Marcos Ramírez

Functional gap junctions facilitate melanoma antigen transfer and cross-presentation between human dendritic cells.

Previously, we found that human dendritic cells (hDCs) pulsed with a melanoma cell lysate (MCL) and stimulated with TNF-α (MCL/TNF) acquire a mature phenotype in vitro and are able to trigger tumor-specific immune responses when they are used in melanoma immunotherapy in patients. In this study, we describe that MCL/TNF induces gap junction (GJ)-mediated intercellular communications and promotes melanoma Ag transfer between ex vivo produced hDCs from melanoma patients. hDCs also exhibit increased expression of the GJ-related protein connexin 43, which contributes to GJ plaque formation after MCL/TNF stimulation. The addition of GJ inhibitors suppresses intercellular tumor Ag transfer between hDCs, thus reducing melanoma-specific T cell activation. In summary, we demonstrate that MCL/TNF-stimulated hDCs can establish functional GJ channels that participate in melanoma Ag transfer, facilitating Ag cross-presentation and an effective dendritic cell-mediated melanoma-specific T cell response. These results suggest that GJs formed between hDCs used in cancer vaccination protocols could be essentials for the establishment of a more efficient antitumor response.

Functional Gap Junctions Accumulate at the Immunological Synapse and Contribute to T Cell Activation

Gap junction (GJ) mediates intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models, we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was Ag specific and time dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43-specific inhibitors were used to prove that intercellular communication between T cells and dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca2+ responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the dendritic cell–T cell signaling machinery that regulates T cell activation.

Tumores gastrointestinales estromales (GIST): Experiencia del Servicio de Cirugía del Hospital Clínico de la Universidad de Chile entre 1999 y 2005

BACKGROUND Gastrointestinal stromal tumors (GIST) are the most common mesenchymatous tumors of the digestive tract. The pathological diagnosis is based on microscopy and immunohistochemistiy. AIM To review the experience of our surgical unit in patients with GIST MATERIAL AND METHODS: Review of medical records of 15 patients (aged 66+/-13 years, 11 women), with a pathological diagnosis of GIST, treated between 1999 and 2005. RESULTS The main presenting symptoms were melena in 40%, hematemesis in 20%, abdominal pain in 60% and anemia in 13%. In only one patient, the tumor appeared as an incidentaloma. All patients underwent upper gastrointestinal endoscopy A CAT scan was done in 87%, a barium swallow in 60% and a digestive endosonography in 20%. Thirteen tumors were located in the stomach and two in the small bowel. Mean tumor diameter was 5.3+/-1.7 cm. Surgical management was a tumor resection in 40%, a partial gastrectomy in 27%, a total gastrectomy in 20% and an intestinal excision in the rest. Mean hospital stay was 6.9+/-4.2 days. No postoperative complications were recorded. CONCLUSIONS The main clinical presentation of GIST in this retrospective series was an upper gastrointestinal bleeding. Surgical treatment was devoid of complications.

Melanocortin 1 Receptor-derived peptides are efficiently recognized by cytotoxic T lymphocytes from melanoma patients

BACKGROUND Melanocortin 1 Receptor (MC1R) is expressed in a majority of melanoma biopsies and cell lines. We previously demonstrated that three hydrophobic low-affinity HLA-A2-restricted MC1R-derived peptides: MC1R291-298, MC1R244-252 and MC1R283-291 can elicit cytotoxic T-lymphocytes (CTL) responses from normal donor peripheral blood lymphocytes (PBL). Moreover, peptide-specific CTL recognized a panel of MHC-matched melanomas, demonstrating that human melanoma cell lines naturally present MC1R epitopes. However, the natural presence of MC1R-specific T cells in melanoma patients tumour and blood remains unknown. METHODS The presence of anti-MC1R specific CD8(+) T cells was established in a population of melanoma-specific T cells derived from peripheral blood mononuclear cells (PBMC) and tumour-infiltrating lymphocytes (TIL) from HLA-A2(+) melanoma patients. RESULTS CTLs specific for the three MC1R-derived peptides that lysed allogeneic HLA-A2(+)MC1R(+) melanomas were elicited from PBMC, demonstrating the existence of an anti-MC1R T cell repertoire in melanoma patients. Moreover, TILs also recognized MC1R epitopes and HLA-A2(+) melanoma cell lines. Finally, HLA-A2/MC1R244-specific CD8(+) T cell clones derived from TILs and a subset of MC1R291 specific TILs were identified using HLA-A2/MC1R tetramers. CONCLUSION Our results demonstrate that MC1R-derived peptides are common immunogenic epitopes for melanoma-specific CTLs and TILs, and may thus be useful for the development of anti-melanoma immunotherapy.

Desafíos en el diagnóstico de enfermedad de Creutzfeldt-Jakob: Caso clínico

Creutzfeldt-Jakob disease has a higher incidence in Chile than in other countries. The postmortem pathological characterization of brain tissue is necessary to reach a definitive diagnosis. We report a 73 years old man with a history compatible with of a rapidly progressive dementia, in which the first electroencephalographic study showed a pattern consistent with non-convulsive status epilepticus. Besides discarding this diagnosis, it was necessary to rule out other causes of rapidly progressive dementia such as Hashimoto encephalopathy. Finally, the sustained clinical deterioration with no response to anticonvulsants and corticosteroids, the imaging studies, a serial electroencephalographic monitoring study and the detection of 14-3-3 protein in cerebrospinal fluid were the keys to achieve the diagnosis of the disease.

Correlation of Ca15-3 elevation with number and site of metastasis in breast cancer patients

875 Background: The tumor marker ca153 has been widely used in the follow up of breast cancer patients providing a good method for early detection of relapse. The objective of our study is to correlate, if possible, the value of the marker with number and site of metastases, in order to reduce the imaging studies and cost of treatment. METHODS We reviewed the clinical laboratory ca15-3 test results from 2000 to April of 2003, and for those that were elevated above 30 u., the clinical record was obtained. Data of clinical stage, levels of ca153, number and site of metastases were recorded and analyzed with stata 7.0. RESULTS We included 503 patients that had at least one elevation. Of those, 307 ( 61%) presented metastasis or loco-regional recurrence and we classified them as group 1. The rest, 196 patients never developed metastasis or recurrence constituted group 2. Mean age of all the patients was 49.7 years (22-86). Maximum level for group 1 had a mean of 435.7 (30.5-4327) and for group 2 57.9 u. (30-624) with a significant difference (p=0.0000). A gradual increase was also observed in the means according to clinical stage: Stage 1: 82.6; Stage 2 491.4; stage 3: 595.8; 4: 480.5 and non classified 352.7 . The same gradual elevation was observed with positive lymph nodes: No positive nodes mean of 167.9, one to three positive nodes 270.2, four to nine 389.2, 10 and more 491.5 The number of metastasis was also important one metastasis had a mean of 255.6, two sites 412.6, three 635.6, four 759.9 and five 2061 ( p=0.000). The site of recurrence had no significant difference between soft tissues, means under 100 p=0.20. For only lung metastasis mean level was of 398.7, bones 307.8, brain 264.6, liver 309. 3. (p= 0.001) with higher values for bone and liver. CONCLUSIONS We observed that the most significant parameters were clinical stage, nodal status, number of affected sites and higher increase with bone and liver metastasis. No significant financial relationships to disclose.