An unusual case of melanoma metastasis in the buccal space: learning by mistakes to distinguish it from salivary neoplasms.

BACKGROUND: The buccal space is an unusual location of malignancies. We report here the case of a woman with a melanoma metastasis in buccal fat pad, to evaluate the imaging features which might lead to the correct, although uncommon, diagnosis. CASE PRESENTATION: A 71-year-old woman presented with a painless visible swelling of the left cheek. MRI revealed the presence of a solid lesion located in the buccal fat pad with features suggestive of malignancy. It showed T1 hyperintensity and T2 hypointensity, and restriction of diffusion. Histological examination showed neoplastic cells compatible with melanoma. DISCUSSION: The lesion features (T1 hyperintensity and T2 hypointensity) initially lead our team to believe that there was a hemorrhagic component, possibly a residue of the biopsy. However, when associated with other malignancy features, such as low apparent diffusion coefficient (ADC) values and contrast enhancement, they should evoke the suspect of melanoma, provided that no biopsy was performed and no trauma occurred in the 3-7 days before.

Spontaneous pneumothorax in cocaine users.

BACKGROUND: Pneumothorax is one of the respiratory toxic effects of cocaine inhalation. The literature counts several cases, some associated to other respiratory conditions such as pneumomediastinum, haemoptysis and others not requiring surgical treatment. AIM: We present a series of nonHIV cocaine-inhaler subjects who underwent video-assisted thoracoscopic surgery (VATS) for isolated spontaneous pneumothorax. DESIGN: Nine subjects, with a mean age of 24 ± 4 years, admitting cocaine inhalation, developed spontaneous pneumothorax and underwent 10 surgical treatments by means of VATS, at our Institution. RESULTS: Previous pneumothorax occurred in six cases episodes ranged from 0 to 5 (mean 1.6 ± 1.6). Chest computed tomography (CT) scan showed abnormalities in seven cases. All subjects underwent lung apicectomy, apical pleurectomy and mechanical pleurodesis. Seven subjects had also bullectomy. In all cases the visceral pleura was partially covered by fibrinous exudate. Histology of the lung showed small foreign body granulomatous inflammation in fibrotic and/or emphysematous pulmonary parenchyma. Relapse of pneumothorax occurred in one subject at 60 days and it was surgically treated. Mean follow-up was 150 ± 38 months (range 120-239). All subjects are now well, with no evidence of pneumothorax. CONCLUSIONS: Spontaneous pneumothorax in cocaine-inhaler subjects is a reality of which physicians need to be aware. Chest CT scan might not reveal abnormalities. Macroscopically the lung might presents bullae and/or peculiar visceral pleura. Foreign body granulomas observed in the specimens suggest that the particulate component of inhaled substances can injure the lung. Surgical treatment of the bullous disease and mechanical pleurodesis can provide a long-term follow-up without relapse of pneumothorax.

Proteome Analysis of Urticating Setae From Thaumetopoea pityocampa (Lepidoptera: Notodontidae).

Thaumetopoea pityocampa (Denis & Schiffermüller) (Lepidoptera: Notodontidae) is harmful to conifer trees because of defoliation and to public health because of the release of urticating setae from the caterpillars. Contact with setae by humans and domestic animals induces dermatitis, usually localized to the exposed areas. Recent studies demonstrated the presence of a complex urticating mechanism where proteins present in the setae may play a role as activators of immune responses. Yet, limited information is available at present about the proteins occurring in the setae of T. pityocampa. Using a refined method for protein extraction from the setae, and a combination of liquid chromatography tandem-mass spectrometry (LC-MS/MS), de novo assembly of transcriptomic data, and sequence similarity searches, an extensive data set of 353 proteins was obtained. These were further categorized by molecular function, biological process, and cellular location. All the 353 proteins identified were found to match through BLAST search with at least one Lepidoptera sequence available in databases. We found the previously known allergens Tha p 1 and Tha p 2 described from T. pityocampa, as well as enzymes involved in chitin biosynthesis, one of the principal components of the setae, and serine proteases that were responsible for inflammatory and allergic reactions in other urticating Lepidoptera. This new proteomic database may allow for a better understanding of the complexity of allergenic reactions due to T. pityocampa and to other Lepidoptera sharing similar defense systems.

A case of endobronchial paraganglioma.

Paragangliomas are rare lung tumours; endobronchial localisation is even more rare. This report describes the case of a 59-year-old patient with a symptomatic endobronchial paraganglioma successfully resected by means of pulmonary lobectomy. Recognition of this uncommon tumour can lead to a correct diagnosis and therapeutic strategy.

Leaf apoplastic proteome composition in UV-B treated Arabidopsis thaliana mutants impaired in extracellular glutathione degradation.

In plants, environmental perturbations often result in oxidative reactions in the apoplastic space, which are counteracted for by enzymatic and non-enzymatic antioxidative systems, including ascorbate and glutathione. However, the occurrence of the latter and its exact role in the extracellular space are not well documented. In Arabidopsis thaliana, the gamma-glutamyl transferase isoform GGT1 bound to the cell wall takes part in the so-called gamma-glutamyl cycle for extracellular glutathione degradation and recovery, and may be implicated in redox sensing and balance. In this work, oxidative conditions were imposed with UV-B radiation and studied in redox altered ggt1 mutants. Elevated UV-B has detrimental effects on plant metabolism, plasma membranes representing a major target for ROS generated by this harmful radiation. The response of ggt1 knockout Arabidopsis leaves to UV-B radiation was assessed by investigating changes in apoplastic protein composition. We then compared the expression changes resulting from the mutation and from the UV-B treatment. Rearrangements occurring in apoplastic protein composition suggest the involvement of hydrogen peroxide, which may ultimately act as a signal. Other important changes related to hormonal effects, cell wall remodeling, and redox activities are also reported. We argue that oxidative stress conditions imposed by UV-B and by disruption of the gamma-glutamyl cycle result in similar stress-induced responses, to some degree at least. Data shown here are associated with the article from Trentin et al. (2015) [1]; protein data have been deposited to the PRIDE database (Vizcaíno et al., 2014) [2] with identifier PXD001807.

Investigation on PLK2 and PLK3 substrate recognition.

Analyses of human phosphoproteome based on primary structure of the aminoacids surrounding the phosphor Ser/Thr suggest that a significant proportion of phosphosites is generated by a restricted number of acidophilic kinases, among which protein kinase CK2 plays a prominent role. Recently, new acidophilic kinases belonging to the Polo like kinase family have been characterized, with special reference to PLK1, PLK2, and PLK3 kinases. While some progress has been made in deciphering the PLK1-dependent phosphoproteome, very little is known about the targets of PLK2 and PLK3 kinases. In this report by using an in vitro approach, consisting of cell lysate phosphorylation, phosphoprotein separation by 2D gel electrophoresis and mass spectrometry, we describe the identification of new potential substrates of PLK2 and PLK3 kinases. We have identified and validated as in vitro PLK2 and PLK3 substrates HSP90, GRP-94, ß-tubulin, calumenin, and 14-3-3 epsilon. The phosphosites generated by PLK3 in these proteins have been identified by mass spectrometry analysis to get new insights about PLKs specificity determinants. These latter have been further corroborated by an in silico analysis of the PLKs substrate binding region.

MassUntangler: a novel alignment tool for label-free liquid chromatography-mass spectrometry proteomic data.

Liquid chromatography-mass spectrometry (LC-MS) has become an important analytical tool for quantitative proteomics and biomarker discovery. In the label-free differential LC-MS approach computational methods are required for an accurate alignment of peaks extrapolated from the experimental raw data accounting for retention time and m/z signals intensity, which are strongly affected by sample matrix and instrumental performance. A novel procedure "MassUntangler" for pairwise alignment has been developed, relying on a pattern-based matching algorithm integrated with filtering algorithms in a multi-step approach. The procedure has been optimized employing a two-step approach. Firstly, low-complexity LC-MS data derived from the enzymatic digestion of two standard proteins have been analyzed. Then, the algorithm's performance has been evaluated by comparing the results with other achieved using state-of-the-art alignment tools. In the second step, our algorithm has been used for the alignment of high-complexity LC-MS data consisting of peptides obtained by an Escherichia coli lysate available from a public repository previously used for the comparison of other alignment tools. MassUntangler gave excellent results in terms of precision scores (from 80% to 93%) and recall scores (from 68% to 89%), showing performances similar and even better than the previous developed tools. Considering the mass spectrometry sensitivity and accuracy, this approach allows the identification and quantification of peptides present in a biological sample at femtomole level with high confidence. The procedure's capability of aligning LC-MS data previously corrected for distortion in retention time has been studied through a hybrid approach, in which MassUntangler was interfaced with the OpenMS TOPP tool MapAligner. The hybrid aligner yielded better results, showing that an integration of different bioinformatic approaches for accurate label-free LC-MS data alignment should be used.

Identification of CXCL13 as a new marker for follicular dendritic cell sarcoma.

The homeostatic chemokine CXCL13 is preferentially produced in B-follicles and is crucial in the lymphoid organ development by attracting B-lymphocytes that express its selective receptor CXCR5. Follicular dendritic cells (FDCs) have been identified as the main cellular source of this chemokine in lymphoid organs. Recently, genome-wide approaches have suggested follicular CD4 T-helper cells (T(H)F) as additional CXCL13 producers in the germinal centre and the neoplastic counterpart of T(H)F (CD4+ tumour T-cells in angioimmunoblastic T-cell lymphoma) retains the capability of producing this chemokine. In contrast, no data are available on CXCL13 expression on FDC sarcoma (FDC-S) cells. By using multiple approaches, we investigated the expression of CXCL13 at mRNA and protein level in reactive and neoplastic FDCs. In reactive lymph nodes and tonsils, CXCL13 protein is mainly expressed by a subset of FDCs in B-cell follicles. CXCL13 is maintained during FDC transformation, since both dysplastic FDCs from 13 cases of Castleman's disease and neoplastic FDCs from ten cases of FDC-S strongly and diffusely express this chemokine. This observation was confirmed at mRNA level by using RT-PCR and in situ hybridization. Of note, no CXCL13 reactivity was observed in a cohort of epithelial and mesenchymal neoplasms potentially mimicking FDC-S. FDC-S are commonly associated with a dense intratumoural inflammatory infiltrate and immunohistochemistry showed that these lymphocytes express the CXCL13 receptor CXCR5 and are mainly of mantle zone B-cell derivation (IgD+ and TCL1+). In conclusion, this study demonstrates that CXCL13 is produced by dysplastic and neoplastic FDCs and can be instrumental in recruiting intratumoural CXCR5+ lymphocytes. In addition to the potential biological relevance of this expression, the use of reagents directed against CXCL13 can be useful to properly identify the origin of spindle cell and epithelioid neoplasms.

Analysis of a sub-proteome which co-purifies with and is phosphorylated by the Golgi casein kinase.

In an attempt to gain information about the identity of the Golgi apparatus casein kinase(s) (G-CK), responsible for the phosphorylation of caseins in lactating mammary gland, the proteins present in fractions enriched in G-CK activity eluted from DEAE-Sepharose and heparin-Sepharose columns were resolved by two-dimensional electrophoresis and analyzed by mass spectrometry. This led to the identification of 47 proteins altogether, none of which is a bona fide protein kinase. At least 9 of the identified proteins however, are readily phosphorylated by co-purifying G-CK activity, and 7 are physically associated with it to give supramolecular complex(es) of about 500 kDa as judged from Superdex S200 gel fitration and glycerol gradient ultracentrifugation experiments. In contrast, the apparent molecular weight of G-CK estimated from an in gel activity assay after SDSPAGE and renaturation is about 41 kDa. Many of the proteins phosphorylated by and/or associated with G-CK belong to the category of chaperonines, including HSP90, GRP-94 and -78, and various isoforms of protein disulfide isomerases, suggesting a global role of this kinase in the modulation of protein folding.

Protein kinase CK2 phosphorylates and upregulates Akt/PKB.

Treatment of Jurkat cells with specific inhibitors of protein kinase CK2 induces apoptosis. Here we provide evidence that the anti-apoptotic effect of CK2 can be at least partially mediated by upregulation of the Akt/PKB pathway. Such a conclusion is based on the following observations: (1) inhibition of CK2 by cell treatment with two structurally unrelated CK2 inhibitors induces downregulation of Akt/PKB, as judged from decreased phosphorylation of its physiological targets, and immunoprecipitate kinase assay; (2) similar results are observed upon reduction of CK2 catalytic subunit by the RNA-interference technique; (3) Akt/PKB Ser129 is phosphorylated by CK2 in vitro and in vivo; (4) such a phosphorylation of activated Akt/PKB correlates with a further increase in catalytic activity. These data disclose an unanticipated mechanism by which constitutive phosphorylation by CK2 may be required for maximal activation of Akt/PKB.

Anthracycline-based chemotherapy as primary treatment for intravascular lymphoma.

BACKGROUND: Optimal therapeutic management of intravascular lymphoma (IVL) lacks precise guidelines. PATIENTS AND METHODS: The clinico-pathological features of 38 HIV-negative patients with IVL were reviewed to define efficacy of chemotherapy in these malignancies. Clinical characteristics of 22 patients treated with chemotherapy and of 16 untreated patients were compared in order to understand better the impact and causes of potential patient selection. RESULTS: Median age was 70 years (range 34-90), with a male/female ratio of 0.9; 23 (61%) patients had Eastern Cooperative Oncology Group performance status (ECOG-PS) > 1; 21 (55%) had systemic symptoms. Cutaneous lesions and anemia were significantly more common among patients treated with chemotherapy; central nervous system (CNS) and renal involvement were significantly more common among untreated patients. Chemotherapy was associated with a response rate of 59% and a 3-year overall survival of 33 +/- 11%. Five of six patients with CNS involvement received chemotherapy: four of them died early; only one patient, treated with adriamycin, cyclophosphamide, vincristine, methotrexate, bleomycin and prednisolone (MACOP-B) followed by high-dose chemotherapy and autologous stem cell transplantation (ASCT), was alive at 19 months. High-dose chemotherapy supported by ASCT was indicated at diagnosis in another patient (43 years of age, stage I), who was alive at 71 months, and at relapse after cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) in two patients who died early after transplantation. PS < or = 1, disease limited to the skin, stage I, and use of chemotherapy were independently associated with better outcome. CONCLUSIONS: Anthracycline-based chemotherapy is the standard treatment for IVL. However, survival is disappointing, with a relevant impact of diagnostic delay and lethal complications. More intensive combinations, containing drugs with higher CNS bioavailability, are needed in cases with brain involvement, and the role of high-dose chemotherapy supported by ASCT should be further investigated in younger patients with unfavorable features.

Primary brain CD30+ ALK1+ anaplastic large cell lymphoma ('ALKoma'): the first case with a combination of 'not common' variants.

BACKGROUND: Primary central nervous system lymphomas (PCNSLs) are rare tumors, mostly represented by diffuse large B cells. PCNSLs with a T phenotype are less frequently reported; even rarer are anaplastic large cell lymphomas (ALCLs). PCNSL ALCLs are commonly represented, like their systemic counterpart, by a variably prevalent amount of large pleomorphic tumor cells ('hallmark cells'), and this feature enhances their recognition. Patient and methods We report the first case of primary brain CD30+ ALK-1+ ALCL with a T-cell phenotype, showing the combination of both the 'lymphohistiocytic' and the 'small cell' variants of the disease. A few elements consistent with 'hallmark cells' were recognizable. However, these cells were never prominent, increasing diagnostic difficulties. Immunohistochemistry results were critical for the correct interpretation. Our findings also differ from the majority of PCNSL ALCLs for the absence of tumor necrosis and the lack of prominent mitotic activity. The neuroimaging picture was not specific. A comparison with literature data concerning the clinical/instrumental features shows a very frequent meningeal involvement in PCNSL ALCLs, in contrast to the majority of PCNSLs. CONCLUSION: The occurrence of such a rare form of ALCL may widen the spectrum of differential diagnoses in PCNSL and their recognition may allow a rapid diagnosis, thus encouraging adequate treatment, which should take into account the high rate of meningeal involvement observed in these cases.

Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas-a possible mechanism for altering the nm23-H1 activity.

PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.

Evaluation of radiological and pathological prognostic factors in surgically-treated patients with bronchoalveolar carcinoma.

OBJECTIVE: The incidence of adenocarcinoma and bronchoalveolar carcinoma has increased in recent years. The aim of this study was to retrospectively evaluate radiological and pathological factors affecting survival in patients with bronchoalveolar carcinoma (BAC) or BAC associated with adenocarcinoma who underwent surgical treatment. METHODS: From May 1988 to September 1999, 49 patients with BAC or BAC and adenocarcinoma underwent surgical treatment. Complete resection was performed in 42 patients. In these patients the impact of the following factors on survival was evaluated: stage, TNM status, radiological and pathological findings (percentage of bronchoalveolar carcinoma in the tumour, presence or absence of sclerosing and mucinous patterns, vascular invasion and lymphocytic infiltration). RESULTS: Twenty-nine patients were male and 20 female. Mean age was 63 years. Five-year survival was 54%. Univariate analysis of the patients who underwent complete resection demonstrated a favourable impact on survival in stages Ia and Ib (P = 0.01) and in the absence of nodal involvement (P = 0.02) and mucinous patterns (P = 0.02). Mucinous pattern was also prognostically relevant at multivariate analysis (P = 0.02). In the 27 patients with stage Ia and Ib disease, univariate analysis demonstrated that the absence of mucinous pattern (P = 0.006) and a higher percentage of BAC (P = 0.01) favourably influenced survival. The latter data were also confirmed by multivariate analysis (P = 0.01). CONCLUSION: Surgical treatment of early-stage BAC and combined BAC and adenocarcinoma is associated with favourable results. However, the definition of prognostic factors is of utmost importance to improve the results of the treatment. In our series tumours of the mucinous subtype and with a lower percentage of BAC had a worse prognosis.

The C-C chemokine receptors CCR4 and CCR8 identify airway T cells of allergen-challenged atopic asthmatics.

In vitro polarized human Th2 cells preferentially express the chemokine receptors CCR3, CCR4, and CCR8 and migrate to their ligands: eotaxin, monocyte-derived chemokine (MDC), thymus- and activation-regulated chemokine (TARC), and I-309. We have studied the expression of chemokines and chemokine receptors in the airway mucosa of atopic asthmatics. Immunofluorescent analysis of endobronchial biopsies from six asthmatics, taken 24 hours after allergen challenge, demonstrates that virtually all T cells express IL-4 and CCR4. CCR8 is coexpressed with CCR4 on 28% of the T cells, while CCR3 is expressed on eosinophils but not on T cells. Expression of the CCR4-specific ligands MDC and TARC is strongly upregulated on airway epithelial cells upon allergen challenge, suggesting an involvement of this receptor/ligand axis in the regulation of lymphocyte recruitment into the asthmatic bronchi. In contrast to asthma, T cells infiltrating the airways of patients with chronic obstructive pulmonary disease and pulmonary sarcoidosis produce IFN-gamma and express high levels of CXCR3, while lacking CCR4 and CCR8 expression. These data support the role of CCR4, of its ligands MDC and TARC, and of CCR8 in the pathogenesis of allergen-induced late asthmatic responses and suggest that these molecules could be considered as targets for therapeutic intervention.

The high mobility group (HMG) boxes of the nuclear protein HMG1 induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells.

HMG1 (high mobility group 1) is a ubiquitous and abundant chromatin component. However, HMG1 can be secreted by activated macrophages and monocytes, and can act as a mediator of inflammation and endotoxic lethality. Here we document a role of extracellular HMG1 in cell migration. HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays. HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells. These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses. Pertussis toxin and the mitogen-activated protein kinase kinase inhibitor PD98059 also blocked HMG1-induced rat smooth muscle cell migration, suggesting that a G(i/o) protein and mitogen-activated protein kinases are required for the HMG1 signaling pathway. We also show that HMG1 can be released by damage or necrosis of a variety of cell types, including endothelial cells. Thus, HMG1 has all the hallmarks of a molecule that can promote atherosclerosis and restenosis after vascular damage.

Maspin and mammaglobin genes are specific markers for RT-PCR detection of minimal residual disease in patients with breast cancer.

BACKGROUND: This study evaluates the specificity of some reverse-transcriptase polymerase chain reaction (RT-PCR) assays for the detection of residual tumor cells in breast cancer patients. The following markers have been analysed: carcinoembryonic antigen (CEA), cytokeratins (CK19 and CK20), polymorphic epithelial mucin (MUC-1), epidermal growth factor receptor (EGFR), maspin, and mammaglobin. RT-PCR was employed to detect breast cancer cells in peripheral blood (PB), bone marrow (BM), and stem cell leukoaphereses (PBPC). PATIENTS AND METHODS: We evaluated the specificity of our RT-PCR assays on a panel of breast cancer specimens (n = 30), on PBPC in patients undergoing high-dose chemotherapy (n = 38), on BM (n = 7) and PB (n = 5) samples obtained from patients with breast cancer. Marrow cells, PB, and PBPC from normal subjects or hematological tumor patients were tested as negative controls. RESULTS: Only maspin and mammaglobin met the criteria of sensitivity and specificity required for the detection of residual disease; they were expressed in 80% and 97% of breast cancer specimens, respectively, and not expressed in normal controls. CK19, CK20. EGFR, MUC-1, and CEA were sometimes expressed in normal blood cells and/or hematological tumors. CONCLUSIONS: Our data support the notion that maspin and mammaglobin are useful markers for RT-PCR detection of minimal residual disease (MRD) in breast cancer patients, and that perspective clinical studies are needed to determine wether RT-PCR assays will be useful in assessing prognosis, tailoring therapy, or developing new strategies for ex vivo purging.