HPyV6, HPyV7 and TSPyV DNA sequences detection in skin disease patients and healthy subjects.

BACKGROUND: The discovery, from 2007, of eight new human polyomaviruses (HPyVs) has revived interest in the Polyomaviridae family and their association with human diseases and cancer. In particular, HPyV6 and HPyV7 were discovered in skin swabs of healthy donors and TSPyV was discovered in a heart transplant recipient affected by virus-associated Trichodysplasia Spinulosa (TS), a rare skin disease, exclusively found in immunocompromised patients. OBJECTIVE: The presence of HPyV6, HPyV7 and TSPyV DNA in skin biopsies from patients affected by different skin diseases (cancers and inflammatory disorders) has been evaluated to confirm their skin tropism and the possible pathological association. METHODS: DNA extracted was amplified with HPyV6, HPyV7 and TSPyV specific PCR real time on Taqman platform with standard profile. RESULTS: HPyV7 and TSPyV sequences were not found in any skin specimen analysed. HPyV6, on the other hand, was detected in 30% of samples from healthy subjects vs. 14.3% of skin cancer patients and 2.9% of inflammatory disorders. HPyV6 sequences have been detected in primary cutaneous T-cell lymphoma (CTCL) patients (in 18.6% out of Mycosis Fungoides (MF) patients and in 16.7% out of CTCL not MF/SS(Sèzary syndrome) but have not been detected in primary cutaneous B-cell lymphoma (CBCL) patients. CONCLUSION: Our preliminary data suggest that these three novel human polyomaviruses seem not to play a significant role neither in the pathogenesis of cutaneous malignancies nor in that of inflammatory disorders but, according to literature, can inhabit the skin. On the basis of our data regarding the HPyV6 DNA presence with decreasing percentages in healthy subjects, skin cancer and inflammatory disorders patients, it could be an intriguing matter to study if the activated innate immune response in inflammatory disorders can suppress the virus. Further investigations are needed to better understand their relationship with the human host and its innate immune system.

Phenotypical characterization of circulating cell subsets in pyoderma gangrenosum patients: the experience of the Italian immuno-pathology group.

BACKGROUND: No data are available as to the phenotype of circulating lymphocyte subsets in pyoderma gangrenosum (PG). AIM: To analyse the expression of different chemokine receptors associated to T-helper (Th)1 (CCR5), Th2 (CCR4) and Th17 (CCR6), as well as the regulatory T-cell subset (Treg) and dendritic cell polarization in the blood of newly diagnosed untreated PG patients. MATERIALS AND METHODS: Multi-parameter flow cytometry was performed on blood samples from 10 PG patients collected at first diagnosis among centres belonging to the Italian Immuno-pathology Group. Blood samples from 10 age- and sex-matched healthy controls (HC) were used as controls. RESULTS: PG patients are characterized by an over-expression in the blood of the CD4+CCR5+ and CD4+CCR6+ and a down-regulation of CD4+CCR4+ counts with respect to healthy subjects. Moreover, they show increased levels of myeloid derived dendritic cells type1 and reduced levels of the Treg CD4+CD25highFOXP3+ subset. CONCLUSIONS: The pattern of chemokine expression argues in favour of a Th1 (CCR5+) and Th17 (CCR6+) polarization with a down-regulation of Th2 (CCR4+).

Flow cytometric analyses of circulating regulatory T cells in patients with dermatitis herpetiformis and other immune mediated dermatoses.

Regulatory T cells (Tregs) play a crucial role by maintaining the peripheral tolerance and inhibiting autoimmunity. In recent years, numerous autoimmune and immune-mediated diseases have been shown to present significant number depletion and/or function impairment of this subset. In the present study, we present a brief overview of the results obtained by our group in association with the centers belonging to the Italian Immunopathology Group, as to the expression levels and biological significance of circulating regulatory CD4+CD25+brightFOXP3+ T cells in a variety of immune-mediated skin diseases (such as psoriasis, scleroderma, bullous pemphigoid and GvHD), together with preliminary results achieved in patients with inflammatory bowel disease-related dermatoses. This review shows that this series of different cutaneous diseases characterised by an immune-mediated pathogenesis, share a significant down-regulation of circulating FOXP3+ Treg cells, whilst the treatment and the achievement of clinical response are generally associated with an opposite phenomenon with up-regulation of Treg cells. Future studies are mandatory to identify the effective role of these modifications in the disease pathogenesis as well as its relationship with the clinical response.

Th1, Th2, Th17 and regulatory T cell pattern in psoriatic patients: modulation of cytokines and gene targets induced by etanercept treatment and correlation with clinical response.

BACKGROUND: Psoriasis is sustained by pro-inflammatory CD4+ T helper cells mainly belonging to the Th1, Th17 and Th22 lineage. OBJECTIVE: To identify whether treatment with the anti-tumour-necrosis-factor antagonist etanercept is able to induce significant modulations in transcription factor and cytokine mRNA gene expressions related to the different T cell immune response polarization (Th1, Th2, Th17 and regulatory T cells, Treg and to correlate them with clinical response. METHODS: The study population included 19 psoriasis patients treated with etanercept and 19 healthy subjects. Blood samples were collected at baseline and every 4 weeks during treatment. Taqman quantitative real-time polymerase chain reaction was applied to analyse the expression of: Stat-4, T-bet, IL-12p35 and IFN-γ (Th1-related); GATA-3, IL-4 (Th2-related); Stat-3, RORγt, IL-23p19 (Th17-related); Foxp3, IL-2 (Treg-related). Flow cytometry was applied to analyse CD4+CD25+(bright)Foxp3+ cells in peripheral blood. RESULTS: Upregulation of Th1 and Th17 and downregulation of Treg subsets was found at baseline. The response to etanercept could be associated with a significant reversal of the Th1/Th17 activation, and a concomitant upregulation of Th2 and Treg subsets. CONCLUSION: Our data may contribute to a better understanding of the mechanisms underlying the achievement of clinical response in psoriasis and could be helpful for the identification of early predictive markers of response.

Reciprocal modulation of circulating CD4+CD25+bright T cells induced by extracorporeal photochemotherapy in cutaneous T-cell lymphoma and chronic graft-versus-host-disease patients.

The mechanisms of action of extracorporeal photochemotherapy (ECP) in cutaneous T-cell lymphoma (CTCL) are poorly understood. Recently, ECP has been shown to induce an increase in regulatory T cell (Treg) expression and functional activities in Graft-versus-host-disease (GvHD), whereas no data are available in CTCL patients. The aim of this study is to evaluate whether ECP is able to modulate the expression levels of the circulating CD4+CD25+bright subset in CTCL patients and whether these modifications are related to the disease course. The patient population included 43 CTCL and 15 chronic GvHD patients treated by ECP at our institutions since 1992. The expression of the circulating CD4+CD25+bright subset was analysed at baseline and sequentially during treatment by flow-cytometry. Fifty healthy donors were used as controls. The baseline circulating CD4+CD25+bright percentage values in CTCL (median: 4.3 percent) were similar to those of healthy donors, whereas GvHD showed significantly lower values (median: 1.5 percent; p<0.001). During treatment, CTCL patients were characterised by an early decrease (from 4.3 percent to 2.4 percent median after 6 months). The CD4+CD25+bright decrease was associated to the disease course, as it occurred in 91.3 percent of responding but in only 25 percent of PD patients (p=0.0001). On the other hand, a significant increase of CD4+CD25+bright cells was observed in GvHD. ECP induces a reciprocal modulation of the circulating CD4+CD25+bright cells in CTCL and GvHD, with a downregulation in CTCL potentially associated with the response mechanisms.

Circulating CD4+CD25 bright FOXP3+ T cells are up-regulated by biological therapies and correlate with the clinical response in psoriasis patients.

BACKGROUND: Regulatory T-cell (T(reg)) modulation is one of the potential mechanisms of anti-tumour-necrosis-factor biological agents. However, literature data on psoriasis patients are lacking. OBJECTIVE: To analyse the circulating CD4+CD25(bright)FOXP3+ subset in 30 patients with psoriasis vulgaris/arthropathic psoriasis treated with biologicals and to investigate its relationship with the clinical response. METHODS: The CD25(bright)FOXP3+ expression within the CD4+ subset was determined by multi-parameter flow cytometry at baseline and during treatment. FOXP3 mRNA expression was analysed by real-time reverse transcription PCR. RESULTS: A response was obtained in 16/17 patients (91.1%) with increased CD25(bright)FOXP3+ values and in only 3/11 patients (27.3%) who showed a CD25(bright)FOXP3+ decrease during biological treatment (p = 0.0001). Responders showed significantly higher values than did non-responders as from the first 2 months of treatment (p = 0.0032). A significantly higher posttreatment expression of mRNA FOXP3 was observed in responders compared to non-responders. CONCLUSION: Biological drugs induce a circulating T(reg) up-regulation in a significant percentage of patients; such an increase is an early predictive marker of response.

Pluripotent stem cells isolated from human amniotic fluid and differentiation into pancreatic beta-cells.

Human amniotic fluid (HAF) contains multipotent stem cells [amniotic fluid-derived stem cells (AFSC)] which can differentiate into a variety of different cell types. Recently, we demonstrated that obestatin, a peptide encoded by the ghrelin gene, exerts anti-apoptotic effects in pancreatic beta-cells and human islets and increases the expression of genes involved in beta-cells differentiation. We investigated whether: 1) AFSC would differentiate into pancreatic beta-cells and 2) obestatin would increase beta-cells differentiation from AFSC. Fluorescence-activated cell sorting analysis and immunocytochemical staining showed the presence of mesenchymal and endothelial markers in AFSC. Real-time PCR evidenced the expression of Octamer binding transcription factor 4 (OCT-4), a marker of pluripotency, during the early differentiation phase. However, the beta-cells differentiation marker duodenal homeobox factor-1 (PDX-1) could not be detected. Obestatin increased OCT-4 expression but had no effect on beta-cells differentiation. These results suggest that, at least under the experimental conditions used in this study, AFSC do not differentiate into beta-cells and obestatin has no additional effect.

Human herpesvirus 7 detection by quantitative real time polymerase chain reaction in primary cutaneous T-cell lymphomas and healthy subjects: lack of a pathogenic role.

BACKGROUND: Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of lymphomas where the tumour population emerges within a multiple subclone pattern. Mycosis fungoides (MF) and Sézary syndrome (SS) are characterized by the expansion of clonal CD4+/CD45RO+ memory T cells. Lymphomatoid papulosis (LyP) is a chronic, lymphoproliferative disorder included in the CD30+ primary CTCL spectrum. Several studies have suggested a role of viral infection for super-antigenic activation of T lymphocytes; however, evidence of their association with CTCLs is still lacking. Human herpesvirus (HHV) 7 is a CD4+ T-lymphotropic herpesvirus; its restricted cellular tropism and the ability to induce cytokine production in infected cells could make it an important pathogenic cofactor in lymphoproliferative disorders. OBJECTIVES: To investigate the presence of HHV7 DNA on CTCL and healthy skin donors (HD). METHODS: We used quantitative real time polymerase chain reaction to evaluate the potential pathogenic role of HHV7. RESULTS: Twenty-seven of 84 (32.1%) HD were positive for HHV7 DNA. Twenty-one of 148 (14.2%) patients with CTCLs were positive for HHV7 DNA: nine of 39 (23.1%) SS, six of 14 (42.9%) CD30+ CTCLs and six of 24 (25.0%) LyP, and HHV7 DNA was negative in all 71 patients with MF. CONCLUSIONS: These results seem to exclude a pathogenic role of HHV7 in CTCLs, suggesting the possibility of skin as a latency site.

Heterogeneity of circulating CD4+ memory T-cell subsets in erythrodermic patients: CD27 analysis can help to distinguish cutaneous T-cell lymphomas from inflammatory erythroderma.

BACKGROUND: Chronic dermatoses, as well as Sézary syndrome (SS), the erythrodermic and leukaemic cutaneous T-cell lymphoma, display a T-cell memory pattern. Recent findings suggest that different memory T-cell subsets can be recognized based on CD27 and CD45RO/RA expression. No data are reported as to CD27 expression in SS. OBJECTIVES: To evaluate different memory T-cell subsets, i.e. central memory (TCM), effector memory (TEM) and terminally differentiated cells in SS and inflammatory erythroderma (IE). MATERIALS AND METHODS: Forty SS and 137 IE patients were included. CD27 and CD45RO/CD45RA expression was analysed by flow cytometry on peripheral blood lymphocytes and immunohistochemistry. RESULTS: A significantly higher expression of the CD4+CD27+CD45RA- TCM subset was observed in SS whilst IE patients were characterized by increased CD4+CD27-CD45RA- TEM levels. The Vbeta-restricted population was homogeneously CD4+CD26-CD27+ in the SS subjects. CONCLUSIONS: SS and IE are characterized by a different memory T-cell subset expression; CD27 expression could be used as an additional diagnostic tool in the differential diagnosis.

In vitro effects of cholesteryl butyrate solid lipid nanospheres as a butyric acid pro-drug on melanoma cells: evaluation of antiproliferative activity and apoptosis induction.

Literature data show that butyric acid derivatives bear a dose-dependent differentiative anti-proliferative activity on cancer cell lines and that apoptosis induction may play a major role. Although it was recently shown that solid lipid nanospheres (SLNs) are a suitable tool for several in vivo drug administration routes, there is little available information on melanoma cell lines. This study was aimed at evaluating the anti-proliferative and apoptotic in vitro effects of cholesteryl butyrate (chol-but) SLNs on melanoma cells. Increasing concentrations of chol-but SLNs were used to test two melanoma cell lines. Both cell lines were treated with Na-butyrate (Na-but) and chol-but SLNs for viability. Those tested with chol-but SLNs were more effective than Na-butirate (3 to 72 h). The apoptotic effects of chol-but SLNs were evaluated between 3 and 72 h by annexin-V (ANX-V)/propidium iodide (PI) staining and the antiproliferative effect by PI staining. Apoptosis anti-proliferative-regulatory proteins as bcl-2, Fas/APO1 (CD95) and PCNA (PC10) were also investigated. Flow cytometric analyses evidenced a G(0/1)-S transition block and a 'sub-G(0/1)' apoptotic peak from 0.5 to 1.0 mM butyric acid. In ANX-V/PI flow cytometric staining, a dose- and time-dependent increase in the apoptotic cell percentage (ANX-V+) coupled with a down-regulation of PC10 and bcl-2 and a parallel up-regulation of Fas/APO1 (CD95) were found in both lines started after 3 to 24 h of chol-but SLNs treatment. Results show that chol-but SLNs exerts a dose/time-dependent effect in melanoma cell apoptosis induction between 3 and 24 h and a dose but not time-dependent effect after 24 h of treatment.

An immunoenzyme technique for the identification of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors using digoxigenated-GM-CSF.

A method for detecting granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors has been devised using human macrophages and a GM-CSF/IL-3-dependent human megakaryoblastic leukemia cell line (M-07e). Recognition of the factor-binding site was accomplished by linking recombinant human (rh) unglycosylated GM-CSF previously labeled with digoxigenated compounds. Digoxigenates were able to link amino and sulphydryl groups of the soluble factor and an immunoperoxidase technique using monoclonal anti-digoxigenin antibody was employed to demonstrate the interaction. To support morphological data cross-linking analysis was performed with M-07e cells using digoxigenated-rh-GM-CSF. Macrophages and M-07e cells incubated with digoxigenated-rh-GM-CSF showed intense positivity by the immunoperoxidase technique. In cross-linking, M07e cells showed a 96 kDa band corresponding to receptor plus bound factor. This technique permits a high degree of specificity in the detection of GM-CSF receptors with good morphological preservation of cellular detail.

Long-term survival of thymoma patients by histologic pattern and proliferative activity.

We performed DNA flow cytometry and analysis of the argyrophilic nucleolar organizer regions (AgNORs) in formalin-fixed, paraffin-embedded sections from 60 surgically resected thymomas. The results were correlated with histologic pattern, stage, associated clinical features, and survival to assess which parameters could best predict prognosis. On univariate analysis, the 10-year survival rates were 86% for predominantly lymphocytic type but only 42% for predominantly epithelial, mixed lymphoepithelial, or spindle cell thymomas (p = 0.006); survival rates were 85% for noninvasive but only 34% for invasive thymomas (p = 0.0002); 73% for diploid but only 38% for aneuploid cases (p = 0.005); 88% for thymomas with 5.75 AgNORs per cell or fewer but only 34% for thymomas with more than 5.75 AgNORs per cell (p < 0.0001). On multivariate survival analysis, tumor stage (p < 0.001) and AgNOR counts (p = 0.009) retained independent prognostic significance. The 16 patients with predominantly lymphocytic type and 5.75 AgNORs per cell or fewer were all alive at the end of the observation period. In conclusion, the histologic type of the American classification and the proliferative activity evaluated by AgNOR analysis are the best predictors of long-term survival for patients with thymoma. Both predictors can be easily evaluated in the same histologic section, are highly reproducible, and permit identification of a group of patients with a favorable outcome regardless of other clinicopathological features.

DNA ploidy and p53 expression correlate with survival and cell proliferative activity in male breast carcinoma.

DNA flow cytometry and the monoclonal antibody DO7 were applied in formalin-fixed, paraffin-embedded specimens from 34 primary male breast carcinomas to verify whether DNA ploidy and p53 expression were associated with survival and proliferative activity. They were compared with tumor clinicopathologic features, sex steroid hormone receptors and cell proliferative activity, assessed by the counts of the argyrophilic nucleolar organizer regions (AgNORs), the monoclonal antibody PC10 against the proliferating cell nuclear antigen and the monoclonal antibody MIB-1. A significant correlation was found between survival and tumor ploidy (median survival, 77 months for diploid but only 38 months for aneuploid cases; P = .03) and p53 expression (median survival, 95 months for cases with p53 scores < or = 14.06% versus 33 for cases with P53 scores > 14.06%; P = .0004; median survival, 99 months for p53 negative vs 39 for positive cases; P = .007). Tumor histological grade (P = .006), AgNOR counts (P = .0001), PC10 scores (P = .002), and MIB-1 scores (P = .001) were also associated with prognosis. In the multivariate analysis, only p53 scores (P = .001) or p53 immunopositivity (P = .003) and AgNOR counts (P = .022) retained an independent prognostic significance. Aneuploid tumors had higher AgNOR counts (P = .002), PC10 (P = .007), MIB-1 (P = .006), and p53 scores (P = .01) than diploid cases. A linear relationship was observed between p53 scores and AgNOR counts (r = .41; P = .014), PC10 (r = .46; P = .005), and MIB-1 scores (r = .44; P = .011). These results indicate that DNA ploidy and p53 expression are associated with survival and cell proliferative activity in male breast carcinoma. Quantitative parameters, such as DNA ploidy, p53 scores, AgNOR counts, PC10, and MIB-1 scores substantially improve the prognostic significance of the traditional parameters in male breast carcinoma.

MIB-1, Ki67, and PCNA scores and DNA flow cytometry in intermediate grade malignant lymphomas.

AIMS: To verify the correlation between MIB-1, Ki67, and proliferating cell nuclear antigen (PCNA-PC10) scores and S-phase fraction in intermediate grade non-Hodgkin's lymphomas (Working Formulation F); and their reliability in differently processed tissues. METHODS: Forty one non-Hodgkin's lymphomas were classified as (F) intermediate grade malignant lymphomas according to the Working Formulation; mitotic counts and percentage of large cells were assessed for each case. Sections from formalin fixed, paraffin wax embedded tissues were stained with anti MIB-1 monoclonal antibody, after microwave oven processing, and anti-PCNA (PC10) monoclonal antibody using an avidin-biotin immunoperoxidase (ABC) method. One thousand cells from 10 representative fields were scored. Frozen sections from surgical specimens were stained with Ki67 monoclonal antibody using the ABC method; the fraction of Ki67 positive cells was calculated scoring 1000 cells. Flow cytometry analysis (FCM) was performed on cell suspensions from fresh tissues. Correlations between data were estimated using linear regression. RESULTS: A linear correlation was found between MIB-1 and Ki67 scores (r = 0.92; p < 0.00001); between MIB-1 and PCNA scores (r = 0.79; p < 0.00001); and between MIB-1 score and S-phase fraction (r = 0.51; p = 0.0006). A linear correlation was also found between Ki67 and PCNA scores (r = 0.85; p < 0.00001); between Ki67 score and S-phase fraction (r = 0.6; p = 0.0002); and between PCNA score and S-phase fraction (r = 0.74; p < 0.00001). A correlation was found between mitotic counts and MIB-1 (r = 0.56; p = 0.0001), PCNA (r = 0.51; p = 0.0007), or Ki67 scores (r = 0.47; p = 0.002); between the percentage of large cells and MIB-1 (r = 0.49; p = 0.0009), PCNA (r = 0.6; p = 0.00003), and Ki67 scores (r = 0.53; p = 0.0003) and S-phase fraction (r = 0.55; p = 0.0002). CONCLUSION: MIB-1, Ki67, and PCNA (PC10) scores and S-phase fraction are highly correlated and equally well represent the proliferative activity of intermediate grade non-Hodgkin's lymphomas in differently processed material. MIB-1 and PCNA stains can be applied even on small biopsy specimens. MIB-1 produces homogenous staining without background; it also strongly stains mitotic figures. It can be performed on routinely processed tissues, permitting the simultaneous evaluation of the morphology and tumour cell kinetics. The wide standard deviations of the proliferative indices found for intermediate grade NHL suggest that this category probably includes various degrees of malignancy.

Biological heterogeneity of diffuse mixed small and large cell non-Hodgkin's lymphomas assessed by DNA flow cytometry and Ki67.

The cell proliferative activity of the clinico-pathologically heterogeneous non-Hodgkin's lymphomas (NHL) included in the intermediate grade F category of the Working Formulation (WF) was investigated. S-phase fraction with flow cytometry on cell suspensions, and Ki67 on frozen tissue sections were performed in 42 F NHL. An avidin-biotin immunocomplex method was used and 1000 cells from 10 representative fields were counted. DNA content, S-phase and Ki67 were also detected in 194 NHL covering the whole spectrum of the WF. DNA content anomalies were found in 52 of 194 NHL. Their incidence, like that of S-phase fraction and Ki67 positive cells, progressively increased from low- to high-grade. A linear correlation was found between Ki67 and S-phase (r = .59). Using the median value of proliferating cells obtained with both procedures as a cut off, two very different groups of lymphomas could be distinguished within a series of 42 F-intermediate NHL: with low and high proliferative cell activity (p < .0001) that were termed F(low) and F(high), respectively. A intermediate group was placed between them. It differed significantly from the others if Ki67 was used but only from the F(high) group if the S-phase fraction analysis was applied. No significant differences were seen when comparing F(low) with the single categories of low-grade NHL and F(high) with H high-grade NHL; no significant differences were found between F(high) and G, and between G and H categories. The existence of distinct groups of NHL in the F category, as defined by biological parameters assessing the cell proliferative activity, indicates that this category includes biologically heterogeneous lymphoma subtypes with different grades of aggressiveness. The results also indicate that the G intermediate category displays proliferation indices similar to those of H high grade category.

"Physiological" and "pharmacological" dialysate sodium concentrations.

Forty three uremic patients on regular hemodialysis treatment (4 hours 3 times/week) were dialyzed for a period of 28.95 +/- 14.46 months with a dialysate sodium concentration (NaD) of 142 mEq/l, similar to their plasma water sodium concentration corrected for the Donnan factor ("physiological" NaD). Blood pressure (BP) and body weight (BW) dropped significantly. During two following periods, lasting 18 and 14 months, with NaD 148 mEq/l, similar to the patients' plasma water sodium concentration ("pharmacologically high" NaD), cardiovascular stability improved and BP did not show significant increase. Using "physiological" and "pharmacologically high" NaD the removal of water and sodium by convection improves the cardiovascular stability and the patients' well being, without bringing about the feared long-term cardiovascular side effects, if an appropriate dry body weight is achieved, because of better correction of the cellular overhydration.

Radiofrequency catheter ablation of atrioventricular nodal reentry tachycardia: selective approach to the slow pathway via the superior vena cava.

Selective radiofrequency catheter ablation of the slow atrioventricular nodal pathway is currently considered the first-line therapy for patients suffering from recurrent symptomatic atrioventricular nodal reentry tachycardia. In most cases slow pathway conduction may be selectively eliminated or modified by the application of radiofrequency current at the posterior portion of Koch's triangle. The ablation site is usually targeted by careful mapping of this area performed using an ablation catheter advanced via the inferior vena cava approach. In this report we describe 2 cases in which the conventional approach to the target site was either impossible owing to the presence of an atresic inferior vena cava (case 1), or contraindicated in view of a history of common femoral vein thrombosis, subsequently extended up to the inferior vena cava (case 2). In both patients a superior vena cava approach was utilized and the slow pathway was successfully ablated. In case of arrhythmias necessitating slow pathway mapping and ablation, such an approach may be considered as a feasible and safe alternative whenever, owing to the presence of anomalies and/or diseases of the inferior vena cava, the conventional approach cannot be employed.

[Fat necrosis in the newborn infant associated with hypercalcemia and dyslipidemia (description of a case)]. / Adiponecrosi del neonato associata a ipercalcemia e dislipidemia (descrizione di un caso).

A case of subcutaneous fat necrosis (S.F.N.) with hypercalcemia and hyperlipemia in a newborn infant is reported. On the basis of previous reports it is impossible to definite the pathogenesis of hypercalcemia and hyperlipemia in subcutaneous fat necrosis. Moreover the Authors point out that in a newborn with S.F.N. plasma levels of calcium and lipids and, if it is possible, urinary prostaglandin E and serum PTH and 250HD3 should be determined.

Circulating CD4+ CD25brightFOXP3+ regulatory T-cells are significantly reduced in bullous pemphigoid patients.

Bullous pemphigoid (BP) is the most frequent autoimmune bullous skin disease, characterised by auto-antibodies against the hemidesmosome complex. Recently, regulatory T cells (Tregs) have been implicated in the development of several autoimmune diseases; few data are available in BP, failing to demonstrate a role of this subset in disease pathogenesis. The aim of this study was to investigate the expression and phenotypes of different Tregs (CD4+ CD25brightFOXP3+ and CD8+ CD28- cells) in BP to clarify whether the depletion of this subset constitutes one mechanism of tolerance loss. The CD4+ CD25brightFOXP3 and CD8+ CD28- circulating subsets were determined by flow-cytometry in 26 untreated BP patients and compared with a group of age- and sex-matched healthy controls (HC, n = 30). Absolute and percentage values of the CD4+ CD25brightFOXP3+ cells were significantly reduced in BP compared with HC (median CD25brightFOXP3+ expression within CD4+ cells: 1.8 vs. 3.5%, p = 0.002); conversely, BP patients were characterised by a significant expansion of the CD25brightFOXP3- "activated" T-cell subset. CCR4 and CD62L were expressed on the majority of CD4+ CD25brightFOXP3+ cells (75.2 and 82.3%, respectively). No differences in the CD8+ CD28- subset were found between BP and HC. This is the first report showing a significant reduction of circulating CD4+ CD25brightFOXP3+ Treg frequency in BP patients.