María I. Rodríguez García

The cell cycle inhibitor p21 controls T-cell proliferation and sex-linked lupus development.

Here we show that the cell-cycle regulator p21 is involved in immune system function. T lymphocytes from p21−/− mice exhibit significant proliferative advantage over wild-type cells following prolonged stimulation, but not after primary activation. Consistent with this, p21-deficient mice accumulate abnormal amounts of CD4+ memory cells, and develop loss of tolerance towards nuclear antigens. Similar to human lupus, female p21-deficient mice develop antibodies against dsDNA, lymphadenopathy, and glomerulonephritis, leading to decreased viability. These data demonstrate a specialized role for p21 in the control of T-cell proliferation, tolerance to nuclear antigens, and female-prone lupus. These findings could be the basis for new therapeutic approaches to lupus.

Leukocyte attraction through the CCR5 receptor controls progress from insulitis to diabetes in non-obese diabetic mice

Lymphocyte infiltration to pancreatic islets is associated to chemoattraction, as are other inflammatory autoimmune processes. We examined whether development of insulitis and diabetes dependson chemoattraction of lymphocytes via the CCR5 chemokine receptor. In non‐obese diabetic (NOD) mice, a substantial fraction of peripheral T cells and virtually all B cells expressed high CCR5 levels. CCR5 expression characterized the effector T cell phenotype, suggesting their potential involvement in disease development. In view of these findings and the CCL5 (RANTES, the CCR5 ligand) expression by pancreatic islets, we treated NOD mice with a neutralizing anti‐CCR5 antibody. This did not influence peri‐insulitis advancement, but inhibited β‐cell destruction and diabetes. These data demonstrate a role of CCR5‐dependent chemoattraction in insulitis progression to islet destruction, suggesting the potential value of therapeutic intervention by CCR5 targeting.

Autocrine Production of IFN-γ by Macrophages Controls Their Recruitment to Kidney and the Development of Glomerulonephritis in MRL/lpr Mice

Anti-DNA autoantibody production is a key factor in lupus erythematosus development; nonetheless, the link between glomerular anti-DNA autoantibody deposition and glomerulonephritis development is not understood. To study the inflammatory and destructive processes in kidney, we used IFN-γ+/− MRL/lpr mice which produce high anti-DNA Ab levels but are protected from kidney disease. The results showed that defective macrophage recruitment to IFN-γ+/− mouse kidney was not caused by decreased levels of monocyte chemoattractant protein-1, a chemokine that controls macrophage migration to MRL/lpr mouse kidney. To determine which IFN-γ-producing cell type orchestrates the inflammation pathway in kidney, we transferred IFN-γ+/+ monocyte/macrophages or T cells to IFN-γ−/− mice, which do not develop anti-DNA autoantibodies. The data demonstrate that IFN-γ production by infiltrating macrophages, and not by T cells, is responsible for adhesion molecule up-regulation, macrophage accumulation, and inflammation in kidney, even in the absence of autoantibody deposits. Therefore, in addition to monocyte chemoattractant protein-1, macrophage-produced IFN-γ controls macrophage migration to kidney; the degree of IFN-γ production by macrophages also regulates glomerulonephritis development. Our findings establish the level of IFN-γ secretion by macrophages as a link between anti-DNA autoantibody deposition and glomerulonephritis development, outline the pathway of the inflammatory process, and suggest potential treatment for disease even after autoantibody development.

p21CIP1/WAF1 Controls Proliferation of Activated/Memory T Cells and Affects Homeostasis and Memory T Cell Responses

Development of autoantibodies and lupus-like autoimmunity by 129/Sv × C57BL/6 p21−/− mice has established that cell cycle deregulation is one the defective pathways leading to break of tolerance. Memory T cell accumulation is thought to be related to tolerance loss in murine lupus models. We studied T cell memory responses in C57BL/6 p21−/− mice that develop lupus-like disease manifestations. p21 did not affect primary proliferation of naive T cells, and was required for cycling control, but not for apoptosis of activated/memory T cells. When we induced apoptosis by secondary TCR challenge, surviving memory T cells depended on p21 for proliferation control. Under conditions of secondary T cell stimulation that did not cause apoptosis, p21 was also needed for regulation of activated/memory T cell expansion. The requirement for p21 in the control of T cell proliferation of activated/memory T cells suggests that in addition to apoptosis, cycling regulation by p21 constitutes a new pathway for T cell homeostasis. Concurring with this view, we found accumulation in p21−/− mice of memory CD4+ T cells that showed increased proliferative potential after TCR stimulation. Furthermore, OVA immunization of p21−/− mice generated hyperresponsive OVA-specific T cells. Overall, the data show that p21 controls the proliferation of only activated/memory T cells, and suggest that p21 forms part of the memory T cell homeostasis mechanism, contributing to maintenance of tolerance.

Regulation of macrophage activation and septic shock susceptibility via p21(WAF1/CIP1)

p21 is a cell‐cycle inhibitor that is also known to suppress autoimmunity. Here, we provide evidence of a novel role for p21 as an inhibitor of macrophage activation. LPS stimulation of p21‐deficient peritoneal macrophages induced increased activation compared with controls, with elevated production of proinflammatory mediators such as TNF‐α and IL‐1β. The enhanced activity of LPS‐stimulated p21‐deficient macrophages correlated with increased activity of the transcription factor NF‐κB. LPS stimulation of p21‐deficient macrophages led to increased IκBα kinase activity, and increased IκBα phosphorylation and degradation, resulting in elevated NF‐κB activity. The effect of p21 in macrophage activation was independent of its cell‐cycle inhibitory role. p21−/− mice showed greater sensitivity to LPS‐induced septic shock than did WT mice, indicating that p21 contributes to maintenance of a balanced response to inflammatory stimuli and suggesting biological significance for the role of p21 in macrophage activation. Our findings project a role for p21 in the control of NF‐κB‐associated inflammation, and suggest that therapeutic modulation of p21 expression could be beneficial in inflammation‐associated diseases.

Comparison between different bone treatments on areal bone mineral density (aBMD) and bone microarchitectural texture as assessed by the trabecular bone score (TBS).

PURPOSE The objective of the study was to assess longitudinal effects of different osteoporosis treatments on TBS and aBMD at lumbar spine. METHOD We analyzed 390 patients (men: 72; women: 318; age>40 years; mean follow-up of 20 months and BMI<37 kg/m(2)). We stratified the cohort by treatments: Naive of treatment (Naive, n=67), Calcium and Vitamin D (CaVitD, n=87), Testosterone (Te, n=36), Alendronate (AL, n=88), Risedronate (Ri, n=39), Denosumab (Dmb, n=43) and Teriparatide (PTH, n=30). The follow-up changes from baseline were normalized at 24 months. RESULTS After 24 months, Naive group TBS decreased by 3.1% (p<0.05) whereas a non-significant increase was observed for spine aBMD (Δ=+0.5%). Compared to the Naive group, significant improvement (p<0.05) was observed in both TBS and aBMD for Te, AL, Ri, Dmb and PTH groups and in the CaVitD group for TBS. At the end of the follow-up, significant improvement have been observed for aBMD in Te (+4.4%), AL (+4.1%), Ri (+4.8), D (+8.8%) and PTH (+8.8%) groups. Significant improvement was observed only in the AL (+1.4%), Dmb (+2.8%) and PTH (+3.6%) groups for TBS. CONCLUSION As expected, TBS of Naive subjects decreased with age. As expected a TBS preservation has been observed under AL and Ri. Te and CaVitD effects on TBS were evaluated for the first time: a similar preservation effect has been observed. A significant TBS increase was observed under Denosumab and PTH. TBS could be a useful tool to monitor treatment effects.

Whole-Organ analysis of calcium behaviour in the developing pistil of olive (Olea europaea L.) as a tool for the determination of key events in sexual plant reproduction

BackgroundThe pistil is a place where multiple interactions between cells of different types, origin, and function occur. Ca2+ is one of the key signal molecules in plants and animals. Despite the numerous studies on Ca2+ signalling during pollen-pistil interactions, which constitute one of the main topics of plant physiology, studies on Ca2+ dynamics in the pistil during flower formation are scarce. The purpose of this study was to analyze the contents and in situ localization of Ca2+ at the whole-organ level in the pistil of olive during the whole course of flower development.ResultsThe obtained results showed significant changes in Ca2+ levels and distribution during olive pistil development. In the flower buds, the lowest levels of detectable Ca2+ were observed. As flower development proceeded, the Ca2+ amount in the pistil successively increased and reached the highest levels just after anther dehiscence. When the anthers and petals fell down a dramatic but not complete drop in calcium contents occurred in all pistil parts. In situ Ca2+ localization showed a gradual accumulation on the stigma, and further expansion toward the style and the ovary after anther dehiscence. At the post-anthesis phase, the Ca2+ signal on the stigmatic surface decreased, but in the ovary a specific accumulation of calcium was observed only in one of the four ovules. Ultrastructural localization confirmed the presence of Ca2+ in the intracellular matrix and in the exudate secreted by stigmatic papillae.ConclusionsThis is the first report to analyze calcium in the olive pistil during its development. According to our results in situ calcium localization by Fluo-3 AM injection is an effective tool to follow the pistil maturity degree and the spatial organization of calcium-dependent events of sexual reproduction occurring in developing pistil of angiosperms. The progressive increase of the Ca2+ pool during olive pistil development shown by us reflects the degree of pistil maturity. Ca2+ distribution at flower anthesis reflects the spatio-functional relationship of calcium with pollen-stigma interaction, progamic phase, fertilization and stigma senescence.

Pollen and pistil features involved in the reproductive biology of the olive (Olea europaea L.)

Resumen de la comunicacion presentada al 28o International Horticultural Congress celebrado del 22 al 27 de 2010 en Lisboa.-- Sm09.028.