The timing of immunomodulation induced by mesenchymal stromal cells determines the outcome of the graft in experimental renal allotransplantation

The immunomodulatory characteristics of mesenchymal stromal cells (MSCs) may lead to multifaceted strategies in rejection of organ transplantation. This study was designed to investigate, first, the effect of the donortype MSCs from Wistar rats on the immune system of immunocompetent Lewis rats and, second, the rejection responses in a renal transplantation model of Wistar to Lewis. In the first experimental model, MSCs from the bone marrow induced a systemic immune response in the immunocompetent Lewis rats, characterized by two different phases. In the initial phase (days 1-3 after MSCs infusion), the main findings were a decrease in the percentage of the main peripheral blood (PB) lymphocyte subpopulations [T cells, B cells, and natural killer (NK) cells], an increase in the FOXP3 MFI in Tregs, and an elevated concentration of circulating proinflammatory cytokines (IL-1 beta and TNF-alpha). In the late phase (days 4-6), the percentage of T cells, B cells, and NK cells returned to baseline levels; the concentration of circulating IL-1b and TNF-a decreased; and the level of anti-inflammatory cytokines (IL-10 and IL-4) increased with respect to the initial phase. In the allogeneic kidney transplantation model, rats were randomized into four groups: nontreated, cyclosporine oral administration, and two groups of rats treated with two different schedules of MSC infusion: 4 days (MSCs-4) and 7 days (MSCs-7) before kidney transplantation and in both a further infusion at the day of transplantation. Both MSC treatments decreased the percentage of T, B, and NK cells in PB. Creatinine levels, survival, and histological parameters were better in MSCs-7 than in MSCs-4. We can conclude that MSCs, by themselves, produce changes in the immune system; they do not need a pathological condition to produce immunomodulatory responses. In the renal allograft model, the optimal time schedule for MSC infusion before grafting was 7 days to prevent acute rejection

JAK3-STAT pathway blocking benefits in experimental lupus nephritis

Es va publicar un erratum de l'article a: Arthritis Research & Therapy, 2016, vol. 18 , num. 1, p. 152Background: Lupus nephritis (LN) is a complex chronic autoimmune disease of unknown etiology characterized by loss of tolerance against several self-antigens. Cytokines are known to be central players in LN pathogenesis. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is one important pathway that mediates signal transduction of several cytokines. In this study, we examined the pathogenic role of this pathway and how CP-690,550 treatment influences LN outcome. Methods: Six-month-old NZB/NZWF1 mice were divided into two different treatment groups: (1) control animals given vehicle treatment, cyclophosphamide, and mycophenolate mofetil treatment as positive controls of the therapy and (2) mice treated with CP-690,550, a JAK3 inhibitor. Mice were treated for 12 weeks. We evaluated renal function, anti-double-stranded DNA (anti-dsDNA) antibody, renal histology changes, kidney complement and immunoglobulin G (IgG) deposits, T-cell and macrophage infiltration, kidney inflammatory gene expression, and circulating cytokine changes. Results: CP-690,550 treatment significantly reduced proteinuria and improved renal function and histological lesions of the kidney. Compared with vehicle-treated animals, those undergoing CP-690,550 treatment showed significantly diminished anti-dsDNA antibody and complement component C3 and IgG deposition in glomeruli. We also observed a significant reduction of T-cell and macrophage infiltration. Kidney gene expression revealed a reduction in inflammatory cytokines and complement and related macrophage-attracting genes. Circulating inflammatory cytokines were also reduced with treatment. Conclusions: On the basis of our results, we conclude that the JAK-STAT pathway is implicated in the progression of renal inflammation in NZB/WF1 mice and that targeting JAK3 with CP-690,550 is effective in slowing down the course of experimental LN. Thus, CP-690,550 could become a new therapeutic tool in LN and other autoimmune diseases

Update on controls for isolation and quantification methodology of extracellular vesicles derived from adipose tissue mesenchymal stem cells

The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV.The aim of our study was to incorporate new controls for the detection and quantification of EV derived from ASC and to analyze the applicability and limitations of the available techniques. ASC were cultured in medium supplemented with 5% of vesicles-free fetal bovine serum. The EV were isolated from conditioned medium by differential centrifugation with size filtration (0.2 mu m). As a control, non-conditioned culture medium was used (control medium).To detect EV, electron microscopy, conventional flow cytometry, and western blot were used. The quantification of the EV was by total protein quantification, ExoELISA immunoassay, and Nanosight. Cytokines and growth factors in the EV samples were measured by multiplex bead array kit. The EV were detected by electron microscope. Total protein measurement was not useful to quantify EV as the control medium showed similar protein contents as the EV samples. The ExoELISA kits had technical troubles and it was not possible to quantify the concentration of exosomes in the samples. The use of Nanosight enabled quantification and size determination of the EV. It is, however, not possible to distinguish protein aggregates from EV with this method. The technologies for quantification and characterization of the EV need to be improved. In addition, we detected protein contaminants in the EV samples, which make it difficult to determine the real effect of EV in experimental models. It will be crucial in the future to optimize design novel methods for purification and characterization of EV

Mesenchymal Stem Cell Therapy Prevents Interstitial Fibrosis and Tubular Atrophy in a Rat Kidney Allograft Model

In solid organ transplantation, mesenchymal stem cell (MSC) therapy is strongly emerging among other cell therapies due to the positive results obtained in vitro and in vivo as an immunomodulatory agent and their potential regenerative role. We aimed at testing whether a single dose of MSCs, injected at 11 weeks after kidney transplantation for the prevention of chronic mechanisms, enhanced regeneration and provided protection against the inflammatory and fibrotic processes that finally lead to the characteristic features of chronic allograft nephropathy (CAN). Either bone marrow mononuclear cells (BMCs) injection or no-therapy (NT) were used as control treatments. A rat kidney transplantation model of CAN with 2.5 h of cold ischemia was used, and functional, histological, and molecular parameters were assessed at 12 and 24 weeks after transplantation. MSC and BMC cell therapy preserves renal function at 24 weeks and abrogates proteinuria, which is typical of this model (NT24w: 68.9 +/- 26.5mg/24 h, MSC24w: 16.6 +/- 2.3mg/24 h, BMC24w: 24.1 +/- 5.3mg/24 h, P < 0.03). Only MSC-treated animals showed a reduction in interstitial fibrosis and tubular atrophy (NT24w: 2.3 +/- 0.29, MSC24w: 0.4 +/- 0.2, P < 0.03), less T cells (NT: 39.6 +/- 9.5, MSC: 8.1 +/- 0.9, P < 0.03) and macrophages (NT: 20.9 +/- 4.7, MSC: 5.9 +/- 1.7, P < 0.05) infiltrating the parenchyma and lowered expression of inflammatory cytokines while increasing the expression of anti-inflammatory factors. MSCs appear to serve as a protection from injury development rather than regenerate the damaged tissue, as no differences were observed in Ki67 expression, and kidney injury molecule-1, Clusterin, NGAL, and hepatocyte growth factor expression were only up-regulated in nontreated animals. Considering the results, a single delayed MSC injection is effective for the long-term protection of kidney allografts

CD40 gene silencing reduces the progression of experimental lupus nephritis modulating local milieu and systemic mechanisms

Lupus nephritis (LN) is an autoimmune disorder in which co-stimulatory signals have been involved. Here we tested a cholesterol-conjugated-anti-CD40-siRNA in dendritic cells (DC) in vitro and in a model of LPS to check its potency and tissue distribution. Then, we report the effects of Chol-siRNA in an experimental model of mice with established lupus nephritis. Our in vitro studies in DC show a 100%intracellular delivery of Chol-siRNA, with a significant reduction in CD40 after LPS stimuli. In vivo in ICR mice, the CD40-mRNA suppressive effects of our Chol-siRNA on renal tissue were remarkably sustained over a 5 days after a single preliminary dose of Chol-siRNA. The intra-peritoneal administration of Chol-siRNA to NZB/WF1 mice resulted in a reduction of anti-DNA antibody titers, and histopathological renal scores as compared to untreated animals. The higher dose of Chol-siRNA prevented the progression of proteinuria as effectively as cyclophosphamide, whereas the lower dose was as effective as CTLA4. Chol-siRNA markedly reduced insterstitialCD3+ and plasma cell infiltrates as well as glomerular deposits of IgG and C3. Circulating soluble CD40 and activated splenic lymphocyte subsets were also strikingly reduced by Chol-siRNA. Our data show the potency of our compound for the therapeutic use of anti-CD40-siRNA in human LN and other autoimmune disorders

Specific metabolomics adaptations define a differential regional vulnerability in the adult human cerebral cortex

Brain neurons offer diverse responses to stresses and detrimental factors during development and aging, and as a result of both neurodegenerative and neuropsychiatric disorders. This multiplicity of responses can be ascribed to the great diversity among neuronal populations. Here we have determined the metabolomic profile of three healthy adult human brain regions¿entorhinal cortex, hippocampus, and frontal cortex¿using mass spectrometry-based technologies. Our results show the existence of a lessened energy demand, mitochondrial stress, and lower one-carbon metabolism (particularly restricted to the methionine cycle) specifically in frontal cortex. These findings, along with the better antioxidant capacity and lower mTOR signaling also seen in frontal cortex, suggest that this brain region is especially resistant to stress compared to the entorhinal cortex and hippocampus, which are more vulnerable regions. Globally, our results show the presence of specific metabolomics adaptations in three mature, healthy human brain regions, confirming the existence of cross-regional differences in cell vulnerability in the human cerebral cortex. Keywords: energy metabolism, mammalian target of rapamycin (mTOR), metabolomics, methionine cycle, mitochondrial stress, nucleotide metabolism, one-carbon metabolism, selective neuronal vulnerabilit

Silenciament gènic de CD40 en al•lo i autoimmunitat. Estudi de la resposta immuno inflamatòria en models animals

[cat]Tots els estudis presentats en aquest treball tenen com a objectiu ampliar el coneixement del gen CD40 i el seu paper en l’activació del sistema immune. Per estudiar aquesta funció es va aplicar un silenciament gènic mitjançant la tecnologia RNAi en models experimentals, tant in vitro com in vivo; en un model de malaltia autoimmune, com la nefritis lúpica, i un de malaltia al•loimmune, com és el cas del trasplantament renal. El senyal de coestimulació de CD40-CD40L juga un paper molt important en l’activació del sistema immunitari. En el cas del trasplantament renal se sap que CD40 juga un paper molt important en el reconeixement de l’al•loantigen i la inflamació provocada per la isquèmia reperfusió. En el cas de la nefropatia lúpica es dóna un reconeixement autoimmune que també inicia una cascada inflamatòria. La teràpia gènica contra CD40 permetrà un tractament més específic i eficient d’aquesta diana, amb una tecnologia d’aplicació futura probablement més efectiva que les teràpies convencionals. La teràpia gènica contra CD40 utilitzant la tecnologia de RNA interferència, permetria evitar la sobre activació del sistema immunitari tant en un context al•loimmune com autoimmune i controlar el dany inflamatori derivat. El nostre primer estudi es va proposar per a veure com afecta la isquèmia freda sobre la funció renal en un model de rebuig agut mediat per anticossos, veure com modifica l’estructura molecular, tissular i cel•lular de l’empelt renal. Analitzar també quin paper juga CD40 en aquest procés. No se saben en profunditat els mecanismes pels quals la isquèmia accelera el rebuig agut i preveu una pitjor evolució de l’empelt. Els nostres resultats apunten també cap a l’alteració de la barrera de filtració glomerular com a una causa important de deteriorament de l’empelt i la seva evolució. Es va poder comprovar que la isquèmia provocada en un procés de trasplantament renal augmenta la síntesis de col•lagen IV i el seu dipòsit en la capsula de Bowman. També provoca l’activació de la immunitat innata i augmenta l’expressió de CD40. Tots aquests mecanismes i els consecutius mediadors inflamatoris alliberats, indueixen una major amplificació del dany i activació de la resposta immune adaptativa, perpetuant així, el cicle de dany i reparació de l’empelt que podria donar lloc a la seva disfunció crònica. Un cop posat el model a punt i caracteritzat, tenint en compte el paper de la senyal coestimuladora CD40 en l’activació de la resposta immune; es va proposar un segon estudi on es va dissenyar un siRNA anti CD40 específic de rata. L’objectiu principal era estudiar el paper i el potencial terapèutic del fàrmac administrat de forma local, en un model de resposta de tipus al•logènica com és el cas del trasplantament renal. En aquest model en el qual l’administració del fàrmac era local intra-arterial, es va administrar el siRNA acomplexat amb un vector de tipus liposoma. En aquest estudi vam observar que el silenciament local intra-renal, previ al trasplantament, amb siRNA anti CD40 amb una dosis única en un model de rebuig humoral va causar un canvi de patró en el tipus de rebuig, es va aconseguir reduir els anticossos donant específics de l'hoste desprès del trasplantament i es va millorar la supervivència. El tractament local de l'òrgan aïllat va aconseguir bloquejar l’expressió gènica de CD40 a l’empelt, donant lloc a un canvi en l’ambient immunològic, els grups de silenciament de CD40 van experimentar un canvi de tipus de rebuig cap a un rebuig cel•lular, més fàcilment abordable des d’un punt de vista terapèutic. La histologia dels empelts del grup no tractat mostrava lesions típiques del rebuig humoral. La majoria dels empelts del grup de tractament amb siRNA anti CD40 mostraven un patró de rebuig cel•lular. Els animals no tractats tenien dipòsits de complement als capil•lars peritubulars, tant C4d com IgG, mentre que el grup de tractament amb siRNA anti CD40, aquests es van veure reduïts. Una altra evidència de la disminució de l’activació del sistema del complement, a conseqüència del silenciament de CD40, també va ser la reducció de l’expressió gènica de proteïnes reguladores de complement, com són CFH i CFI. Es va confirmar l’aparició d’anticossos donant específics a la majoria d’animals del grup no tractat, que eren absents prèviament al trasplantament, així doncs, hi ha una formació massiva d’aquests degut a una ràpida activació de les cèl•lules B, en el nostre estudi, aquest anticossos donant específic es van reduir parcialment en el grup de tractament amb rapamicina, siRNA anti CD40, i al grup de combinació de teràpia. Per tant en aquest treball es demostra que el silenciament de CD40 com a tractament local previ a la implantació de l’empelt renal és una estratègia efectiva i abordable per prevenir el rebuig humoral. Aquest resultats preliminars obren les portes a aplicar aquest tipus de teràpia gènica en altres models de trasplantament o altres malalties inflamatòries o autoimmunes on les cèl•lules B hi tinguin un paper important. Així doncs es va proposar un tercer treball l’objectiu del qual era analitzar el paper de CD40 en aquest model de nefritis lúpica, de caràcter autoimmune. Inicialment es va fer un estudi in vitro utilitzant un cultiu de cèl•lules dendrítiques derivades de moll d’os. Primer vam comparar l’entrada cel•lular del siRNA modificat químicament amb un siRNA nu sense modificar mitjançant citometria de flux; vam observar que ambdós molècules tenien un 100% d’entrada, però que el siRNA modificat tenia una MFI més elevada. Per microscòpia confocal vam confirmar una localització citoplasmàtica del siRNA i que l’entrada era rapida. Posteriorment es va estimular les cèl•lules dendrítiques amb LPS durant 12h i, un cop comprovat que l’estímul fos eficaç, vam observar que el siRNA anti CD40 era capaç d’inhibir de manera evident l’augment d’expressió de CD40 induït per LPS. Per conèixer millor l’eficiència de l’administració sistèmica del siRNA es va escollir un model amb ratolins ICR en el qual vam comprovar la cinètica d’expressió de CD40 després d’un estímul de LPS en diferents teixits. Vam observar que l’estímul de LPS induïa un augment de l’expressió de CD40, tenint un pic màxim passades 4h de la injecció del LPS, i que aquesta tornava a nivells basals en 24-48h. Un cop caracteritzada, es van fer grups de tractament amb el siRNA per valorar el potencial del fàrmac i vam observar que el siRNA era capaç de reduir significativament aquesta expressió durant tres o quatre dies Es va avaluar la biodistribució del compost injectant un siRNA marcat amb un fluorocrom (Cy5.5) a ratolins ICR i vam observar que la distribució del fàrmac a l’organisme era major en fetge, melsa i ronyó independentment de si l’administració era intravenosa o intraperitoneal, ja que no hi havia diferències entre aquestes. 30 minuts després de l’administració ja vam observar una internalització del siRNA a les cèl•lules dels teixits. Es va comprovar que en un teixit patològic, com és el cas d’un ronyó afectat per nefritis lúpica, la distribució del siRNA era semblant com en el cas d’un teixit sa, per tant, no hi ha diferències entre l’alliberament de la molècula en un teixit sa i patològic. Aquest resultats ens van permetre establir un protocol de tractament dels animals amb nefropatia lúpica, es va decidir administrar el siRNA intraperitonealment i dos cops per setmana. El model animal que es va escollir per l’experiment de nefritis lúpica va ser NZB/W F1, un dels models més utilitzats i ben establert experimentalment. En el nostre estudi, els resultats obtinguts van demostrar que el silenciament de CD40 en aquest model muri millora la supervivència i frena l’evolució de la malaltia. Clínicament s’observa una disminució de la proteïnúria, albuminúria i dels anticossos circulants anti dsDNA. A part del grup tractat amb ciclofosfamida, que és un control positiu de tractament, també es va incloure el CTLA4, com a control de bloqueig de coestimulació: Aquest últim tot i mostrar diversos graus d'activitat en diversos assajos, només va resultar ser tan potent com el tractament amb una dosi única setmanal de siRNA anti CD40 per aturar la progressió de la proteïnúria, clarament una dosi sub-terapèutica en comparació amb els efectes del tractament amb siRNA anti CD40 dos cops per setmana. També vam observar una millora de les lesions histològiques típiques de la malaltia, els grups de bloqueig de coestimulació van ser els més eficaços en prevenir les lesions histològiques, destacant el grup tractat amb siRNA dos cops per setmana on l’infiltrat intersticial, l’atròfia tubular, i l’infiltrat intersticial eren absents. També es va observar una reducció de la infiltració renal per cèl•lules T i cèl•lules que segreguen immunoglobulines. En la patologia de la nefritis lúpica se sap que el dany renal és iniciat amb els dipòsits glomerulars d’immunocomplexes, aquests juguen un paper molt important i són predominantment anticossos contra ssDNA, dsDNA, histones, proteïnes de complement, etc. Aquests immunocomplexes localitzats tant al mesangi com de forma subendotelial, contribueixen al reclutament de cèl•lules inflamatòries. En el nostre estudi vam observar que els ratolins no tractats tenien dipòsits d’immunoglobulina G en el parènquima renal, mentre que tots els tractaments aconseguien reduir-los, cal remarcar que els grups de tractament amb bloqueig de coestimulació van ser més efectius que el grup tractat amb CYP. L’anàlisi de l’expressió gènica de CD40 demostra, com era previsible, una disminució significativa en el parènquima renal. El silenciament de CD40 també va induir una disminució de citosines pro inflamatòries com és el cas de la IL6; gens relacionats amb l’inflammosoma es mostren elevats en el grup no tractat, mentre que en els grups de tractament estan disminuïts. Tot això suggereix també un efecte de modulació local de la resposta inflamatòria. Podem concloure que els nostres resultats evidencien el potencial terapèutic i els efectes del silenciament específic de CD40 mitjançant el siRNA com una forma de disminuir i modular l'activació del sistema immune en els ronyons inflamats, tant en el cas del trasplantament d’òrgans sòlids com en el cas de malalties autoimmunes.[eng]All studies presented in this paper are intended to expand the knowledge of the gene CD40 and its role in activating the immune system. To study this function we applied gene silencing by RNAi technology in experimental models, both in vitro and in vivo, in a model of autoimmune disease such as lupus nephritis, and loimmune allergic disease, such as the transplantation. The signal of CD40-CD40L costimulation plays an important role in activating the immune system. In the case of kidney transplantation is known that CD40 plays an important role in recognizing the reference alloantigen and inflammation caused by ischemia-reperfusion. In the case of lupus nephritis occurs an autoimmune recognition that also initiates an inflammatory cascade. Gene therapy against CD40 allow a more efficient and specific for this target application technology with future probably more effective than conventional therapies. Gene therapy against CD40 using RNA interference technology would prevent the activation of the immune system both in the context al•loimmune and autoimmune inflammatory damage and control derivative. Our first study was proposed to see the affect of cold ischemia on renal function in a model of acute rejection mediated by antibodies, see how modifying the molecular structure, tissue and cell graft. Also analyze what role CD40 plays in this process. Mechanisms by which ischemia accelerates acute rejection and predicts a worse graft outcome are not well known. Our results also point to the disruption of the glomerular filtration barrier as a major cause of deterioration of the graft and its evolution. It was found that ischemia caused in the process of kidney transplantation increases the synthesis of collagen IV and its deposit in the Bowman's capsule. It also causes the activation of innate immunity and increases the expression of CD40. All these mechanisms and consecutive inflammatory mediators released, induce a greater amplification of damage and activation of the adaptive immune response, thus perpetuating the cycle of damage and repair of the graft that could lead to chronic dysfunction. Once the model set up, taking into account the role of the CD40 costimulatory signal in the activation of the immune response, proposed a second study where we designed a siRNA specific rat anti-CD40. The main objective was to study the role and therapeutic potential of the drug administered locally in a model of type allogeneic response such as renal transplantation. In this model drug administration was local intra-arterial with a siRNA liposome vector type. In this study, we observed that silencing local intra-renal pre-transplant anti-CD40 siRNA with a single dose in a model of humoral rejection caused a change of pattern in the type of rejection was reduced donor antibodies specific host after transplantation and improved survival. Local treatment of the isolated organ was able to block CD40 gene expression in the graft, resulting in a change in the environment immune silencing CD40 groups showed a shift towards a type of rejection cellular rejection more easily approachable from a therapeutic point of view. The histology of the grafts in the untreated group showed lesions typical of humoral rejection damage. Most of the grafts treated with siRNA group showed a pattern of anti CD40 cell rejection. Non treated animals presented deposits in peritubular capillaries, both C4d and IgG, while the group treated with anti-CD40 siRNA, these were reduced. Further evidence of decreased activation of the complement system, due to the silencing of CD40 was also reduced gene expression of complement regulatory proteins such as CFH and CFI. We confirmed the appearance of donor-specific antibodies in the majority of animals not treated group, which were absent prior to transplant, so there is a massive formation of these due to a rapid activation of cells B, in our study, this donor specific antibodies were partially reduced in the group treated with rapamycin, anti-CD40 siRNA, and the combination therapy group. Therefore in this paper we show that the silencing of CD40 as a local treatment prior to implantation of the graft is an effective and affordable strategy to prevent humoral rejection. The preliminary results open the door to apply this type of gene therapy in other models of transplantation and other autoimmune or inflammatory diseases where B cells have an important role there. So he proposed a third work whose aim was to analyze the role of CD40 in this model of lupus nephritis, autoimmune in nature. Initially we performed a study using an in vitro dendritic cell culture of derived from bone marrow. We first compared the cell entry of chemically modified siRNA with a naked unmodified siRNA by flow cytometry, we found that both molecules had 100% input, but the modified siRNA had a higher MFI. For confocal microscopy we confirm a cytoplasmic localization of siRNA and the entrance was quick. Later stimulate dendritic cells with LPS for 12 hours and, once confirmed that the stimulus was effective, we observed that anti-CD40 siRNA was able to clearly inhibited the increased expression of CD40 induced LPS. To better understand the efficiency of systemic administration of siRNA molecule we perform an study with ICR mice in which we found the kinetics of expression of CD40 after LPS stimulation in different tissues. We observed that LPS stimulation induced an increased expression of CD40, having a peak past 4 hours after the injection of LPS, and this returned to baseline levels within 24-48h. Once characterized, were the groups treated with siRNA to assess the potential of the drug was observed that the siRNA was able to significantly reduce this expression for three or four days. We evaluated the biodistribution of the compound injected siRNA labeled with a fluorochrome (Cy5.5) and ICR mice, we observed that the distribution of the drug in the body was greater in liver, spleen and kidney whether intravenous administration was or intraperitoneally, as there were no differences between them. 30 minutes after the administration we observed internalization of siRNA in cells of tissues. It was found that in a pathological tissue, such as a kidney affected by lupus nephritis, the distribution of siRNA was similar as in the case of healthy tissue, so there is no difference between the release of molecule in healthy and pathological tissue. These results allowed us to establish a protocol for treatment of the animals with lupus nephritis; it was decided to administer siRNA and intraperitoneally twice a week. The animal model chosen for the experiment was lupus nephritis NZB / W F1, one of the models used and well established experimentally. In our study, the results showed that silencing of CD40 in the murine model improves survival and slows disease progression. A decrease of proteinuria, albuminuria and anti dsDNA antibodies was seen. In the group treated with cyclophosphamide, a positive control treatment was also included CTLA4 as a costimulation blockade control: The latter even showed varying degrees of activity in several tests, proved to be just as powerful treatment with a single dose of weekly anti CD40 siRNA to stop the progression of proteinuria clearly a sub-therapeutic dose compared with the effects of treatment with anti-CD40 siRNA twice a week. We also observed an improvement of histological lesions typical of the disease in groups with costimulation blockade treatment, treatment was most effective in preventing histological lesions, highlighting the group treated with siRNA twice a week where the interstitial infiltrate, atrophy tubular and interstitial infiltrates were absent. We also observed a reduction in renal infiltration by T cells and cells that secrete immunoglobulins. In the pathology of lupus nephritis is known that kidney damage is initiated glomerular deposits of immunocomplexes, they play an important role and are predominantly antibodies against ssDNA, dsDNA, histone protein complement, etc. These immunocomplexes located either in the subendothelial mesangium contribute to the recruitment of inflammatory cells. In our study we found that the non treated mice had deposits of IgG in the renal parenchyma, while all treatments were able to reduce them, we should note that the treatment groups with costimulation blockade was more effective than group treated with CYP. The analysis of gene expression of CD40 demonstrated, as expected, a significant decrease in the renal parenchyma. Silencing CD40 also induced a decrease in pro-inflammatory cytokines such as IL6, genes related inflammosoma are elevated in the untreated group, whereas treatment groups are diminished. This also suggests an effect of modulation of the local inflammatory response. In conclusion, our results demonstrate the potential therapeutic effects of CD40-specific silencing using siRNA as a way to reduce and modulate the activation of the immune system in inflamed kidneys, in the case of solid organ transplants as in the case of autoimmune disease

Datasets for the validation of the 'in vivo' siRNA-silencing of CD40 and for the detection of new markers of aterosclerosis progression in ApoE-deficient mice

Data presented in this Data in Brief article correspond to the article 'in vivo' silencing of CD40 reduces progression of experimental atherogenesis through a NFκB/miR-125b axis and reveals new potential mediators in the pathogenesis of atherosclerosis' (M. Hueso, L. De Ramon, E. Navarro, E. Ripoll, J.M. Cruzado, J.M. Grinyo, J. Torras, 2016) [1]. Here, we describe the validation of the silencing of CD40 expression with a specific siRNA in ApoE−/− mouse aortas, and its systemic effects on splenic lymphocytic subpopulations as well as on the infiltration of aortic intima by F4/80+, galectin-3+ macrophages or by NF-κB+ cells. We also show the output of a Gene Ontology and TLDA analysis which allowed the detection of potential mediators of atherosclerosis progression. We provide the scientific community with a set of genes whose expression is increased during atherosclerosis progression but downregulated upon CD40 silencing