NK and NKT-like cells in granulomatous and fibrotic lung diseases.

ABTRACT: Background The pathogenetic and regulatory roles of natural killer (NK) and natural killer T-like cells in interstitial lung diseases (ILDs), fibrotic and granulomatous of unknown etiology are unclear. Objectives Here we investigated NK and NKT-like cells in peripheral blood (PB) and Bronchoalveolar lavage (BAL) from patients with ILDs. Method 190 patients (94 male mean age 61 ± 14.3 years) and 8 controls undergoing bronchoscopy for ILD diagnostic work-up were enrolled consecutively; 115 patients sarcoidosis, 24 chronic fibrotic hypersensitivity pneumonitis and 43 patients other ILDs [32 idiopathic pulmonary fibrosis (IPF) and 11 non-specific interstitial pneumonia (NSIP)]. PB and BAL were processed by flow cytometry using monoclonal antibodies to differentiate NK and NKT-like cells. Results NK% in BAL was significantly different among ILDs (p = 0.02). Lower NK% was observed in BAL from sarcoidosis than other ILDs (p < 0.05). Similar findings were observed for NKT-like, whereas no differences were found for PB NK%. Difference of NK% was observed between BAL and PB in all groups (p < 0.001). Sarcoidosis patients reported the best area under the curve for NKT-like (AUC = 0.678, p = 0.0015) and NK cells (AUC = 0.61, p = 0.001). In the IPF-NSIP subgroup, NK% cell was inversely correlated with FVC% (r = - 0.34, p = 0.03) and DLCO% (r = - 0.47, p = 0.0044). Conclusions NK and NKT-like were expressed differently in BAL from patients with different ILD and were significantly depleted in sarcoidosis respect to other ILDs. This suggests that these cells may play a protective role in the pathogenesis of sarcoidosis.

Dermoscopic features and patterns of poromas: a multicentre observational case-control study conducted by the International Dermoscopy Society.

BACKGROUND: Poromas are benign cutaneous sweat gland tumours that are challenging to identify. The dermoscopic features of poromas are not well characterized. OBJECTIVE: To determine the clinical-dermoscopic features of poromas. METHODS: Cross-sectional, observational study of 113 poromas and 106 matched control lesions from 16 contributors and eight countries. Blinded reviewers evaluated the clinical and dermoscopic features present in each clinical and dermoscopic image. RESULTS: Poromas were most commonly non-pigmented (85.8%), papules (35.4%) and located on non-acral sites (65.5%). In multivariate analysis, dermoscopic features associated with poroma included white interlacing areas around vessels (OR: 7.9, 95% CI: 1.9-32.5, P = 0.004), yellow structureless areas (OR: 2.5, 95% CI: 1.1-6.0, P = 0.04), milky-red globules (OR: 3.9, 95% CI: 1.4-11.1, P = 0.01) and poorly visualized vessels (OR: 33.3, 95% CI: 1.9-586.5, P = 0.02). The presence of branched vessels with rounded endings was positively associated with poromas but did not reach statistical significance (OR: 2.4, 95% CI: 0.8-6.5, P = 0.10). The presence of any of these five features was associated with a sensitivity and specificity of 62.8% and 82.0%, respectively. CONCLUSION: We identified dermoscopic features that are specific to the diagnosis of poroma. Overall, however, the prevalence of these features was low. Significant clinical and dermoscopic variability is a hallmark of these uncommon tumours, which are most prevalent on non-acral sites.

Comparative proteomic analysis of bronchoalveolar lavage of familial and sporadic cases of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by progressive deterioration of the alveolar integrity. Among IPF identified phenotypes, that of familial (f-)IPF is usually associated with several gene mutations which are seldom observed in sporadic (s-)IPF. This study aimed at investigating the molecular patterns and variability in f-IPF and s-IPF patients through a differential proteomic analysis. Protein patterns of bronchoalveolar lavage fluid (BALF) samples from 10 familial and 17 sporadic IPF patients were compared using 2D electrophoresis and mass spectrometry. Principal component analysis (PCA) was applied to proteomic data and an enrichment analysis was also performed to characterize specific pathogenic mechanisms and to identify potential biomarkers. BALF samples from f-IPF showed 87 protein spots differentially expressed than those from s-IPF samples; once identified, these spots revealed 22 unique proteins. The functional analysis showed that the endothelial reticulum stress probably plays a central pathogenetic role in f-IPF with an up-regulation of proteins involved in wounding and immune responses, coagulation system, and ion homeostasis. Up-regulated proteins in the s-IPF group were those involved in the oxidative stress response. PCA analysis of differentially expressed proteins clearly distinguished f-IPF from s-IPF patients, and in agreement with radiological and histological patterns, pointed out a higher heterogeneity in f-IPF than s-IPF samples. The 'Slit/Robo signaling', 'clathrin-coated vesicle' and 'cytoskeleton remodelling', were extrapolated by 'pathways analysis' and the results of 'diseases (by biomarkers)' highlighted a 'connective tissue and autoimmune disease', two aspects of increasing interest in IPF.

A functional proteomics approach to the comprehension of sarcoidosis.

Pulmonary sarcoidosis (Sar) is an idiopathic disease histologically typified by non-caseating epitheliod cell sarcoid granulomas. A cohort of 37 Sar patients with chronic persistent pulmonary disease was described in this study. BAL protein profiles from 9 of these Sar patients were compared with those from 8 smoker (SC) and 10 no-smoker controls (NSC) by proteomic approach. Principal Component Analysis was performed to clusterize the samples in the corresponding conditions highlighting a differential pattern profiles primarily in Sar than SC. Spot identification reveals thirty-four unique proteins involved in lipid, mineral, and vitamin Dmetabolism, and immuneregulation of macrophage function. Enrichment analysis has been elaborated by MetaCore, revealing 14-3-3ε, α1-antitrypsin, GSTP1, and ApoA1 as "central hubs". Process Network as well as Pathway Maps underline proteins involved in immune response and inflammation induced by complement system, innate inflammatory response and IL-6signalling. Disease Biomarker Network highlights Tuberculosis and COPD as pathologies that share biomarkers with sarcoidosis. In conclusion, Sar protein expression profile seems more similar to that of NSC than SC, conversely to other ILDs. Moreover, Disease Biomarker Network revealed several common features between Sar and TB, exhorting to orientate the future proteomics investigations also in comparative BALF analysis of Sar and TB.

Dermoscopy of tumours arising in naevus sebaceous: a morphological study of 58 cases.

BACKGROUND: Naevus sebaceous is a congenital hamartoma commonly associated with the development of secondary neoplasms. There are sparse data relating to the dermoscopy of tumours arising in naevus sebaceous. OBJECTIVES: To evaluate the dermoscopic features of a large series of neoplasms arising in naevus sebaceous. METHODS: Digital dermoscopic images of 58 histopathologically confirmed cases of secondary neoplasms arising in naevus sebaceous collected from 11 hospitals in Spain, France, Italy and Austria were evaluated for the presence of dermoscopic structures and patterns. RESULTS: The most frequent tumours collected were: trichoblastoma (39.6%), basal cell carcinoma (20.7%) and syringocystadenoma papilliferum (15.6%). The most remarkable findings were: (i) the pattern composed of asymmetrical large blue-grey ovoid nests was more common in basal cell carcinomas than in trichoblastoma (58.3% vs. 4.3%; P < 0.001) and the pattern composed of a symmetrical total large blue-grey ovoid nest (a blue-grey homogeneous area which occupies the whole lesion) was more common in trichoblastoma (82.6% vs. 16.6%; P < 0.001); (ii) the most frequent pattern associated with syringocystadenoma papilliferum was a symmetric, erythematous lesion with exophytic papillary structures, ulceration and vessels (55.5%); (iii) the most common pattern associated with apocrine hidrocystomas was a total symmetric homogeneous area and arborizing telangiectasias (60%). CONCLUSIONS: Benign adnexal tumours associated with naevus sebaceous are dermoscopic mimickers of basal cell carcinomas. A pigmented nodule arising in a naevus sebaceous with a total blue large ovoid nest on dermoscopy could be a trichoblastoma, basal cell carcinoma, hidrocystoma or hidradenoma. Dermoscopy can be a useful diagnostic tool which generates a more accurate preoperative diagnosis.

Melanoma detection in Italian pigmented lesion clinics.

AIM: Accuracy in melanoma detection is important to recognize early curable melanomas and to minimize the unnecessary excision of benign lesions. The aim of this paper was to evaluate melanoma screening accuracy of Italian pigmented lesion clinics in terms of number needed to excise (NNE), melanoma thickness, and number of melanomas diagnosed during patient follow-up. METHODS: Information on all skin tumors excised in 2011 were extracted from the databases of the participating centers. Information whether the lesion was excised at the baseline examination or during patient follow-up was recorded, as well as the overall number of patients examined in each center in 2011. RESULTS: After e-mail solicitation, 22 of 40 centers agreed to participate. A total of 8229 excised lesions were collected. The overall number of examined patients was 86.564, thus 9.5% of screened patients had a lesion removed. Of the excised lesions, 866 were diagnosed as melanoma (1% of examined patients) and 5311 (88.9%) were melanocytic nevi. Three NNE were calculated giving values of 7.9 excised lesions to find 1 melanoma, 7.1 melanocytic lesions to find 1 melanoma, and 3.7 lesions to find 1 skin malignancy. The median melanoma thickness was 0.6 mm, with only 15.1% of melanomas ≥ 1 mm of thickness. Melanomas detected over time were 96 (11.1%; mean thickness, 0.3 mm), with 15.6% of lesions excised after short-term follow-up and 84.4% after long-term follow-up. CONCLUSION: The NNE values comparable to those achieved in specialized clinical settings and the high number of early melanomas diagnosed at the baseline examination or during patient follow-up indicate a high level of accuracy in melanoma screening achieved by Italian pigmented lesion clinics.

Towards a functional proteomics approach to the comprehension of idiopathic pulmonary fibrosis, sarcoidosis, systemic sclerosis and pulmonary Langerhans cell histiocytosis.

Bronchoalveolar lavage fluid of patients with four interstitial lung diseases (sarcoidosis, idiopathic pulmonary fibrosis, pulmonary Langerhans cell histiocytosis, fibrosis associated to systemic sclerosis) and smoker and non smoker control subjects were compared in a proteomic study. Principal component analysis was used to statistically verify the association between differentially expressed proteins and the conditions analyzed. Pathway and functional analysis by MetaCore and DAVID software revealed possible regulatory factors involved in specific "process networks" like regulation of stress and inflammatory responses. Immune response by alternative complement pathways, protein folding, Slit-Robo signaling and blood coagulation were "pathway maps" possibly associated with interstitial lung diseases pathogenesis. Four interesting proteins plastin 2, annexin A3, 14-3-3ε and S10A6 (calcyclin) were validated by Western blot analysis. In conclusion, we identified proteins that could be directly or indirectly linked to the pathophysiology of the different interstitial lung diseases. Multivariate analysis allowed us to classify samples in groups corresponding to the different conditions analyzed and based on their differential protein expression profiles. Finally, functional and pathway analysis defined the potential function and relations among identified proteins, including low abundance molecules present in the MetaCore database. BIOLOGICAL SIGNIFICANCE: This is the first study where different interstitial lung diseases such as sarcoidosis, idiopathic pulmonary fibrosis, pulmonary Langerhans cell histiocytosis, fibrosis associated to systemic sclerosis and smoker and non smoker control subjects were compared in a proteomic study to highlight their common pathways. We decided to report not only principal component analysis, used to statistically verify the association between differentially expressed proteins and the conditions analyzed, but also functional analysis general results, considering all differential proteins potentially involved in these conditions, to speculate about possible common pathogenetic pathways involved in fibrotic lung damage.

Analysis of serum amyloid A in sarcoidosis patients.

A crucial pathogenetic role of serum amyloid A (SAA) in granulomatous inflammation of sarcoidosis has recently been reported. In this study we analyzed SAA expression in detail, starting from proteomic analysis of serum of sarcoidosis patients. We also used the faster ELISA method that enabled us to examine a greater number of samples. Serum concentrations of SAA were significantly higher in sarcoidosis patients than controls (p<0.001), inversely correlated with FEV(1) and significantly higher in patients with subacute onset requiring prolonged and multiple steroid treatments (class 6 SCAC) than in patients with subacute onset not requiring therapy (class 4 SCAC) (p<0.001). Our results suggest that serum amyloid A could be a suitable marker of sarcoidosis: its serum concentrations are significantly higher in sarcoidosis patients than controls, the protein is only expressed in gels of sarcoidosis patients and not in healthy subjects, and the SAA1 isoforms could match the unidentified biomarker of sarcoidosis reported in a previous proteomic study by another group. The effectiveness of SAA as a clinical biomarker of sarcoidosis should now be investigated in a large prospective study.

Meat quality and intramuscular fatty acid composition of Sanfratellano and Haflinger foals.

The goal of the trial was to evaluate meat quality, with an emphasis on intramuscular fatty acid composition, of Sanfratellano foals, compared to that from Haflinger foals,both slaughtered at 18 months of age. Thirty foals, half of Sanfratellano breed and half of Haflinger breed, naturally weaned at 7-8 months, were divided into two homogeneous groups at 15 months of age and fed a finishing diet based on polyphite hay and concentrate. The finishing period lasted three months. Sanfratellano foals showed higher slaughter weight (P<0.05) as well carcass weight (P<0.05) compared to Haflinger foals. Meat physical and proximate analyses did not discriminate the two groups. Normal pH values (5.6-5.7) measured at 4-6 day post mortem were recorded in meat from both groups. Shear force values accounted (range 55-58N) for a favourable tenderness in both groups. The intramuscular fat level was low in both groups (<2.5%) supporting the healthy image of this meat. The proportion of linolenic acid was higher (P<0.01) in Haflinger meat than in Sanfratellano one, thus causing a higher (P<0.05) total n-3 fatty acid content. Overall meat from both groups showed a favourable repartition among saturated (36-37% total FAME's), monounsaturated (33% total FAME's) and polyunsaturated fatty acids (30-31% total FAME's).

The sentinel node biopsy in melanoma patients.

A palpable node is the first and most frequent sign of regional metastasis of malignant melanoma (MM). Nevertheless, the role of the elective lymph node dissection in patients with cutaneous melanoma remains one of the most debated topics of surgical oncology. Lymphatic mapping and sentinel node (SN) biopsy are supported as the standard of surgical care of MM by the World Health Organization and the Sunbelt Melanoma Clinical Trial. The only way to identify patients harboring microscopic nodal metastases is the elective complete regional lymphadenectomy or, as preferred today for its high specificity and minimal morbidity, the SN biopsy, which provides a histologic representative sample of the entire basin.

Sentinel lymph node biopsy in patients with primary cutaneous melanoma: study of 455 cases.

BACKGROUND: The role of elective lymph node dissection in the treatment of patients with primary melanoma is a debated topic in surgical oncology. However, recent data assure a survival improvement with this technique only for patients harbouring nodal metastases. The emergence of a new procedure of lymphatic mapping permits the identification of the sentinel lymph node (SLN), the first draining node from the site of cutaneous melanoma, which has demonstrated to be predictive of staging of the entire regional lymphatic basin and useful in selecting for lymph node dissection only those patients who have early micrometastases. OBJECTIVES: To verify in a large series of cases whether a combination of preoperative lymphoscintigraphy and intraoperative mapping with both vital blue dye and a hand-held gamma probe would permit an increase of the rate of successful SLN localization up to 100%; to check the utility of a wider application of SLN biopsy in patients with thin melanomas owing to a favourable risk-benefit ratio; to determine the predictive value of SLN biopsy by performing regional lymphadenectomy in patients who have pathological evidence of metastases in the SLN; to observe whether the use of SLN technique and selective lymphadenectomy might improve the clinical evolution of patients and favour low rates of recurrence. METHODS: In 425 AJCC stage I or II melanoma patients, preoperative lymphoscintigraphy by intracutaneous injection of Tc99m-labelled albumin nanocolloids around the tumour or the tumour's excision scar was combined with the intraoperative use of a hand-held gamma probe and patent blue V mapping technique, in order to identify and harvest the SLN. In five cases the blue dye was voluntarily not used because of previous allergic reactions. In other 25 preliminary cases the procedure was performed using the blue dye alone (10 cases) or combined with a preoperative lymphoscintigraphy (15 cases). A wide excision of the primary site was then undertaken in all cases. SLNs were sent to the pathologist for serial sectioning and permanent preparations with histological and immunohistochemical examination. Patients with pathological evidence of metastatic disease in SLN returned for regional lymphadenectomy. RESULTS: The combined use of lymphoscintigraphy, blue dye and gamma probe allowed us to identify one or more SLNs in all cases except for two (99.5% rate of success). In 70 melanomas less than 0.76 mm thick, SLNs were negative for metastases, whereas in 380 patients with thicker tumours micrometastases were demonstrated in 75 cases (19.7%). In patients with SLN metastases who underwent regional lymph node dissection, no other metastases were found three times out of four. After a median follow-up period of 18 months the rate of recurrence of the disease in 335 patients with SLN free of metastasis was low (5.4%) with a very low regional nodal recurrence (1.2%). Moreover, the worsening of the disease did not exceed 18.5% of cases with metastasis in SLN. CONCLUSIONS: Our data confirm in a large series of cases that the SLN biopsy is extremely selective and useful to find early micrometastases and to identify patients needing regional lymphadenectomy and adjuvant immunotherapy. Patients with intermediate thickness melanoma (0.76-4.0 mm) should be informed on the availability of such a revolutionary procedure, which represents a new opportunity in primary melanoma surgery.

Malignant blue nevus of the scalp.

A 70-year-old woman had noticed, at the age of 30, a single blue nodule of about 1 cm in size on her scalp. The lesion remained stable until 1991, when it became larger and ulcerated and, because of the sudden onset of additional macules and nodules around it, the patient presented at our Dermatological Division in August 1992. Physical examination showed a blue-black plaque, 2 x 2 cm in size, on the left parietal area of the scalp, surrounded by several blue-grey pigmented nodules and macules (Fig. 1). Chest X-ray, abdomen scan, and a total body computed tomography (CT) scan were negative for metastatic disease. A wide resection of the scalp lesion was performed. The histologic evaluation revealed a dense collection of spindled melanocytes in the dermis and in the subcutaneous fat. Nuclear and cytoplasmic pleomorphism, some mitotic figures, and necrosis foci were present (Figs 2 and 3). Pictures of cellular blue nevus were found in the surrounding lesions. Ten months later, new blue macular and papular lesions appeared in proximity to the surgical scar. The patient refused any additional surgery, and so was treated with dacarbazine (DTIC) 800 mg intravenously (every 20 days) and 2 alpha interferon (3 million units subcutaneously, three times weekly). The growth of the lesions slowed down for a few weeks, and then increased again to become a wide, blue-black vegetating mass (Fig. 4). In June 1995, a total body CT scan revealed multiple focal nodules on the lungs and two metastatic masses on the eighth segment of the liver. A palliative polychemotherapy, with vindesin 3 mg/m2 and DTIC 400 mg/m2, was started, but did not stop the progression of the disease, and the patient died in December 1996.