Cisplatin induces changes in the matrix metalloproteinases and their inhibitors in the developing rat cerebellum.

The role of matrix metalloproteinases (MMP-3 and MMP-9), tissue inhibitor of MMP (TIMP-2), and GAP-43 (growth-associated-protein) in neocerebellar vermis lobules during postnatal histogenesis was studied after challenge with cisplatin (cisPt). CisPt is one of the most effective and widely used cytotoxic agents in the treatment of a variety of malignancies, in both children and adult patients. A single injection of cisPt to 10-day-old rats altered the spatiotemporal MMP/TIMP expression balance and provoked a decrease in GAP43 immunoreactivity. The imbalance appeared one day (PD11) after cisPt injection, producing disorder of cerebellum histogenesis processes in which MMPs might be involved, i.e. genesis of granule cells, Purkinje cell differentiation and synaptogenesis. Following the early injury, a simultaneous increase in MMP and TIMP expression in the ML was noticed at PD17, likely initiating recovery of Purkinje cell dendrite growth and remodelling processes. However, disturbances at the beginning of recovery phase had emerged, probably due to the down-regulation of GAP-43 after cisPt treatment. The data provide further support for the usefulness of cisPt as a tool for the study of morphological and functional changes in the CNS during postnatal development.

Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells.

Tumor-promoted constraints negatively affect cytotoxic T lymphocyte (CTL) trafficking to the tumor core and, as a result, inhibit tumor killing. The production of reactive nitrogen species (RNS) within the tumor microenvironment has been reported in mouse and human cancers. We describe a novel RNS-dependent posttranslational modification of chemokines that has a profound impact on leukocyte recruitment to mouse and human tumors. Intratumoral RNS production induces CCL2 chemokine nitration and hinders T cell infiltration, resulting in the trapping of tumor-specific T cells in the stroma that surrounds cancer cells. Preconditioning of the tumor microenvironment with novel drugs that inhibit CCL2 modification facilitates CTL invasion of the tumor, suggesting that these drugs may be effective in cancer immunotherapy. Our results unveil an unexpected mechanism of tumor evasion and introduce new avenues for cancer immunotherapy.

Autofluorescence properties of rat cerebellum cortex during postnatal development.

BACKGROUND AND OBJECTIVES: The multilayered structure of rat neocerebellum cortex (VI-VIII lobules of the vermis) during postnatal development undergoes rearrangements, which in turn are affected by treatment with the anti-tumoral drug cisplatin. The dependence of autofluorescence emission properties on the tissue structural and molecular features has been investigated. STUDY DESIGN/MATERIALS AND METHODS: Autofluorescence analysis was performed at defined time points of cerebellar histogenesis--11, 17, and 30 postnatal days- under normal conditions or after 5 microg/g body weight cisplatin treatment at 10 postnatal day. Autofluorescence signal was analyzed in vivo at the surface of intact lobules of cerebellum vermis by means of fiber optic spectrofluorometry, or on tissue sections by means of microspectrofluorometry and fluorescence imaging. RESULTS: In vivo spectroscopy showed changes of autofluorescence signal both during normal histogenesis and after cisplatin treatment. External granular layer (EGL) and molecular layer (ML), that is, the more external layers were found to be interested by structural alterations, and showed the greatest changes in signal amplitude, accounting for the in vivo results. Fitting analysis indicated that changes in spectral shape reflected an increase in oxidative damages induced by cisplatin treatment. CONCLUSIONS: The results confirm the relationship of the autofluorescence emission properties with histological and biochemical features of biological tissue.

Interaction between the cellular prion (PrPC) and the 2P domain K+ channel TREK-1 protein.

The cellular prion protein (PrP(C)) is a highly conserved protein throughout the evolution of mammals and therefore is thought to play important cellular functions. Despite decades of intensive researches, the physiological function of PrP(C) remains enigmatic. Differently, in particular pathological contexts, generally referred as transmissible spongiform encephalopathies, a conformational isoform of PrP(C), i.e., PrP(Sc), is considered the causative agent of these diseases. In this study, we investigated putative PrP(C) cellular functions through the identification of PrP(C) protein interactants. Using a bacterial two-hybrid approach, we identified a novel interaction between PrP(C) and a two-pore potassium channel protein, TREK-1. This interaction was further verified in transfected eukaryotic cells using co-immunoprecipitation and confocal microscopic analysis of the fluorescent transfected proteins. Importantly, in the cerebellar cortex, the endogenous PrP(C) and TREK-1 proteins exhibited co-localization signals in correspondence of the Purkinje cells. Furthermore, a deletion mapping study defined the carboxyl-terminal regions of the two proteins as the possible determinants of the PrP(C)-TREK-1 interaction. Our results indicated a novel PrP(C) interacting protein and suggested that this complex might be relevant in modulating a variety of electrophysiological-dependent cellular responses.

Proliferation and migration of granule cells in the developing rat cerebellum: cisplatin effects.

We evaluated the relationship among proliferation, death and migration of granule cells in lobules VI-VIII of vermis, in comparison with lobule III, during cerebellar development. To this aim, a single injection of cisplatin, i.e., a cytostatic agent that is known to induce death of proliferating granule cells, was given to 10-day-old rats. Histochemical markers of proliferating (PCNA immunoreaction) and apoptotic (TUNEL staining) cells were used; the variations of the external granular layer (EGL) thickness were evaluated in parallel. After PCNA and TUNEL reactions, evident changes of the whole EGL were found on PD11 (1 day after treatment), when a reduction of the thickness of this layer was found in treated rats, mainly in consequence of the high number of apoptotic cells in all the cerebellar lobules. On PD17 (7 days after treatment), a thick layer of proliferating cells was observed in lobules VI-VIII of treated rats, while the peculiar pattern of the normal development showed a thin EGL. At the same time, in treated rats, the number of apoptotic cells in EGL was low. In all developmental stages of treated rats, after GFAP immunoreaction, glial fibers appeared twisted, thickened, and with an irregular course; intensely labeled end-feet were present. The damage of radial glia suggests an alteration of migratory processes of granule cells, which is also evidenced by the decreased thickness of the premigratory zone of the EGL. Injured radial glia fibers were restricted to lobules VI-VIII and they persisted at PD30, leading to the presence of ectopic granule cells in the molecular layer, as we previously described.