular stimuli into appropriate functional responses.

Cost-effectiveness of active versus conservative colposcopic management of mild dyskaryosis.

Management of mild dyskaryosis remains controversial. In this study, we compared the cost-effectiveness of active versus conservative colposcopic management of women presenting with mild dyskaryosis in two different hospital settings. All women presenting in 2001 with a mild dyskaryotic smear and requiring colposcopy were studied in two different clinical settings (70 women at Darent Valley Hospital (DVH) and 327 at St George's Hospital (SGH)). At DVH, treatment is offered should there be any evidence of cervical intraepithelial neoplasia (CIN). On the other hand, a more conservative approach of cytological and colposcopical follow-up is offered to patients with evidence of low-grade disease at SGH. The outcome of both groups of patients was determined in terms of the number of colposcopy visits per patient, the risk of missing disease as a consequence of patients lost to follow-up and hospital costs as well as costs to patient over a four-year period. The majority (70%) of DVH patients had 1-2 colposcopy visits whereas the majority (60%) of SGH patients had 3-7 visits. At SGH 44% of untreated patients were lost to follow-up and an unknown number of those might have had high-grade disease. Active management is more cost-effective compared with conservative management ( pound323 and pound589 as cost per patient effectively treated in the two hospitals respectively). In conclusion, active management of low-grade disease is associated with lower hospital and patient costs compared with the conservative strategy.

Primary gallbladder sarcoma: a clinicopathologic study of 15 cases, heterogeneous sarcomas with poor outcome, except pediatric botryoid rhabdomyosarcoma.

BACKGROUND: Primary gallbladder sarcoma (PGBS) is rare, with only 39 documented cases, with the predominant type being leiomyosarcoma. DESIGN: Cases recorded as "gallbladder sarcoma" were retrieved from our files; the clinicopathologic features were reviewed and recorded. Only primary gallbladder wall mesenchymal tumors were included. Epithelial tumors, mixed tumors (carcinosarcoma or sarcomatoid carcinoma), tumors extending into the gallbladder from the abdomen, or sarcoma with other known primaries were excluded. RESULT: PGBS occurred in 4 males and 11 females with the adult median age of 68.5 (range: 24 to 88 y, n=12) and 3 children ages 1.5 to 3 years, the latter all with botryoid embryonal rhabdomyosarcoma. Patients presented with acute and/or chronic cholecystitis, abdominal pain, weight loss, pruritus, elevated alkaline phosphatase and bilirubin, and leukocytosis. The median tumor size was 4.5 cm, mean tumor size 5.7 cm, and range 2.0 to 14.0 cm. Most PGBS involved the entire wall and ulcerated the mucosa. PGBSs were diagnosed as 7 myxofibrosarcomas [malignant fibrous histiocytoma, storiform pleomorphic to myxoid, 2 with an unusual fibromyxoid sarcoma-like (Evans-like), and pleomorphic hyalinizing angiectatic tumor-like mixture], 2 leiomyosarcomas, 1 gastrointestinal stromal tumor-like (GIST-like), 3 botryoid embryonal rhabdomyosarcomas (RMS), and 2 epithelioid angiosarcomas. Diagnosis was based on morphology and immunohistochemistry. A diagnosis of LMS required myoid-intersecting fascicles and diffuse, strong immunoreactivity for smooth muscle actin +/- desmin. RMS revealed myxoid grape-like hypocellular tumor with stellate cells, mild atypia, mitoses and desmin, and myoregulatory protein (MyoD1) and skeletal muscle-specific myogenin (Myf4) reactivity. The GIST-like sarcoma was palisaded and myoid-like but failed to stain for CD34 or CD117. Angiosarcomas demonstrated an extravascular proliferation of atypical epithelioid endothelial cells, and mitotic activity. All cases were negative for S100 protein, HMB45, keratins, and CK18. All patients received cholecystectomy and 6 known adjuvant therapy. Follow-up of 12 revealed that 7 patients died of disease within 3 weeks to 1 year and 4 months after diagnosis, 3 died of unknown causes, and 2, both adjuvant therapy treated botryoid RMS in young children, were alive without disease 11 and 27 years later. CONCLUSIONS: PGBSs are rare. Carcinosarcoma, spindle cell carcinoma (by use of keratins and CK18), and melanoma must first be excluded. A variety of sarcoma types are found, yet malignant fibrous histiocytoma is the predominant variant, more common than LMS. GIST is a controversial sarcoma in gallbladder; angiosarcoma can rarely occur in this location. PGBS mainly occur in older female adults and have overall poor prognosis. A subgroup of adjuvant therapy-treated botryoid embryonal RMS in the gallbladder of young children, although rare, can have excellent prognosis.

Expression, localisation and functional activation of NFAT-2 in normal human skin, psoriasis, and cultured keratocytes.

Ciclosporin A (CsA) is widely utilized for the treatment of inflammatory skin diseases such as psoriasis. The therapeutic effects of CsA are thought to be mediated via its immunosuppressive action on infiltrating lymphocytes in skin lesions. CsA and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor Nuclear Factor of Activated T cells (NFAT). As calcineurin and NFAT 1 have been shown to be functionally active in cultured human keratocytes, expression of other NFAT family members such as NFAT-2 and possible functional activation was investigated in human keratocytes. RT-PCR and Western Analysis were used to investigate the presence of NFAT-2 mRNA and protein in human keratocytes. Tissue culture of human keratocytes and immunostaining of cells on coverslips and confocal microscopy were used to assess the degree of nuclear localisation of NFAT-2 in cultured cells. Keratome biopsies were taken from patients with psoriasis (lesional and non-lesional skin) and normal skin and immunohistochemistry was used to assess the NFAT-2 localisation in these biopsies using a well characterized anti-NFAT-2 antibody. The NFAT-2 mRNA and protein expression was demonstrated using RT-PCR and Western blotting. Moreover, the expression of NFAT-2 in normal skin, non-lesional and lesional psoriasis showed a striking basal staining suggesting a role for NFAT-2 in keratocytes proliferation. A range of cell types in the skin express NFAT-2. The expression of NFAT-2 in human keratocytes and response to different agonists provides perhaps a unique opportunity to examine the regulation, subcellular localization and kinetics of translocation of different NFATs in primary cultured human cells. In these experiments the author assessed the expression, localization of NFAT-2 in cultured human keratocytes and measured the degree of nuclear localisaion of NFAT-2 using immunofluorescence and confocal microscopy and whether CsA and tacrolimus inhibit NFAT-2 nuclear translocation. As with NFAT 1, differentiation-promoting agents that increase intracellular calcium concentration induced nuclear translocation of NFAT-2 in cultured keratocytes but with different kinetics. These data provide the first evidence of that NFAT-2 is expressed in normal skin, psoriasis and that NFAT-2 functionally active in human keratocytes and that nuclear translocation of NFAT-2 in human skin cells has different kinetics than NFAT 1 suggesting that NFAT-2 may play an important role in regulation of keratocytes proliferation and differentiation at a different stage. Inhibition of this pathway in human epidermal keratocytes many account, in part for the therapeutic effects of CsA and tacrolimus in skin disorders such as psoriasis. Thus, supporting our previous work data that calcineurin/NFAT is functionally active not only in T cells, but in skin cells.