Jose A. Garcia-Sanz

Translation control: bridging the gap between genomics and proteomics?

mRNA profiling enables the expression levels of thousands of transcripts in a cell to be monitored simultaneously. Nevertheless, analyses in yeast and mammalian cells have demonstrated that mRNA levels alone are unreliable indicators of the corresponding protein abundances. This discrepancy between mRNA and protein levels argues for the relevance of additional control mechanisms besides transcription. As translational control is a major mechanism regulating gene expression, the use of translated mRNA in profiling experiments might depict the proteome more closely than does the use of total mRNA. This would combine the technical potential of genomics with the physiological relevance of proteomics.

T Cell Activation In Vivo Targets Diacylglycerol Kinase α to the Membrane: A Novel Mechanism for Ras Attenuation

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid, leading to decreased and increased levels, respectively, of these two lipid messengers that play a central role in T cell activation. Nine DGK isoforms, grouped into five subtypes, are found in higher organisms; all contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. In this study, we have researched in vivo the regulation of DGKα, using a transgenic mouse model in which injection of an antigenic peptide activates the majority of peripheral T cells. We demonstrate that DGKα, highly expressed in resting T lymphocytes, is subject to complex control at the mRNA and protein levels during in vivo T cell activation. Subcellular fractionation of T lymphocytes shortly after in vivo engagement of the TCR shows rapid translocation of cytosolic DGKα to the membrane fraction. At early time points, DGKα translocation to the membrane correlates with rapid translocation of Ras guanyl nucleotide-releasing protein (RasGRP), a nucleotide exchange activator for Ras that associates to the membrane through a diacylglycerol-binding domain. To demonstrate a causal relationship between DGKα activity and RasGRP relocation to the membrane, we determined RasGRP translocation kinetics in a T cell line transiently transfected with constitutive active and dominant-negative DGKα mutants. We show that membrane localization of DGKα is associated with a negative regulatory signal for Ras activation by reversing RasGRP translocation. This study is the first demonstration of in vivo regulation of DGKα, and provides new insight into the functional role of a member of this family of lipid kinases in the regulation of the immune response.

Expression of proinflammatory cytokines in osteoarthritis of the temporomandibular joint.

OBJECTIVE This study reports the expression of proinflammatory cytokines in temporomandibular joint (TMJ) of patients affected with temporomandibular osteoarthritis (OA). DESIGN In twelve OA of the TMJ (OA-TMJ) affected patients and in six healthy volunteer subjects studied as control, the expression of IL1beta (interleukin-1beta), IL2, IL4, IL5, IL6, IL10, IL12p35, IL12p40, IL17, IFNgamma (interferon-gamma), TNFalpha (tumor necrosis factor-alpha), and TNFbeta mRNAs was evaluated. Using quantitative real-time RT-PCR technique, the cytokine levels, reported as Ct (cycle threshold), DeltaCt (Ct cytokine-Ct 18S rRNA) and RQ (relative quantification), in patient and control groups were compared. RESULTS Expression of IL1beta, IL2, IL12p35, IL12p40, IL17, TNFalpha, TNFbeta, and IFNgamma mRNAs was significantly higher in patients as compared with controls. In particular, IL12 was the predominant cytokine expressed in patients (IL12p35 RQ=30.2 and IL12p40 RQ=29.0). Conversely, IL10 mRNA levels were higher in controls (RQ=1.8). CONCLUSIONS These data suggest that not only IL1beta, IFNgamma, and TNFalpha but also IL10, IL12, and IL17 are involved in the OA-TMJ pathogenesis. Furthermore, an inflammatory response characterised by the predominant expression of IL12 mRNA and down-regulated expression of IL10 mRNA is associated with the degenerative changes observed in OA-TMJ.

Differential cytokine expression by human dendritic cells in response to different Porphyromonas gingivalis capsular serotypes.

AIM Capsular polysaccharides play an important role in the virulence of Gram-positive and Gram-negative bacteria. In Porphyromonas gingivalis, six serotypes have been described based on capsular antigenicity and its pathogenicity has been correlated both in vitro and in animal models. This study aimed to investigate the differential response of human dendritic cells (DCs) when stimulated with different P. gingivalis capsular serotypes. MATERIALS AND METHODS Using different multiplicity of infection (MOI) of the encapsulated strains K1-K6 and the non-encapsulated K(-) strain of P. gingivalis, the mRNA expression levels for interleukin (IL)-1beta, IL-2, IL-5, IL-6, IL-10, IL-12, IL-13, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, and TNF-beta in stimulated DCs were quantified by real-time reverse transcription-polymerase chain reaction. RESULTS All P. gingivalis capsular serotypes induced a T-helper type 1 (Th1) pattern of cytokine expression. K1- and K2-stimulated DCs expressed higher levels of IL-1beta, IL-6, IL-12p35, IL-12p40, and IFN-gamma and at lower MOI than DCs stimulated with the other strains. CONCLUSIONS These results demonstrate a differential potential of P. gingivalis capsular serotypes to induce DC responses and a higher capacity of strains K1 W83 and K2 HG184 than other K serotypes to trigger cytokine expression.

Chemokine Receptor-Specific Antibodies in Cancer Immunotherapy: Achievements and Challenges

The 1990s brought a burst of information regarding the structure, expression pattern, and role in leukocyte migration and adhesion of chemokines and their receptors. At that time, the FDA approved the first therapeutic antibodies for cancer treatment. A few years later, it was reported that the chemokine receptors CXCR4 and CCR7 were involved on directing metastases to liver, lung, bone marrow, or lymph nodes, and the over-expression of CCR4, CCR6, and CCR9 by certain tumors. The possibility of inhibiting the interaction of chemokine receptors present on the surface of tumor cells with their ligands emerged as a new therapeutic approach. Therefore, many research groups and companies began to develop small molecule antagonists and specific antibodies, aiming to neutralize signaling from these receptors. Despite great expectations, so far, only one anti-chemokine receptor antibody has been approved for its clinical use, mogamulizumab, an anti-CCR4 antibody, granted in Japan to treat refractory adult T-cell leukemia and lymphoma. Here, we review the main achievements obtained with anti-chemokine receptor antibodies for cancer immunotherapy, including discovery and clinical studies, proposed mechanisms of action, and therapeutic applications.

CCL2 inhibits the apoptosis program induced by growth factor deprivation, rescuing functional T cells.

The precise mechanisms involved in the switch between the clonal expansion and contraction phases of a CD8+ T cell response remain to be fully elucidated. One of the mechanisms implicated in the contraction phase is cytokine deprivation, which triggers apoptosis in these cells. CCR2 chemokine receptor is up-regulated following IL-2 deprivation, and its ligand CCL2 plays an essential role preventing apoptosis induced by IL-2 withdrawal not only in CTLL2 cells, but also in mouse Ag-activated primary CD8+ T cells because it rescued functional CD8+ T cells from deprivation induced apoptosis, promoting proliferation in response to subsequent addition of IL-2 or to secondary antigenic challenges. Thus, up-regulation of the CCR2 upon growth factor withdrawal together with the protective effects of CCL2, represent a double-edged survival strategy, protecting cells from apoptosis and enabling them to migrate toward sites where Ag and/or growth factors are available.

Distinct human T-lymphocyte responses triggered by Porphyromonas gingivalis capsular serotypes

AIM Porphyromonas gingivalis can synthesize an extracellular capsule and different serotypes have been described based on capsular antigenicity. On dendritic cells (DCs), the type of capsule present plays a role on the strength of the developed immune response. This study aimed to investigate the T-lymphocyte responses when stimulated with autologous mature DCs exposed to different P. gingivalis K-serotypes. MATERIALS AND METHODS Naïve CD4(+) T-lymphocytes were obtained from healthy subjects and stimulated with autologous DCs primed with increasing multiplicity of infections of the different P. gingivalis K-serotypes. The Th1, Th2, Th17 and T-regulatory cytokines and transcription factor levels were quantified. RESULTS Distinct types of response were detected when T-lymphocytes were stimulated by DCs primed with the different P. gingivalis K-serotypes. T-lymphocytes stimulated by K1 or K2-primed DCs elicited higher levels of Th1 and Th17-associated cytokines, T-bet and RORC2 than T-lymphocytes stimulated with DCs primed with the other serotypes. Conversely, the serotypes K3-K5 induced higher levels of Th2-associated cytokines and GATA-3 than the others. CONCLUSIONS These results demonstrate that DCs primed with the different P. gingivalis K-serotypes elicited distinct T-cell responses. Strains K1 (W83) and K2 (HG184) induced a Th1/Th17 pattern of immune response and K3 (A7A1-28), K4 (ATCC(®49417™) ), and K5 (HG1690) a Th2 response.

Live Imaging of Mouse Endogenous Neural Progenitors Migrating in Response to an Induced Tumor

Adult neurogenesis is restricted to specific brain regions. Although involved in the continuous supply of interneurons for the olfactory function, the role of neural precursors in brain damage-repair remains an open question. Aiming to in vivo identify endogenous neural precursor cells migrating towards a brain damage site, the monoclonal antibody Nilo2 recognizing cell surface antigens on neuroblasts, was coupled to magnetic glyconanoparticles (mGNPs). The Nilo2-mGNP complexes allowed, by magnetic resonance imaging in living animals, the in vivo identification of endogenous neural precursors at their niche, as well as their migration to a lesion site (induced brain tumor), which was fast (within hours) and orderly. Interestingly, the rapid migration of neuroblasts towards a damage site is a characteristic that might be exploited to precisely localize early damage events in neurodegenerative diseases. In addition, it might facilitate the study of regenerative mechanisms through the activation of endogenous neural cell precursors. A similar approach, combining magnetic glyconanoparticles linked to appropriate antibodies could be applied to flag other small cell subpopulations within the organism, track their migration, localize stem cell niches, cancer stem cells or even track metastatic cells.

Activation of RANKL‐induced osteoclasts and memory T lymphocytes by Porphyromonas gingivalis is serotype dependant

AIM Destructive periodontitis is associated with a Th1-Th17 immune response and activation of RANKL-induced osteoclasts. In addition, Porphyromonas gingivalis K1 and K2 serotypes induce a strong Th1-Th17 response. This study aimed to investigate whether these P. gingivalis serotypes induce higher osteoclasts activation, by increased Th17-associated RANKL production, and an antigen-specific memory T-lymphocyte response. MATERIAL AND METHODS The RANKL production and TRAP(+) osteoclast induction were quantified on naïve T lymphocytes stimulated with dendritic cells primed with the P. gingivalis serotypes. The T-bet, GATA-3, RORC2 and Foxp3 expression was correlated with RANKL production. The frequency of proliferating memory T lymphocytes in response to P. gingivalis serotypes was determined in both periodontitis and healthy subjects. RESULTS T lymphocytes stimulated by K1 or K2-primed dendritic cells elicited higher levels of RANKL and TRAP(+) osteoclasts than cells stimulated with the other serotypes. RANKL positively correlated with RORC2. Whereas periodontitis patients had a higher frequency of memory T lymphocytes responding to K1 or K2, healthy subjects had a higher frequency of memory T lymphocytes responding to K4 or K(-) . CONCLUSIONS P. gingivalis serotypes K1 and K2, but not others, are associated with an increased production of the osteoclastogenesis-related factor RANKL. This important information suggests that these serotypes could elicit a greater bone resorption in vivo and have a role in the periodontitis pathogenesis.

An adaptation of the LMS method to determine expression variations in profiling data

One of the major issues in expression profiling analysis still is to outline proper thresholds to determine differential expression, while avoiding false positives. The problem being that the variance is inversely proportional to the log of signal intensities. Aiming to solve this issue, we describe a model, expression variation (EV), based on the LMS method, which allows data normalization and to construct confidence bands of gene expression, fitting cubic spline curves to the Box–Cox transformation. The confidence bands, fitted to the actual variance of the data, include the genes devoid of significant variation, and allow, based on the confidence bandwidth, to calculate EVs. Each outlier is positioned according to the dispersion space (DS) and a P-value is statistically calculated to determine EV. This model results in variance stabilization. Using two Affymetrix-generated datasets, the sets of differentially expressed genes selected using EV and other classical methods were compared. The analysis suggests that EV is more robust on variance stabilization and on selecting differential expression from both rare and strongly expressed genes.