Ana Torres

The Chemokine Receptor CCR7 Activates in Dendritic Cells Two Signaling Modules That Independently Regulate Chemotaxis and Migratory Speed

CCR7 is necessary to direct dendritic cells (DCs) to secondary lymphoid nodes and to elicit an adaptative immune response. Despite its importance, little is known about the molecular mechanisms used by CCR7 to direct DCs to lymph nodes. In addition to chemotaxis, CCR7 regulates the migratory speed of DCs. We investigated the intracellular pathways that regulate CCR7-dependent chemotaxis and migratory speed. We found that CCR7 induced a Gi-dependent activation of MAPK members ERK1/2, JNK, and p38, with ERK1/2 and p38 controlling JNK. MAPK members regulated chemotaxis, but not the migratory speed, of DCs. CCR7 induced activation of PI3K/Akt; however, these enzymes did not regulate either chemotaxis or the speed of DCs. CCR7 also induced activation of the GTPase Rho, the tyrosine kinase Pyk2, and inactivation of cofilin. Pyk2 activation was independent of Gi and Src and was dependent on Rho. Interference with Rho or Pyk2 inhibited cofilin inactivation and the migratory speed of DCs, but did not affect chemotaxis. Interference with Rho/Pyk2/cofilin inhibited DC migratory speed even in the absence of chemokines, suggesting that this module controls the speed of DCs and that CCR7, by activating its components, induces an increase in migratory speed. Therefore, CCR7 activates two independent signaling modules, one involving Gi and a hierarchy of MAPK family members and another involving Rho/Pyk2/cofilin, which control, respectively, chemotaxis and the migratory speed of DCs. The use of independent signaling modules to control chemotaxis and speed can contribute to regulate the chemotactic effects of CCR7.

Cilastatin Attenuates Cisplatin-Induced Proximal Tubular Cell Damage

A major area in cancer therapy is the search for protective strategies against cisplatin-induced nephrotoxicity. We investigated the protective effect of cilastatin on cisplatin-induced injury to renal proximal tubular cells. Cilastatin is a specific inhibitor of renal dehydrodipeptidase I (DHP-I), which prevents hydrolysis of imipenem and its accumulation in the proximal tubule. Primary cultures of proximal cells were treated with cisplatin (1–30 μM) in the presence or absence of cilastatin (200 μg/ml). Apoptosis and mitochondrial injury were assessed by different techniques. Cisplatin uptake and DNA binding were measured by inductively coupled plasma spectrometry. HeLa cells were used to control the effect of cilastatin on the tumoricidal activity of cisplatin. Cisplatin increased cell death, apoptotic-like morphology, caspase activation, and mitochondrial injury in proximal tubular cells in a dose- and time-dependent way. Concomitant treatment with cilastatin reduced cisplatin-induced changes. Cilastatin also reduced the DNA-bound platinum but did not modify cisplatin-dependent up-regulation of death receptors (Fas) or ligands (tumor necrosis factor α, Fas ligand). In contrast, cilastatin did not show any effects on cisplatin-treated HeLa cells. Renal DHP-I was virtually absent in HeLa cells. Cilastatin attenuates cisplatin-induced cell death in proximal tubular cells without reducing the cytotoxic activity of cisplatin in tumor cells. Our findings suggest that the affinity of cilastatin for renal dipeptidase makes this effect specific for proximal tubular cells and may be related to a reduction in intracellular drug accumulation. Therefore, cilastatin administration might represent a novel strategy in the prevention of cisplatin-induced acute renal injury.

The neuronal protein Kidins220 localizes in a raft compartment at the leading edge of motile immature dendritic cells

Kidins220, a protein predominantly expressed in neural tissues, is the first physiological substrate for protein kinase D (PKD). We show that Kidins220 is expressed in monocyte‐derived and in peripheral blood immature dendritic cells (im DC). Immature DC (im DC) migrate onto extracellular matrices changing cyclically from a highly polarized morphology (monopolar (MP) stage) to a morphologically symmetrical shape (bipolar (BP) stage). Kidins220 was localized on membrane protrusions at the leading edge or on both poles in MP and BP cells, respectively. CD43, CD44, ICAM‐3 and DC‐SIGN,and signaling molecules PKD, Arp2/3 were found at the leading edge in MP or on both edges in BP cells, showing an intriguing parallelism between morphology and localization of molecular components on the poles of the motile DC. F‐actin co‐localized and it was necessary for Kidins220 localization on the membrane in MP and BP cells. Kidins220 was also found in a raft compartment. Disruption of rafts with methyl‐β‐cyclodextrin induced rounding of the cells, inhibition of motility and lost of Kidins220 polarization. Our results describe for the first time the molecular components of the polesof motile im DC and indicate that a novel neuronal protein may be an important component among these molecules.

Automatic quantification of viability in epithelial cell cultures by texture analysis

Quantification of live cells in phase contrast microscopy images allows in vivo assessment of the viability of cultured cells. An automatic screening procedure seems advisable because of the large number of cells that must be counted to achieve reasonable accuracy. This paper presents a method that quantifies necrosis in cell cultures by texture analysis of microscope images.

Protective Effects of Cilastatin against Vancomycin-Induced Nephrotoxicity

Vancomycin is a very effective antibiotic for treatment of severe infections. However, its use in clinical practice is limited by nephrotoxicity. Cilastatin is a dehydropeptidase I inhibitor that acts on the brush border membrane of the proximal tubule to prevent accumulation of imipenem and toxicity. The aim of this study was to investigate the potential protective effect of cilastatin on vancomycin-induced apoptosis and toxicity in cultured renal proximal tubular epithelial cells (RPTECs). Porcine RPTECs were cultured in the presence of vancomycin with and without cilastatin. Vancomycin induced dose-dependent apoptosis in cultured RPTECs, with DNA fragmentation, cell detachment, and a significant decrease in mitochondrial activity. Cilastatin prevented apoptotic events and diminished the antiproliferative effect and severe morphological changes induced by vancomycin. Cilastatin also improved the long-term recovery and survival of RPTECs exposed to vancomycin and partially attenuated vancomycin uptake by RPTECs. On the other hand, cilastatin had no effects on vancomycin-induced necrosis or the bactericidal effect of the antibiotic. This study indicates that cilastatin protects against vancomycin-induced proximal tubule apoptosis and increases cell viability, without compromising the antimicrobial effect of vancomycin. The beneficial effect could be attributed, at least in part, to decreased accumulation of vancomycin in RPTECs.

Automatic detection of cellular necrosis in epithelial cell cultures

Automatic discrimination and quantification of alive and dead cells in phase contrast microscopy images allows in vivo analysis of the viability of cultured cells without staining. Unsupervised segmentation, based on texture analysis, classifies each image region into three groups: live cells, necrotic cells and background. The segmentation is based on three discriminant functions, built using a total of 12 parameters derived from the histogram and the co-occurrence matrix. These parameters were selected performing a discriminant analysis on a training set that included images from three different cultures. Once images are automatically segmented, the approximate number of live and dead cells is obtained by dividing each area by the average size of each cell type. The number and percentage of live and necrotic cells have been obtained for primary cellular cultures in intervals of 48 hr. during two weeks. The results have been compared with the figures given by an experienced human observer, showing a very good correlation (Pearsons coefficient 0.95, kappa 0.87). A reliable and easy-to-use tool has been developed. It provides quantitative results on phase contrast microscopy images of cell cultures, with preliminary results showing accuracy similar to that provided by an expert, allowing to count a higher number of fields.

Cilastatin protection against cyclosporin A-induced nephrotoxicity: clinical evidence.

ABSTRACT Background: Several studies have documented the nephroprotective effect of cilastatin co-administered with imipenem in subjects treated with cyclosporin A. However, no large clinical studies are available to confirm this observation. Here the quality of the evidence on cilastatin nephroprotection against cyclosporin-induced nephrotoxicity is evaluated. Methods: The results of all studies where cyclosporin was used alone or combined with imipenem/cilastatin (Tienam*) on the same clinical setting were systematically reviewed. Primary outcome was the reduction in serum creatinine concentration. Secondary outcome included development of acute renal failure. Medline was searched using three different retrieval systems (Pubmed, Silver Platter, Knowledge Finder) from January 1966 to February 2006. Attempts were made to contact authors of relevant studies to obtain additional data. Five clinical studies were found, including 125 patients under cyclosporin plus imipenem/cilastatin and 104 under cyclosporin alone. Results: Cyclosporin increased serum creatinine in all the studies. Average reduction of serum creatinine in cilastatin-treated versus untreated patients was ∆ = –0.53 mg/dL (95%CI: –0.90 to –0.17) (Z = 2.84, p = 0.004). Variability between studies was large (from ∆ = –0.21 to ∆ = –1.59 mg/dL) and heterogeneity pronounced (χ2 = 8.760, df = 4; p = 0.067). Meta-regression of serum creatinine reduction versus baseline serum creatinine explained 84% of this variability, by the variation in basal serum creatinine. When randomized and observational clinical studies were analyzed separately, conclusions were the same: serum creatinine in cilastatin treated patients was reduced by ∆ = –0.98 mg/dL (95%CI: –1.57 to –0.38) in randomized studies (Z = 3.213, p = 0.001) and ∆ = –0.32 mg/dL (95%CI: –0.63 to –0.01) in observational studies (Z = 2.013, p = 0.044). Odds Ratio for developing acute renal failure was 0.24 (95% CI: 0.11–0.53, p < 0.0001) on patients simultaneously treated with cyclosporin plus imipenem/cilastatin compared to patients treated with cyclosporin alone. Conclusions: Administration of cilastatin may reduce acute cyclosporin nephrotoxicity.

THETA STATE AND COLLAPSE OF OFF-LATTICE CHAINS IN TWO DIMENSIONS

We have performed a Monte Carlo study of dimensions for two dimensional linear chains of different lengths. These chains are composed of Gaussian units which interact through a 6‐12 Lennard‐Jones potential. From this study, the theta state for this model has been characterized. Scaling curves have been obtained and different universal exponents, such as the theta point exponent ν, νθ, and the cross‐over exponent Φt have been numerically evaluated. The results are compared with theoretical predictions and with the values corresponding to simulations in lattice models. The results for ν and νθ agree with the theory, but our best estimation for the cross‐over exponent is closer to the simple mean field estimation.