A Differential and Timed Contribution of Identified Hippocampal Synapses to Associative Learning in Mice.

Although it is generally assumed that the hippocampus is involved in associative learning, the specific contribution of the different synapses present in its intrinsic circuit or comprising its afferents and efferents is poorly defined. We studied here activity-dependent changes in synaptic strength of 9 hippocampal synapses (corresponding to the intrinsic hippocampal circuitry and to its main inputs and outputs) during the acquisition of a trace eyeblink conditioning in behaving mice. The timing and intensity of synaptic changes across the acquisition process was determined. The evolution of these timed changes in synaptic strength indicated that their functional organization did not coincide with their sequential distribution according to anatomical criteria and connectivity. Furthermore, we explored the functional relevance of the extrinsic and intrinsic afferents to CA3 and CA1 pyramidal neurons, and evaluated the distinct input patterns to the intrinsic hippocampal circuit. Results confirm that the acquisition of a classical eyeblink conditioning is a multisynaptic process in which the contribution of each synaptic contact is different in strength, and takes place at different moments across learning. Thus, the precise and timed activation of multiple hippocampal synaptic contacts during classical eyeblink conditioning evokes a specific, dynamic map of functional synaptic states in that circuit.

Expression of ERp5 and GRP78 on the membrane of chronic lymphocytic leukemia cells: association with soluble MICA shedding.

MICA is a ligand of the activating receptor NKG2D, expressed by NK and T cells. MICA expression is induced in cancer cells favoring their elimination by the immune system; however, many advanced tumors shed soluble MICA (sMICA), which impairs NKG2D-mediated cytotoxicity. ERp5 and GRP78 are endoplasmic reticulum-resident proteins that are translocated to the surface of epithelial tumor cells where they interact with MICA and are involved in sMICA shedding. In this study, we analyze the role of ERp5 and GRP78 in sMICA shedding in chronic lymphocytic leukemia (CLL). Immunofluorescence and flow cytometry analyses showed that ERp5 and GRP78 were significantly expressed on the surface of B cells and leukemia cells, but they were not expressed on T cells. The expression of ERp5 and GRP78 was significantly higher in leukemia cells than in B cells from controls. ERp5 and GRP78 co-localized with MICA on the surface of leukemia cells and the levels of expression of ERp5 and GRP78 correlated with the level of expression of membrane-bound MICA in CLL patients. Associated with higher expression of membrane-bound ERp5 and GRP78, serum sMICA levels were approximately threefold higher in patients than in controls. Elevated sMICA levels in CLL patients were associated with the down-modulation of NKG2D surface expression on CD8 T cells. Finally, pharmacological inhibition of B cell lines and stimulated leukemia cells showed that ERp5 activity is involved in sMICA shedding in CLL. In conclusion, these results uncover a molecular mechanism which regulates MICA protein shedding and immune evasion in CLL.

Patients with psoriatic arthritis may show differences in their clinical and genetic profiles depending on their age at psoriasis onset.

OBJECTIVES: The age of psoriasis onset has an important impact on the clinical expression and heritability of psoriasis. Psoriasis characteristics according to the age at disease onset have been extensively studied. However, the impact of the age of psoriasis onset on psoriatic arthritis (PsA) features has not been analysed in depth. The aim of the present paper is to analyse whether the age of psoriasis onset may have an impact on the clinical and genetic characteristics in a cohort of PsA patients. METHODS: The study included 110 PsA patients classified in accordance with the CASPAR criteria. Patients were divided into early (onset age <30 years) and late (onset age >30 years) onset psoriasis, and clinical features were studied in accordance to this stratification. Distribution of several genes within the MHC region were analysed in accordance with the prior stratification, and their frequencies compared to that of 110 healthy matched blood donors. RESULTS: Compared to patients with late-onset disease, PsA patients with early-onset psoriasis showed more frequently: a longer psoriasis-arthritis latency period (9.9±6 years vs. 3.8±4 years, p=0.0001), a positive family history of disease (60.3% vs. 20.5%, OR 6.1, 95% CI: 2.5-15.0, p=0.0001), severe psoriasis (PASI 8.2±4 vs. 3.6±2.2, p=0.0001), clinical enthesitis (37.7% vs. 22.4%, OR 2.09, 95% CI: 0.9-4.9, p=0.08), and oligoarthritis (47.5% vs. 28.6%, OR 2.26, 95% CI: 1.02-5.02, p=0.04). MICA-A9 was associated with susceptibility in both early-onset (60.7% vs. 30%, p=0.0002) and late-onset patients (59.2% vs. 30%, p=0.0008). However, HLA-Cw*0602 was significantly increased in patients with early-onset psoriasis (73.8% vs. 17%, p<0.0001), whereas the allele 384 of the microsatellite C1_4_4, located 34 kb telomeric to HLA-C locus, was increased only in late-onset cases (49% vs. 21%, p=0.001). CONCLUSIONS: Clinical and genetic features of PsA may differ depending on the age at psoriasis onset. This type of stratification should be considered in future genetic and epidemiological studies of PsA.

Rapid erasure of hippocampal memory following inhibition of dentate gyrus granule cells.

The hippocampus is critical for the acquisition and retrieval of episodic and contextual memories. Lesions of the dentate gyrus, a principal input of the hippocampus, block memory acquisition, but it remains unclear whether this region also plays a role in memory retrieval. Here we combine cell-type specific neural inhibition with electrophysiological measurements of learning-associated plasticity in behaving mice to demonstrate that dentate gyrus granule cells are not required for memory retrieval, but instead have an unexpected role in memory maintenance. Furthermore, we demonstrate the translational potential of our findings by showing that pharmacological activation of an endogenous inhibitory receptor expressed selectively in dentate gyrus granule cells can induce a rapid loss of hippocampal memory. These findings open a new avenue for the targeted erasure of episodic and contextual memories.

Pleiotropic Anti-Angiogenic and Anti-Oncogenic Activities of the Novel Mithralog Demycarosyl-3D-ß-D-Digitoxosyl-Mithramycin SK (EC-8042).

Demycarosyl-3D-ß-D-digitoxosyl-mithramycin SK (DIG-MSK) is a recently isolated analogue of mithramycin A (MTA) that showed differences with MTA in the DNA binding strength and selectivity. These differences correlated with a better therapeutic index and less toxicity in animal studies. Herein, we show that DIG-MSK displays a potent anti-tumor activity against different types of cancer cell lines, ovarian tumor cells being particularly sensitive to this drug. Of relevance, DIG-MSK exerts low toxicity on fibroblasts and peripheral blood mononuclear cells, this toxicity being significantly lower than that of MTA. In correlation with its antitumor activity, DIG-MSK strongly inhibited Sp1-mediated transcription and endogenous Sp1 mRNA expression, which correlated with the inhibition of the expression of key Sp1-regulated genes involved in tumorigenesis, including VEGFA, BCL2L1 (Bcl-XL), hTERT, BRCA2, MYC and SRC in several ovarian cells. Significantly, DIG-MSK was a stronger inhibitor of VEGFA expression than MTA. Accordingly, DIG-MSK also exhibited potent anti-angiogenic activity on microvascular endothelial cells. Likewise, it significantly inhibited the gene expression of VEGFR1, VEGFR2, FGFR, PDGFB and PDGFRA and, additionally, it induced the expression of the anti-angiogenic factors angiostatin and tunstatin. These effects correlated with a pro-apoptotic effect on proliferating microvascular endothelial cells and the inhibition of the formation of endothelial capillary structures. Overall, the pleiotropic activity of DIG-MSK in inhibiting key oncogenic and angiogenic pathways, together with its low toxicity profile, highlight the therapeutic potential of this new drug.

Lenalidomide induces immunomodulation in chronic lymphocytic leukemia and enhances antitumor immune responses mediated by NK and CD4 T cells.

Lenalidomide is an immunomodulatory drug with therapeutic activity in chronic lymphocytic leukemia (CLL). However, it has pleiotropic effects, and the mechanism of action responsible for its therapeutic activity has not been well defined yet. Herein, we show that lenalidomide treatment does not have an effect on the proliferation of leukemia cells, but it increases the proliferation of B cells from healthy donors. Lenalidomide did not exert a direct effect on the apoptosis of leukemia cells obtained from CLL patients, although it indirectly induced their apoptosis through the activation of nonmalignant immune cells. Thus, lenalidomide markedly increased the proliferation of NK and CD4 T cells. The effect of lenalidomide on NK cells was secondary to the induction of IL-2 production by CD4 T cells. Accordingly, depletion of T cells or blockade of IL-2 activity completely abrogated the proliferation of NK cells. Additionally, lenalidomide enhanced NK and NKT-like cell-mediated natural cytotoxicity against leukemia cells from CLL patients. Lenalidomide also upregulated CD20 expression on leukemia cells and, accordingly, it had a synergistic effect with rituximab on promoting antibody-dependent cell-mediated cytotoxicity against primary leukemia cells. Overall, these observations provide a support for combining lenalidomide with rituximab as a treatment in CLL.

The activity of a novel mithramycin analog is related to its binding to DNA, cellular accumulation, and inhibition of Sp1-driven gene transcription.

DIG-MSK (demycarosyl-3D-ß-D-digitoxosyl-mithramycin SK) is a recently isolated compound of the mithramycin family of antitumor antibiotics, which includes mithramycin A (MTA) and mithramycin SK (MSK). Here, we present evidence that the binding of DIG-MSK to DNA shares the general features of other mithramycins such as the preference for C/G-rich tracts, but there are some differences in the strength of binding and the DNA sequence preferentially recognized by DIG-MSK. We aimed at gaining further insights into the DIG-MSK mechanism of action by direct comparison with the effects of the parental MTA. Similar to MTA, MSK and DIG-MSK accumulated rapidly in A2780, IGROV1 and OVCAR3 human ovarian cancer cell lines, and DIG-MSK was a potent inhibitor of both basal and induced expression of an Sp1-driven luciferase vector. This inhibitory activity was confirmed for the endogenous Sp1 gene and a set of Sp-responsive genes, and compared to that of MTA and MSK. Furthermore, DIG-MSK was stronger than MTA as inhibitor of Sp3-driven transcription and endogenous Sp3 gene expression. Differences in the effects of MTA, MSK and DIG-MSK on gene expression may have a large influence on their biological activities.

Expansion of NK cells and reduction of NKG2D expression in chronic lymphocytic leukemia. Correlation with progressive disease.

The immune system may mediate anti-tumor responses in chronic lymphocytic leukemia (CLL) which may affect disease progression and survival. In this study, we analyzed the immune characteristics of 99 consecutive previously diagnosed CLL patients and 50 healthy controls. The distribution of lymphocyte subsets at diagnosis was retrospectively analyzed. Compared with controls, leukemia patients showed an expansion of NK and CD8 T cells at diagnosis. The relative number of CD8 T cells at diagnosis was associated with time to treatment, suggesting that CD8 T cells may modify disease progression. The distribution of lymphocyte subsets was analyzed again when patients were enrolled in this study. The median time since these patients were diagnosed was 277 weeks. Compared with diagnosis, the absolute number of CD8 T cells significantly decreased in these patients, reaching similar values to healthy controls; however NK cells kept significantly elevated overtime. Nevertheless, NK cells showed an impaired expression of NKG2D receptor and a defective cytotoxic activity. This down-regulation of NKG2D expression was further enhanced in patients with advanced and progressive disease. Additionally, membrane NKG2D levels significantly decreased on CD8 T cells, but a significant increase of NKG2D+CD4+ T cells was observed in CLL patients. The cytotoxic activity of NK cells was diminished in CLL patients; however the treatments with IL-2, IL-15, IL-21 and lenalidomide were able to restore their activity. The effect of IL-2 and IL-15 was associated with the increase of NKG2D expression on immune cells, but the effect of IL-21 and lenalidomide was not due to NKG2D up-regulation. The expansion of NK cells and the reversibility of NK cell defects provide new opportunities for the immunotherapeutic intervention in CLL.

Prognostic significance of CD8 and CD4 T cells in chronic lymphocytic leukemia.

The prognostic value of the number of T cells and NK cells at diagnosis in CLL was analyzed in a cohort of 256 patients with CLL diagnosed between 1997 and 2007. Patients with leukemia showed elevated NK cells and T cell populations and CD4/CD8 ratio was inverted in 39.7% cases. Prognostic significance of lymphocytes was analyzed as a ratio of relative number of T cells to the size of the malignant monoclonal B-cell pool (T/NK cells:Malignant monoclonal B-cells ratio). Patients showed higher relative number of CD4 (p = 0.03), CD8 (p = 0.02), and NK cells (p = 0.01) in early Rai stage of disease. The multivariate Cox analysis identified the relative number of CD8 (hazard ratio (HR) = 1.464; p = 0.006) and CD4 T cells (HR = 0.091; p < 0.01) as independent predictors for survival. Additionally, patients with relative CD8 count > 0.074 or CD4 count > 0.1 had higher 10-year overall survival than patients with CD8 count ≤0.074 or CD4 count ≤0.1 (p = 0.002). Higher CD8 count was associated with significantly higher median time of survival of patients (149.33 vs. 82.06 months). Finally, association of the good prognostic factor of leukemia cells (CD38⁻ with high relative CD8 count identified a group of patients with an indolent clinical course with an overall survival probability at 10 years of 95%.