Regions of mouse mammary tumor virus superantigen involved in interaction with the major histocompatibility complex class II I-A molecule.

To study the major histocompatibility complex class II I-E dependence of mouse mammary tumor virus (MMTV) superantigens, we constructed hybrids between the I-E-dependent MMTV(GR) and the I-E-independent mtv-7 superantigens and tested them in vivo. Our results suggest that, although the C-terminal third mediates I-A interaction, additional binding sites are located elsewhere in the superantigen.

Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice.

Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.

Combinatorial peptide library-based identification of peptide ligands for tumor-reactive cytolytic T lymphocytes of unknown specificity.

A novel approach for the identification of tumor antigen-derived sequences recognized by CD8(+) cytolytic T lymphocytes (CTL) consists in using synthetic combinatorial peptide libraries. Here we have screened a library composed of 3.1 x 10(11) nonapeptides arranged in a positional scanning format, in a cytotoxicity assay, to search the antigen recognized by melanoma-reactive CTL of unknown specificity. The results of this analysis enabled the identification of several optimal peptide ligands, as most of the individual nonapeptides deduced from the primary screening were efficiently recognized by the CTL. The results of the library screening were also analyzed with a mathematical approach based on a model of independent and additive contribution of individual amino acids to antigen recognition. This biometrical data analysis enabled the retrieval, in public databases, of the native antigenic peptide SSX-2(41-49), whose sequence is highly homologous to the ones deduced from the library screening, among the ones with the highest stimulatory score. These results underline the high predictive value of positional scanning synthetic combinatorial peptide library analysis and encourage its use for the identification of CTL ligands.

Identification and potential origin of invasive clawed frogs Xenopus (Anura: Pipidae) in Sicily based on mitochondrial and nuclear DNA

African clawed frogs of the widespread polytypic species Xenopus laevis Daudin, 1802 (ranging large parts of sub-Saharan Africa) have been spreading since the 1940s, and have established reproductive populations in Europe, Asia and the Americas, where they can have negative impact as competitors of native amphibians and as disease vectors for chytridomycosis or ranaviruses. Here we use two mitochondrial (cytochrome b, 16S rDNA) and one nuclear (RAG 1: Recombination Associated Gene 1) DNA markers to infer the potential origin of invasive clawed frogs from Sicily that represent the largest invasive population in Europe. Identical mtDNA haplotypes match with those of Xenopus laevis, and Sicilian clawed frogs very probably belong to a lineage from the Cape Region of South Africa, most likely originating from a laboratory stock. Nuclear data support this conclusion. Identical mtDNA sequences (cyt b, 16S) of frogs sampled across their range in Sicily suggest the occurrence of a single source population and a potential bottleneck at their release, but faster evolving multilocus nuclear data (microsatellites, SNPs) on the population genetics would be important in the future to better support this hypothesis

Successful combined minimally invasive direct coronary artery bypass and transapical aortic valve implantation.

Transapical aortic valve implantation is indicated in high-risk patients with aortic stenosis and peripheral vascular disease requiring aortic valve replacement. Minimally invasive direct coronary artery bypass grafting is also a valid, minimally invasive option for myocardial revascularization in patients with critical stenosis on the anterior descending coronary artery. Both procedures are performed through a left minithoracotomy, without cardiopulmonary bypass, aortic cross-clamping, and cardioplegic arrest. We describe a successful combined transapical aortic valve implantation and minimally invasive direct coronary bypass in a high-risk patient with left anterior descending coronary artery occlusion and severe aortic valve stenosis.

Synergistic action of GA-binding protein and glucocorticoid receptor in transcription from the mouse mammary tumor virus promoter.

B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position -139 to -146 from the cap site) and fp2 (at -157 to -164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways.

Isolated limb perfusion with tumor necrosis factor and melphalan for non-resectable soft tissue sarcomas: long-term results on efficacy and limb salvage in a selected group of patients

Background And Objectives: Isolated limb perfusion with TNF-alpha and melphalan (TM-ILP) is a limb salvage therapy for non-resectable soft tissue sarcomas (STS) of the extremities. It is indicated for patients for whom amputation or debilitating surgery is the only alternative. It can be used either as an exclusive therapy (in palliation) or as a neo-adjuvant treatment, followed by marginal resection of tumor remnants with minimal functional impairment. Methods: Between February 1992 and March 2006, 57 TM-ILPs were performed on 51 patients with 88% high grade and 84% advanced stage tumors. Results: Mean follow-up is 38.9 months (4-159, median 22 months). Twenty-one percent patients had significant early complications, with 3 major re-operations, and 23% suffered long-lasting complications. Complete response was observed in 25%, partial response in 42%, stable disease in 14% and progressive disease in 14%. Resection of the tumor remnants was possible in 65%. A complementary treatment was necessary in 31%, mostly radiation therapy. A local recurrence was observed in 35%, after a mean of 20.3 months (2-78), and distant relapse was seen in 45%, after a mean of 13.4 months (5-196). Mean Disease-free survival was 14.9 months, and overall 5-year-survival 43.5%. Amputation rate at 5 years was 24%. Conclusions: TM-ILP is a conservative treatment with a high complications rate, but it can be successful even for the most severe STS of extremities. As a consequence the limb can be spared from amputation or debilitating surgery on the long term in about 75% of patients

MGMT methylation analysis of glioblastoma on the Infinium methylation BeadChip identifies two distinct CpG regions associated with gene silencing and outcome, yielding a prediction model for comparisons across datasets, tumor grades, and CIMP-status.

The methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Recent studies in anaplastic glioma suggest a prognostic value for MGMT methylation. Investigation of pathogenetic and epigenetic features of this intriguingly distinct behavior requires accurate MGMT classification to assess high throughput molecular databases. Promoter methylation-mediated gene silencing is strongly dependent on the location of the methylated CpGs, complicating classification. Using the HumanMethylation450 (HM-450K) BeadChip interrogating 176 CpGs annotated for the MGMT gene, with 14 located in the promoter, two distinct regions in the CpG island of the promoter were identified with high importance for gene silencing and outcome prediction. A logistic regression model (MGMT-STP27) comprising probes cg1243587 and cg12981137 provided good classification properties and prognostic value (kappa = 0.85; log-rank p < 0.001) using a training-set of 63 glioblastomas from homogenously treated patients, for whom MGMT methylation was previously shown to be predictive for outcome based on classification by methylation-specific PCR. MGMT-STP27 was successfully validated in an independent cohort of chemo-radiotherapy-treated glioblastoma patients (n = 50; kappa = 0.88; outcome, log-rank p < 0.001). Lower prevalence of MGMT methylation among CpG island methylator phenotype (CIMP) positive tumors was found in glioblastomas from The Cancer Genome Atlas than in low grade and anaplastic glioma cohorts, while in CIMP-negative gliomas MGMT was classified as methylated in approximately 50 % regardless of tumor grade. The proposed MGMT-STP27 prediction model allows mining of datasets derived on the HM-450K or HM-27K BeadChip to explore effects of distinct epigenetic context of MGMT methylation suspected to modulate treatment resistance in different tumor types.

P2Y1 receptor-evoked glutamate exocytosis from astrocytes: control by tumor necrosis factor-alpha and prostaglandins.

ATP, released by both neurons and glia, is an important mediator of brain intercellular communication. We find that selective activation of purinergic P2Y1 receptors (P2Y1R) in cultured astrocytes triggers glutamate release. By total internal fluorescence reflection imaging of fluorescence-labeled glutamatergic vesicles, we document that such release occurs by regulated exocytosis. The stimulus-secretion coupling mechanism involves Ca2+ release from internal stores and is controlled by additional transductive events mediated by tumor necrosis factor-alpha (TNFalpha) and prostaglandins (PG). P2Y1R activation induces release of both TNFalpha and PGE2 and blocking either one significantly reduces glutamate release. Accordingly, astrocytes from TNFalpha-deficient (TNF(-/-)) or TNF type 1 receptor-deficient (TNFR1(-/-)) mice display altered P2Y1R-dependent Ca2+ signaling and deficient glutamate release. In mixed hippocampal cultures, the P2Y1R-evoked process occurs in astrocytes but not in neurons or microglia. P2Y1R stimulation induces Ca2+ -dependent glutamate release also from acute hippocampal slices. The process in situ displays characteristics resembling those in cultured astrocytes and is distinctly different from synaptic glutamate release evoked by high K+ stimulation as follows: (a) it is sensitive to cyclooxygenase inhibitors; (b) it is deficient in preparations from TNF(-/-) and TNFR1(-/-) mice; and (c) it is inhibited by the exocytosis blocker bafilomycin A1 with a different time course. No glutamate release is evoked by P2Y1R-dependent stimulation of hippocampal synaptosomes. Taken together, our data identify the coupling of purinergic P2Y1R to glutamate exocytosis and its peculiar TNFalpha- and PG-dependent control, and we strongly suggest that this cascade operates selectively in astrocytes. The identified pathway may play physiological roles in glial-glial and glial-neuronal communication.

Cutaneous benign mixed tumor (chondroid syringoma) of the eyelid: clinical presentation and management.

PURPOSE: To describe the clinical presentation of cutaneous benign mixed tumor of the eyelid and its management options. METHODS: Periocular cases of cutaneous benign mixed tumor were gathered from members of an oculoplastics specialty Internet discussion group. A total of 9 patients are described in this retrospective, interventional case series. The clinical presentation, histopathology, and management of these lesions is reviewed. RESULTS: Patients were typically asymptomatic, presenting with a slowly enlarging, nontender nodule of 2 to 8 years' duration. The lesions ranged from 4 mm to 17 mm in greatest dimension. Four of the lesions were on the eyelid margin, three in the sub-brow area of the upper eyelid, and two in the central lids. All six cases not involving the brow were fixed to the tarsus; one brow lesion was believed to be adherent to the skin. None of the lesions was associated with significant changes of the overlying epidermis, although one lesion showed overlying pigmentation. All patients underwent excisional biopsy for diagnostic or cosmetic reasons. On histopathologic examination, the tumors were biphasic, with an epithelial component exhibiting apocrine or hair follicle differentiation and a myxoid, adipocytic, chondroid, and/or fibrous stroma. The pathologic diagnoses were all consistent with cutaneous benign mixed tumor (chondroid syringoma, pleomorphic adenoma). Follow-up ranged from 2 weeks to 12 months, although several patients failed to keep scheduled follow-up appointments. No clinical recurrences were identified. CONCLUSIONS: Cutaneous benign mixed tumor may occur in the eyelid, and, although uncommon, should be included in the differential diagnosis of firm, nodular eyelid tumors. The histopathologic features are similar to those seen in this tumor type arising in other areas of the body. Preoperative consideration of this diagnostic possibility may allow the surgeon to plan for complete excision, thereby reducing the possibility of recurrence or malignant transformation.

In vivo effects of a recombinant vaccinia virus expressing a mouse mammary tumor virus superantigen.

Early after infection, the mouse mammary tumor virus (MMTV) expresses a superantigen (SAg) at the surface of B lymphocytes. Interaction with the T-cell receptor Vbeta domain induces a polyclonal proliferative response of the SAg-reactive T cells. Stimulated T cells become anergic and are deleted from the T-cell repertoire. We have used a recombinant vaccinia virus encoding the MMTV(GR) SAg to dissect the effects of the retroviral SAg during an unrelated viral infection. Subcutaneous infection with this recombinant vaccinia virus induces a very rapid increase of Vbeta14 T cells in the draining lymph node. This stimulation does not require a large Plumber of infectious particles and is not strictly dependent on the expression of the major histocompatibility complex class II I-E molecule, as it is required after MMTV(GR) infection. In contrast to MMTV infection during which B cells are infected, we do not observe any clonal deletion of the reactive T cells following the initial stimulation phase. Our data show that contrary to the case with MMTV, macrophages but not B cells are the targets of infection by vaccinia virus in the lymph node, indicating the ability of these cells to present a retroviral SAg. The altered SAg expression in a different target cell observed during recombinant vaccinia virus infection therefore results in significant changes in the SAg response.