Phenotypic characterization and functional analysis of human tumor immune infiltration after mechanical and enzymatic disaggregation

Multi-parametric flow cytometry analysis is a reliable method for phenotypic and functional characterization of tumor infiltrating immune cells (TIIC). The isolation of infiltrating leukocytes from solid tumors can be achieved through various methods which can be both enzymatic and mechanical; however, these methods may alter cell biology. The aim of this study was to compare the effects of three tissue disaggregation techniques on TIIC biology in breast, kidney and lung tumor specimens. We therefore compared two enzymatic treatments using either collagenase type IA alone or in combination with collagenase type IV and DNase I type II, and one mechanical system (Medimachine?). We evaluated the impact of treatments on cell viability, surface marker integrity and proliferative capacity. We show that cell viability was not significantly altered by treatments. However, enzymatic treatments decreased cell proliferation; specifically collagenases and DNase provoked a significant decrease in detection of surface markers such as CD4, CD8, CD45RA and CD14, indicating that results of phenotypic studies employing these techniques could be affected. In conclusion, mechanical tissue disaggregation by Medimachine? appears to be optimal to maintain phenotypic and functional TIIC features.     

Prefrontal cortex is involved in internal decision of forthcoming saccades

Deciding where to look is mandatory to explore the visual world. To study the neural correlates subserving the cognitive phase of self-initiated eye movements in humans, we tested 12 healthy participants, using event-related functional MRI. Changes in the frontal-cortical activity preceding voluntary saccades were studied when the participants freely decided the direction of a forthcoming saccade, compared with a condition in which they had only to prepare an externally cued saccade. Self-initiation of saccades, before their execution, was specifically associated with frontal-lobe activation in the dorsolateral prefrontal cortex, and in the right presupplementary eye field and frontal eye fields, suggesting the roles of these areas in the decision process of where to look when facing two possible visual targets.     

Topology of protease activities reflects atherothrombotic plaque complexity

The pathological remodeling of the arterial wall in atherosclerosis involves protease activities, which play a major role in complications, through plaque rupture. Here, we investigated the release of active proteases by human carotid plaques in relation to (1) the degree of lesion complexity and (2) their compartmentalization between cap, core and media. Eighty human carotid endarterectomy specimens were dissected into culprit stenosing (CPs) and adjacent non-complicated/non-stenosing plaques (NPs). Thirty-five additional CPs were microdissected into cap, core and media. All specimens were compared to control non-atherosclerotic endarteries for the release of components of the plasminogen/plasmin system and matrix metalloproteinases (MMPs). Results show a greater release of the plasminogen activators (PAs), plasmin and active MMPs by CPs compared to NPs, whereas healthy arteries released even lower levels. Furthermore, we highlight a functional interaction between these proteases in human atherosclerotic tissues and more importantly, we demonstrate that the core constitutes the main source of protease activities within CPs. Together, these results suggest that CPs generate plasmin, mainly in the core, which could in turn participate in MMP activation and the onset of complications.     

Chemotherapy as initial treatment of locally advanced unresectable pancreatic cancer: a valid option?

INTRODUCTION: Radio-chemotherapy is the standard treatment for locally advanced unresectable pancreatic cancer (LAPC). Chemotherapy has been shown to be effective in the treatment of metastatic disease and we therefore evaluated its use as a first-line treatment for LAPC. PATIENTS AND METHODS: We carried out a retrospective analysis of all consecutive patients treated for LAPC (N=33) between July 1997 and April 2005, analysing the results of first-line chemotherapy (CT group) and radio-chemotherapy (RCT group) in this setting. RESULTS: The first-line treatment was RCT in six patients (18.3%) and CT in 26 patients (78.8%). Secondary treatment was administered to nine patients of CT group with well-controlled disease: "closure" radio-chemotherapy for seven patients (26.9%) and secondary resection for three (12%). After a median follow-up of 27 months, 23 patients died (69.7%). Overall survival was 13.8 months [95% CI: 10.1-19.4] for the whole population, 9.5 months [95% CI: 4.6-] for the RCT and 18.0 months [95% CI: 12.4-25.5] for the CT. Overall survival for the CT patients undergoing secondary surgery or "consolidation" radio-chemotherapy was 28.8 months [95% CI: 13.8-]. CONCLUSION: First-line chemotherapy is a valid option for LAPC treatment, making it possible to identify the patients who may benefit from secondary resection or radio-chemotherapy.     

Tissue diffusion and retention of metalloproteinases in ascending aortic aneurysms and dissections

Histopathological alterations in human aneurysms and dissections of the thoracic ascending aorta include areas of mucoid degeneration within the medial layer, colocalized with areas of cell disappearance and disruption of extracellular matrix elastic and collagen fibers. We studied the presence of matrix metalloproteinases in relation to their capacity to diffuse through the tissue or to be retained in areas of mucoid degeneration in aneurysms and dissections of the ascending aorta. Ascending aortas from 9 controls, 33 patients with aneurysms, and 14 with acute dissections, all collected at surgery, were analyzed. The morphological aspect was similar whatever the etiology or phenotypic expression of the pathological aortas, involving areas of extracellular matrix breakdown and cell rarefaction associated with mucoid degeneration. Release of proMMP-2, constitutively expressed by smooth muscle cells, was not different between controls and aneurysmal aortas, whereas the aneurysmal aortas released more of the active form. Release of pro and active MMP-9 was also similar between controls and aneurysmal aortas. Immunohistochemical staining of MMP-2 and MMP-9 was weak in both control and pathological aortas. In contrast, released MMP-7 (matrilysin) and MMP-3 (stromelysin-1) could not be detected in conditioned media but were present in tissue extracts with no detectable quantitative difference between controls and pathological aortas. Immunohistochemical staining of MMP-7 and MMP-3 revealed their retention in areas of mucoid degeneration, and semiquantitative evaluation of immunostaining showed more MMP-7 in pathological aortas than in controls. In conclusion, areas of mucoid degeneration, the hallmark of aneurysms, and dissections of thoracic ascending aortas, whatever their etiology, are not inert and can retain specific proteases.     

Delayed but Complete Response following Oral Temozolomide Treatment in Melanoma Leptomeningeal Carcinomatosis

Isolated leptomeningeal recurrence of melanoma is rare, occurring in 2% of patients with central nervous system involvement secondary to melanoma. The optimal treatment of leptomeningeal carcinomatosis (LMC) in melanoma has not yet been determined and remains a major challenge. We report a melanoma patient who presented with isolated LMC in the form of a new-onset weakness of the lower limbs, paresthesia of the left hand and foot, lumbago and headache. A lumbar puncture and spinal MRI confirmed LMC. The patient was treated with temozolomide 75 mg/m(2)/day on a 4 weeks on/2 weeks off schedule. After an initial transient clinical deterioration, the patient showed a complete radiological response as well as a dramatic improvement in quality of life. The encouraging clinical response reported here suggests that dose-intensified temozolomide might have significant activity in the treatment of leptomeningeal dissemination of melanoma and may be a valid treatment option for patients who have not been previously exposed to this agent. Moreover, this treatment regimen is extremely well tolerated and obviates the need for repeated intrathecal administrations of chemotherapeutic agents, which are often not well tolerated by patients who have significant co-morbidities due to their disease. As illustrated in this case, response to temozolomide may occur in a delayed manner, highlighting the importance of following temozolomide treatment long enough before determining that it is inefficient in a given patient.