Immunologic biomarkers for clinical and therapeutic management of psoriasis.

BACKGROUND: The therapeutic management of psoriasis includes conventional treatments as well as the new generation of highly effective TNF-α inhibitors. However, psoriasis has proven to be a complex therapeutic challenge and treatment failures are not uncommon. Thus, laboratory biomarkers of disease progression/therapeutic efficacy may greatly help in the clinical management of psoriasis. AIMS: To identify laboratory biomarkers for clinical management and therapeutic monitoring of psoriasis. METHODS: An observational study performed on 59 patients, presenting moderate to severe psoriasis, undergoing treatment with anti-TNF-α agents (etanercept, adalimumab, and infliximab). Soluble and cellular immune/inflammatory parameters were assessed at baseline and after 12 and 24 weeks of treatment. RESULTS: Clinical efficacy was achieved in 88% of the subjects at 12 weeks, reaching 90% after 24 weeks. IL-6 and IL-22, which were elevated at baseline, were significantly reduced, in association with a significant decrease of CLA+ T cells and an increase of Treg lymphocytes. T, B, and NK cell subsets and T cell response to recall antigens did not show any evidence of immune suppression. CONCLUSIONS: Immune/inflammatory parameters including IL-6 and IL-22, CLA+ T cells, and Treg lymphocytes may prove to be valuable laboratory tools for the clinical and therapeutic monitoring of psoriasis.

Long-term follow-up of peripheral lymphocyte subsets in a cohort of multiple sclerosis patients treated with natalizumab.

Natalizumab, an anti-alpha4 integrin monoclonal antibody inhibiting the adhesion of lymphocytes to the endothelium, is a widely accepted drug treatment for relapsing-remitting multiple sclerosis (RRMS). A peripheral increase of T and B lymphocytes has already been observed as an early treatment effect. This retrospective observational study was aimed to evaluate the peripheral lymphocyte subsets during a long-term treatment follow-up. We included 23 RRMS patients treated with natalizumab for at least 24-48 months who had pretreatment lymphocyte evaluation. Baseline values of lymphocyte subsets and CD4/CD8 ratio did not differ significantly from the 23 matched healthy subjects. The periodic (every 3-6 months) assessment of immune cell subsets was performed by flow cytometry on peripheral blood collected before drug injection. Therapy with natalizumab was confirmed to be effective during the observational period. For all patients, the increase in lymphocytes during natalizumab therapy compared to baseline at every assessment was significantly higher compared to that of overall white blood cells (2·1- and 1·3-fold, respectively, P < 0·0001). Both T cell subsets were proportionally modified and the CD4/CD8 ratio did not change significantly, while B cells increased significantly compared to T and NK cells (3·2-, 1·88- and 1·92-fold, respectively, P < 0·0001). These changes remained constant throughout the 25-48-month period of therapy. In conclusion, effective natalizumab treatment of RRMS patients was associated with the persistence of its biological effects through a stable increase of peripheral lymphocytes, mainly B cells, and an unchanged proportion of T cell subsets in long-term follow-up.

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.

Effective treatment of Kaposi's sarcoma by electrochemotherapy and intravenous bleomycin administration.

The present prospective study was aimed at evaluating the long-term efficacy of local electrochemotherapy (ECT) with the intravenous administration of bleomycin, on disease progression and viral activity in classic Kaposi's sarcoma (cKS), a vascular tumor related to human herpes virus-8 infection. Eighteen patients affected by isolate or multiple cutaneous lesions, refractory to conventional treatments, although in the absence of visceral involvement, were enrolled in a study. Follow-up visits were performed after 4 weeks and every 6 months for up to 48 months. A more extensive exploration of the immunologic status as well as of virological parameters was performed in nine patients. The results showed a significant clinical improvement in all patients after 4 weeks. A complete regression was observed in 12 patients after the first ECT, while four patients required a second treatment on the residual lesions after 4 weeks from the first intervention. The positive outcome persisted during the subsequent clinical control visits. Two patients, that showed rapidly evolving did not improve and relapsed despite a second round of ECT treatment. Effective treatment was associated with the reduction of viral load to undetectable levels. These data support the conduct of larger studies directed at validating the efficacy of ECT as a first-line therapy for cKS.

High prevalence of latent tuberculosis infection in autoimmune disorders such as psoriasis and in chronic respiratory diseases, including lung cancer.

The early diagnosis and treatment of individuals harboring M. tuberculosis is key to ensuring the effectiveness of health programs aimed at the elimination of tuberculosis (TB). Monitoring for TB also has other important health care implications for the related immune pathology caused by the chronic inflammatory response to M. tuberculosis. Moreover, the recent introduction of biologic therapies for the treatment of several immune-mediated inflammatory diseases has shown unexpected high frequencies of reactivation of latent TB. The present cross-sectional study is aimed at estimating the prevalence of latent tuberculosis infection (LTBI) in different groups of subjects, either undergoing a routine program of screening for TB or a clinical monitoring of autoimmune or lung disorders, by analyzing their immune response in vitro to a pool of different M. tuberculosis antigens through an IFN-gamma-release assay (IGRA). We consecutively tested 1,644 subjects including health care workers (931), healthy immigrants from different countries (93), patients with a diagnosis of psoriasis (405), patients with lung inflammatory disease (60) or lung neoplasia (32) and a group of HIV-1 infected Italian subjects (120). The prevalence of IGRAs positive responses among health care workers was 8.9 percent. In comparison, significantly higher frequencies were found in healthy immigrant subjects (33.3%), similar to those found in inflammatory broncho-pneumopathies (34.5%) or lung cancer (29.6%). Interestingly, an unexpected high prevalence was also found in patients affected by psoriasis (18.0%), while HIV-infected subjects had values comparable to those of health care workers (10.8%). An age cut-off was determined and applied for each group by receiver operating characteristic (ROC) curves in order to perform the statistical analysis among age-comparable groups. Multivariate analysis showed that the age and clinical conditions such as having a diagnosis of psoriasis or a lung inflammatory disease were independent risk factors for developing an IGRA positive response. This study highlights an unprecedented high prevalence of IGRA positive responses among patients affected by psoriasis and emphasizes the need for a preliminary assessment of LTBI before the administration of any biologic therapy based on cytokine antagonists such as anti-TNF-alpha. Moreover, screening for LTBI should be routinely performed in the presence of a chronic pulmonary disease.

Release of palladium from biomechanical prostheses in body fluids can induce or support PD-specific IFNgamma T cell responses and the clinical setting of a palladium hypersensitivity.

The increased use of Palladium (Pd) for biomedical applications, which has more than doubled in the last ten years, appears to be associated with an increased frequency of adverse reactions to Pd. The aim of this study is to investigate the relationship between the implant of a biomechanical apparatus containing Pd and the setting of a hypersensitivity to Pd by determining the levels of the metal released in biological fluids, assessing the effects of Pd on peripheral blood mononuclear cell (PBMC) cytokine production and exploring the clinical setting of skin sensitization. Of a total of 3,093 subjects examined in 2006, sensitization to Pd alone or in association with nickel (Ni) was observed in 1.6% and 13.03% of the individuals, respectively. Of these, a group of six subjects positive to Pd and negative to Ni at patch testing were selected on the basis of the oral clinical symptoms in order to measure both the levels of Pd in biological fluids and the degradation of the dental prostheses. Specific Pd measurements were carried out on salivary fluid, urine and serum samples by High Resolution Inductively Coupled Plasma-Mass Spectrometry. In addition, the degradation of the dental prostheses was assessed by both a leaching test and an analysis of the micro morphology of orthodontic prostheses. The induction of IFN-gamma production by Pd was assessed in PBMC by the ELISpot assay. Skin sensitization to Pd was evaluated by patch testing and clinical examination. Ten healthy subjects were comparatively tested as controls. We found a specific induction of an IFN-gamma response by Pd in PBMC collected from all the subjects positive to Pd at patch testing. On the contrary, control subjects did not show any response to Pd as assessed by IFN-gamma ELISpot assay or by skin testing. Remarkably, the levels of Pd in all biological samples (saliva, sera, urine) were significantly higher in Pd-sensitized patients than in those collected from controls, reaching the highest concentrations in the urine. The leaching studies gave additional evidence that the dental appliances can release measurable levels of Pd in saliva. Oral clinical symptoms in patients with Pd dental prostheses were associated with measurable levels of Pd in the biological fluids, the induction of Pd-specific IFN-gamma responses in PBMC and the clinical evidence of skin sensitization to Pd. These data suggest that dental appliances may represent an active source of Pd in the body, and this, in turn, can favour the clinical setting of a hypersensitivity to this metal.

CD81 expression on CD19+ peripheral blood lymphocytes is associated with chronic HCV disease and increased risk for HCV infection: a putative role for inflammatory cytokines.

The level of CD81 cell surface expression, a cellular co-receptor for hepatitis C virus (HCV), is critical for productive HCV infection of host cells. In addition, the cross-linking of HCV-E2 protein to CD81 can alter the function of T and B lymphocytes as well as that of NK cells by interfering with the activation signalling pathway. The down-regulation of CD81 expression on peripheral blood lymphocytes (PBL) has been associated to effective therapy of HCV infection. The aim of the present study is to quantitatively assess the levels of CD81 expression in PBL from HCV-infected patients compared to subjects at high risk for HCV infection such as HIV-infected individuals or patients with Porphyria Cutanea Tarda (PCT). The expression of CD81 was quantified by flow-cytometry using Phycoerythrin-labelled standard beads. Determination of CD81 was performed on CD3+ and CD19+ lymphocytes from 34 healthy controls, 51 patients with HCV infection and different clinical outcomes [these included HCV-RNA-negative subjects (8), patients with chronic active hepatitis (16), recipients of liver transplantation under immunosuppressive therapy (12), a subgroup with concomitant HIV infection (9) or concomitant PCT (6)]. In addition, 60 HIV-infected subjects and 4 patients with PCT were studied. The putative role of inflammatory cytokines in modulating CD81 was explored in vitro by assessing the effect of IL-6 or IFN-gamma on cultured human hepatocytes. A significant increase of the CD81 expression was found on CD19+ lymphocytes in association with either HIV or HCV infection, as compared to the control group. Immunosuppressive therapy with FK506, subsequent to liver transplantation, restored CD81 expression at normal levels. Data gathered in vitro using the WRL 68 hepatocytic cell line confirmed that inflammatory cytokines can up-regulate CD81 expression in liver cell inclusion. Our data suggest that CD81 up-regulation can increase the risk of HCV infection, particularly in HIV-infected subjects. In addition, the results strongly suggest that the cytokines released by activated lymphocytes at sites of inflammation may play a part in up-regulating CD81 expression.

[IL-2 bronchoscopic istillation and immune cell activation: preliminary results of the BRIIL-2 study for treatment of pulmonary metastasis from renal cancer and melanoma]. / Broncoistillazione diretta di IL-2 in fibrobroncoscopia e attivazione immunitaria: risultati preliminari dello studio clinico BRIIL-2 sul trattamento delle metastasi polmonari da neoplasia renale e melanoma.

BRIIL-2 is a clinical study for evaluation of efficacy and toxicity of third line treatment of pulmonary metastasis from renal cancer and melanoma with flexible bronchoscopic istillation of IL-2. Moreover, we evaluate local (BALT) and peripheral lymphocytic activation during this IL-2 administration. Up today we enrolled two patients with pulmonary metastasis from renal cancer already treated with two lines of molecular therapy, chemotherapy or systemic immunotherapy. Regarding to immunologic stimulation, lymphocytic fraction decreased from 21 to 2% in the first and from 10.5 to 6% in the second patient, indicating lymphocytic enrollment for activation, while TCD4/CD8 ratio is stable. In both patients we also observed a significant increase of HLA-DR in T lymphocytes (CD3) either in BAL or in peripheral blood. No significant major toxicities were observed after broncho-istillation, even if the dose was progressively increased. Thus IL-2 broncho-istillation could represent a valid administration modality to obtain an effective immunologic stimulation either local or systemic.

Serum cytokines and bioumoral immunological characterization of psoriatic patients in long term etanercept treatment.

The purpose of this study is to evaluate blood cytokines and immunological parameters in psoriatic patients during long-term treatment with Etanercept. Forty-five subjects of both sexes affected by psoriasis with or without arthritis entered the study and were treated with Etanercept according to international standard protocols. Biochemical blood analysis was carried out at baseline and during follow-up every second month. In particular, the following parameters were kept under control: antinuclear antibodies, anti-nDNA antibodies, anti-histone antibodies, blood cell count, circulating lymphocyte subtypes (CD3, CD4, CD8, CD16, CD19) and IgE. Cytokine profiles (IL-1-alpha, IL-1-beta, IL-6, IL-8, IL-10, IL-12, INF, TNF-alpha) were also evaluated in blood samples during the treatment up to 1 year of follow-up. A significant decrease in PASI score (p < 0.01) and in several cytokine levels was observed, particularly in IL-1, IL-6, IFN-gamma (p < 0.01) and to a lesser extent in TNF-alpha (p < 0.05). No statistically significant changes were recorded after 1 year of follow-up in blood immunological parameters, in particular in ANA titre, CD4/CD8 ratio, IgE levels, CD16, CD19 and eosinophils count. In conclusion, long-term treatment with Etanercept leads not only to a significant improvement in PASI score, but also to significant changes (reduction) in several proinflammatory and modulatory cytokines involved in the pathogenesis of the disease; on the other hand, there are no effects on immunological or bioumoral parameters showing that etanercept modulates rather than suppresses the physiological responses during psoriasis treatment.

Demographic indicators and risk of infection with human herpesvirus type 8 in Central Italy.

BACKGROUND: A cross-sectional study was conducted in Latium and Abruzzo Regions (Central Italy) to estimate the prevalence of infection with human herpesvirus type 8 (HHV-8) and the association between demographic indicators and risk of HHV-8 infection. PATIENTS AND METHODS: Sera from 416 healthy individuals (>or=45 years of age), originally recruited in a multicentric case-control study on classic Kaposi's sarcoma (KS), were tested for antibodies against HHV-8. The association between demographic indicators (i.e., urban/rural residence, occupation) and HHV-8 seropositivity was assessed by means of multiple logistic regression (MLR) odds ratios (OR) and 95% confidence intervals (CI), adjusted for age and occupation. RESULTS: Overall, 20.4% of the study participants had antibodies against HHV-8, 23.2% of the men and 17.0% of the women (p = 0.15). HHV-8 seropositivity rates significantly increased with age (p = 0.01), from 10.0% in those under 65 years of age to 24.9% in 75 years or older (MLROR = 2.4). By multivariate analysis, a significantly 2-fold higher risk of HHV-8 was found in individuals living in rural areas, as compared to those living in metropolitan/urban areas (MLR-OR = 2.0, 95% CI: 1.1-3.5), and in farmers, as compared to white collars (MLR-OR = 2.1, 95% CI: 1.1-4.1). CONCLUSIONS: The study findings suggest that demographic factors such as age, urban/rural residence, and occupation are associated with HHV-8 seropositivity among adult individuals living in central Italy.

Vascular endothelial growth factor gene polymorphisms increase the risk to develop psoriasis.

We investigated the relationship between eight polymorphisms in the gene encoding for vascular endothelial growth factor (VEGF) (-1540C > A, -1512Ins18, -1451C > T, -460T > C, -160C > T, -152G > A, -116G > A and +405G > C) and plaque-type psoriasis stratified for age at onset, gender and family history of dermatosis. For this purpose, 117 patients with chronic plaque-type psoriasis and 215 healthy subjects were enrolled. We found that being homozygous -1540AA, -1512InsIns, -1451TT, -460CC and -152AA conferred a significant risk in developing psoriasis compared with heterozygous (-1540CA, -1512 + Ins, -1451CT, -460CT and -152AG) and homozygous genotypes (-1540CC, -1512 + +-1451CC, -460TT and -152GG) grouped together [odds ratio (ORs) = 1.73, 1.73, 1.73, 1.77 and 1.87, respectively]. Conversely, having the -116AA or +405GG genotype did not significantly increase the risk of disease expression compared with other genotypes of the same loci. Interestingly, we found that -1540AA, -1512InsIns, -1451TT, -460CC and -152AA homozygous genotypes have a significant two-fold increased risk in developing psoriasis after the age of 40 years (late-onset psoriasis) (ORs = 2.19, 2.19, 2.19, 2.05 and 2.26; P = 0.02, 0.02, 0.02, 0.04 and 0.02, respectively) as compared with controls. On the contrary, we found no phenotype-genotype association of the same magnitude among the patients in whom psoriasis developed at or before the age of 40 years (early-onset psoriasis) compared with controls. Genotype distributions were not significantly different when cases and controls were stratified either by gender or family history of psoriasis. Finally, VEGF plasma concentration was not significantly different between patients and controls and was not correlated with the severity of the disease.

Early but not lasting improvement of recalcitrant subcorneal pustular dermatosis (Sneddon-Wilkinson disease) after infliximab therapy: relationships with variations in cytokine levels in suction blister fluids.

Subcorneal pustular dermatosis (SCPD) is an uncommon disorder, characterized by a chronic relapsing vesiculopustular eruption, mainly involving the trunk and intertriginous areas, and usually seen in women > 40 years old. Various therapies have been reported to be effective in treating SCPD, such as dapsone, systemic glucocorticoids, acitretin, etretinate, infliximab and phototherapy. We report a case of a 54-year-old woman affected by SCPD who after failure of different therapies showed a dramatic but only temporary improvement of her disease during a cycle of therapy with infliximab. In addition, an array of cytokines was simultaneously measured in suction blister fluids obtained from involved or uninvolved skin at various time intervals during the first 12 weeks of observation.

Cytokine profiles during infliximab monotherapy in psoriatic arthritis.

BACKGROUND: Biological therapies are a new breakthrough in the treatment of psoriasis and psoriatic arthritis (PsA). Among these, tumour necrosis factor (TNF)-alpha antagonists such as infliximab and etanercept are the most promising as TNF is considered to be essential in driving cytokine cascade at sites of cutaneous and synovial inflammation in this disease. OBJECTIVES: To evaluate the time-related response of serum cytokine release during infliximab monotherapy and assess serum cytokine levels in order to provide a fast, minimally invasive tool to monitor and/or predict efficacy of anti-TNF-alpha therapy. METHODS: Twenty patients affected by PsA with Psoriasis Area and Severity Index (PASI) score between 0.4 and 42.8 were treated with infliximab for 30-42 weeks. The assessment of arthritis severity was performed using the American College of Rheumatology (ACR) criteria and ultrasonography evaluation. The treatment schedule consisted of infliximab (5 mg kg(-1) intravenously) at 0, 2 and 6 weeks and every 12 weeks on an individual basis determined by therapeutic results and adverse events reported. At baseline and before every infusion blood samples were taken to assess serum cytokine levels [TNF-alpha, interleukin (IL-6), E-selectin, vascular endothelial cell growth factor (VEGF), fibroblast growth factor (FGF), matrix metalloproteinase (MMP-2)]. RESULTS: Eighteen of 20 psoriatic patients achieved > 50% improvement and 14 of 20 patients attained > 75% improvement in the PASI score at 10 weeks. All arthritic patients achieved > 50% improvement (ACR-50) and 16 of 20 patients attained > 75% improvement (ACR-75) at 10 weeks. TNF-alpha did not decrease immediately during the first part of the study. A significant decrease was detected at week 12 (P < 0.01). In contrast, IL-6, VEGF, FGF and E-selectin showed significant decreases after early infliximab infusions. PASI was not correlated with TNF-alpha in the serum but was significantly correlated with FGF, VEGF and MMP-2. Treatment was well tolerated and there were no significant adverse events in most patients, other than an urticarial reaction and an autoimmune hepatitis. CONCLUSIONS: Monotherapy with infliximab has to be considered an efficacious and safe treatment for PsA in comparison with traditional disease-modifying antirheumatic drugs. The resolution of cutaneous and synovial symptoms is not related to TNF-alpha serum levels in the initial phases. Apoptosis may play an important role in the modulation of the inflammatory response.

Development of a novel ELISA system for detection of anti-BP180 IgG and characterization of autoantibody profile in bullous pemphigoid patients.

BACKGROUND: The NC16A immunodominant region of the bullous pemphigoid (BP) antigen BP180 has been used to develop several enzyme-linked immunosorbent assays (ELISAs) as diagnostic tools for BP autoantibody detection. OBJECTIVES: Because BP180 autoantibody reactivity is not restricted to NC16A, we have investigated the possibility of developing an ELISA based on selected epitopes additional to this immunodominant region. METHODS: Initially 78 BP sera were tested using an NC16A ELISA and IgG reactivity was detected in 64 BP sera (82%). The 14 NC16A-negative BP sera were then analysed by immunological screening against seven BP180-specific epitopes. Recombinant phages displaying BP180 epitopes were grown as plaques, blotted onto a nitrocellulose filter and incubated with BP sera. RESULTS: Three and five NC16A-negative BP sera reacted with epitopes AA 1080-1107 and AA 1331-1404 of the BP180 ectodomain, respectively. Thus, a novel ELISA with GST-1080 and GST-1331 (GST-1080/1331) was developed: 32 of 78 BP sera (41%) proved positive by this assay. The combined use of ELISAs with GST-NC16A and GST-1080/1331 detected IgG reactivity in 72 of 78 BP sera, increasing the sensitivity from 82% to 92%. In addition, autoreactivity against the three extracellular epitopes appeared to be related to the presence of both skin and mucosal involvement as assessed by Fisher's exact probability test. CONCLUSIONS: Our findings further characterize the autoimmune response in BP by identifying a subgroup of NC16A-negative patients who react with different BP180 extracellular epitopes. The developed ELISA system appears more sensitive than the ELISA based on NC16A alone and also informative about the epitope profile of BP patients.

Heterogeneity of oral isolates of Candida albicans in HIV-positive patients: correlation between candidal carriage, karyotype and disease stage.

Opportunist infections involving Candida albicans often develop in HIV-positive patients and oral lesions tend to become more frequent as the disease progresses. Previous studies have shown contrasting results concerning the variability of the pulsed-field gel electrophoresis (PFGE) subtypes of C. albicans observed in HIV-positive patients. Carriage of C. albicans was determined by an oral rinse technique; 41 strains of C. albicans (78% serotype A and 22% serotype B) were isolated. There was a direct correlation between candidal load (cfu/ml) and the blood HIV load, whereas there was an inverse correlation with the stage of disease and the CD4 cell counts. The PFGE patterns of isolates were variable with regard to the number and positions of bands. The variability of the band sizes in some run positions showed a Gaussian distribution. Generally, the most frequent size variants were associated with the strains with the highest cfu/ml and lowest CD4 counts (< or =200 cells/microl). These findings suggest a possible strain selection over time during disease progression, especially in HIV-positive subjects with low CD4 counts.

Increased tryptase and myeloperoxidase levels in blister fluids of patients with bullous pemphigoid: correlations with cytokines, adhesion molecules and anti-basement membrane zone antibodies.

Tryptase and myeloperoxidase respectively represent 2 specific markers of activated mast cells or neutrophils. Therefore, establishing the levels of these enzymes may be useful to quantify the cell involvement in the tissues or fluids of different origins and in different pathologies. The aim of this study was to analyse the levels of these 2 markers in both the sera and blister fluids of patients affected with bullous pemphigoid. These levels were then correlated to the concentrations of 19 cytokines and 2 soluble adhesion molecules determined in the same samples and also with the log (anti-basement membrane zone antibody) titres, evaluated in the patients' sera. For these purposes, 15 patients with bullous pemphigoid (10 males and 5 females; median age: 84 years, range 66-87; median disease duration: 0 years, range 0-3: median number of skin lesions: 17, range 14-30; median anti-basement membrane zone antibody titre: 1:320, range 0.0-1:2560) and 15 normal subjects (11 males and 4 females, median age: 81 years, range 59-86) were analysed by means of commercially available kits. Results showed that blister fluid myeloperoxidase and tryptase levels were increased as compared with the respective sera (P<0.01) and several correlations were observed with cytokines and adhesion molecules. In fact, significant correlations of blister fluid tryptase levels were observed with IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, VEGF, RANTES and sICAM-1, while myeloperoxidase was correlated with IL-1beta, IL-13 and IL-15. The blister fluid tryptase levels were also significantly correlated with the anti-basement membrane zone antibody titres (R=0.53, P=0.05). In conclusion, these findings are in accord with an involvement of both mast cells and neutrophils in bullous pemphigoid and their recruitment may be mediated by different biological modulators. Our findings seem to indicate that the cytokine (IL-3, IFN-gamma and OSM) or adhesion molecule (sICAM-1) concentrations in blister fluid are logarithmically related to the anti-basement membrane zone antibody titers.

Identification of a new control region in the genome of the DDP strain of BK virus isolated from PBMC.

The various strains of human polyomavirus BK (BKV) show a marked heterogeneity in the non-coding control region (NCCR), which includes the origin of replication and the regulatory region for early and late transcription. A new BKV strain (DDP, U91605) was identified by direct detection and sequencing of PCR products of BKV-NCCR DNA obtained from PBMC samples of HIV-positive or -negative subjects. The DDP strain NCCR sequence showed an organisation not described previously in vivo with the maximum homology with the archetypal strain (WW) (M34048), as compared with those collected in GenBank. Structurally, P68, Q39, and S68 boxes were perfectly conserved, whereas the R63 box was completely deleted. This deletion involves the loss of sequences able to bind cellular factors essential for the DNA transcription, such as NF1 binding sites, normally present twice in the R box and the modification of SP1. It is possible that these rearrangements represent a cause of the loss of the VP1 region observed in 9/22 PBMC samples and never observed in urine isolates, which are similar to the WW strain.

Cytokines and bullous pemphigoid.

This report reviews the data presented in the literature concerning the presence and levels of different cytokines in sera, lesional tissue or blister fluids of patients with bullous pemphigoid. The list of cytokines analysed includes 21 molecules: interleukins (IL)-1 => 8, IL-10 => 13, IL-15, granulocyte-monocyte-colony stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), oncostatin-M (OSM), regulated upon activation normal T cell expressed and presumably secreted (RANTES), transforming growth factor-beta 1 (TGF-beta 1), tumor necrosis factor-alpha (TNF-alpha) and vascular endothelial growth factor (VEGF). Basic information regarding the functions of these cytokines and their possible involvement in the pathogenetic steps of the disease, such as autoantigen expression, autoantibody induction, complement activation, local cell recruitment and stimulation, resident cell activation, release of various effector molecules and tissue damage are also reported. A specific function for each cytokine in bullous pemphigoid induction cannot be still defined, however, the literature attributes a major role to IL-1, IL-4, IL-5, IL-6, IL-8 and IFN-gamma. On the basis of significant (direct or inverse) correlations found between disease intensity and the blister fluid/serum levels, the following cytokines IL-7, IL-15, RANTES, VEGF and TNF-alpha, besides those previously mentioned, may also be involved in this disease.

Anti-intercellular substance antibody log titres are correlated with serum concentrations of interleukin-6, interleukin-15 and tumor necrosis factor-alpha in patients with Pemphigus vulgaris relationships with peripheral blood neutrophil counts, disease severity and duration and patients' age.

Pemphigus vulgaris is a rare dermatosis of autoimmune origin, characterized by autoantibodies directed against intercellular substance (AICS) and presenting with intra-epidermal blisters and/or erosions of the skin and mucous membranes. The aim of this paper is to analyze the relationships between serum AICS titers (after log transformation) and: patients' age, disease duration and disease activity; serum cytokine (IL-6, IL-7, IL-15 and TNF-alpha) concentrations and peripheral blood cell counts (namely neutrophils, lymphocytes and natural killer cells). Fifteen consecutive subjects affected with PV were enrolled. Diagnosis was supported by histological examination as well as by direct and indirect immunofluorescence tests. Cytokine determinations were made by means of commercially available ELISA kits. This study shows for the first time that AICS titers have a significant correlation with age of PV patients (R=0.57, p=0.031) and with the disease duration (R=0.73, p=0.002). A correlation between blood neutrophils count and log (AICS) titres was observed (R=0.6, p=0.021). Furthermore, significant correlations were observed between log (AICS) titres and serum IL-15 (R=0.54, p=0.048), serum IL-6 (R=0.53, p=0.05) or serum TNF-alpha concentrations (R=0.53, p=0.05). These data, taken together, show that there are several connections between the log (AICS) titres, some proinflammatory cytokines, peripheral blood neutrophil counts and the numbers of individuals' lesions, suggesting a relationship between AICS production and lesion development.