Mesenchymal Stem Cell Engagement Modulates Neuroma Microenviroment in Rats and Humans and Prevents Postamputation Pain.

Postamputation pain is currently managed unsatisfactorily with neuron-targeted pharmacological and interventional therapies. Non-neuronal pain mechanisms have emerged as crucial factors in the development and persistence of postamputation pain. Consequently, these mechanisms offer exciting prospects as innovative therapeutic targets. We examined the hypothesis that engaging mesenchymal stem cells (MSCs) would foster local neuroimmune interactions, leading to a potential reduction in postamputation pain. We utilized an ex vivo neuroma model from a phantom limb pain patient to uncover that the oligodeoxynucleotide IMT504 engaged human primary MSCs to promote an anti-inflammatory microenvironment. Reverse translation experiments recapitulated these effects. Thus, in an in vivo rat model, IMT504 exhibited strong efficacy in preventing autotomy (self-mutilation) behaviors. This effect was linked to a substantial accumulation of MSCs in the neuroma and associated dorsal root ganglia and the establishment of an anti-inflammatory phenotype in these compartments. Centrally, this intervention reduced glial reactivity in the dorsal horn spinal cord, demonstrating diminished nociceptive activity. Accordingly, the exogenous systemic administration of MSCs phenocopied the behavioral effects of IMT504. Our findings underscore the mechanistic relevance of MSCs and the translational therapeutic potential of IMT504 to engage non-neuronal cells for the prevention of postamputation pain. PERSPECTIVE: The present study suggests that IMT504-dependent recruitment of endogenous MSCs within severely injured nerves may prevent post-amputation pain by modifying the inflammatory scenario at relevant sites in the pain pathway. Reinforcing data in rat and human tissues supports the potential therapeutic value of IMT504 in patients suffering postamputation pain.

IMT504 protects beta cells against apoptosis and maintains beta cell identity, without modifying proliferation.

We have demonstrated that oligodeoxynucleotide IMT504 promotes significant improvement in the diabetic condition in diverse animal models. Based on these results, here we evaluated whether these effects observed in vivo could be due to direct effects on ß-cells. We demonstrate by immunofluorescence that IMT504 enters the cell and locates in cytoplasm where it induces GSK-3ß phosphorylation that inactivates this kinase. As GSK-3ß tags Pdx1 for proteasomal degradation, by inactivating GSK-3ß, IMT504 induces an increase in Pdx1 protein levels, demonstrated by Western blotting. Concomitantly, an increase in Ins2 and Pdx1 gene transcription was observed, with no significant increase in insulin content or secretion. Enhanced Pdx1 is promising since it is a key transcription factor for insulin synthesis and is also described as an essential factor for the maintenance ß-cell phenotype and function. Dose-dependent inhibition of H2 O2 -induced apoptosis determined by ELISA as well as decreased expression of Bax was also observed. These results were confirmed in another ß-cell line, beta-TC-6 cells, in which a cytokine mix induced apoptosis that was reversed by IMT504. In addition, an inhibitor of IMT504 entrance into cells abrogated the effect IMT504. Based on these results we conclude that the ß-cell recovery observed in vivo may include direct effects of IMT504 on ß-cells, by maintaining their identity/phenotype and protecting them from oxidative stress and cytokine-induced apoptosis. Thus, this work positions IMT504 as a promising option in the framework of the search of new therapies for type I diabetes treatment.

Modulation of the Inflammatory Response by Pre-emptive Administration of IMT504 Reduces Postoperative Pain in Rats and has Opioid-Sparing Effects.

Despite the available knowledge on underlying mechanisms and the development of several therapeutic strategies, optimal management of postoperative pain remains challenging. This preclinical study hypothesizes that, by promoting an anti-inflammatory scenario, pre-emptive administration of IMT504, a noncoding, non-CpG oligodeoxynucleotide with immune modulating properties, will reduce postincisional pain, also facilitating therapeutic opioid-sparing. Male adult Sprague-Dawley rats with unilateral hindpaw skin-muscle incision received pre-emptive (48 and 24 hours prior to surgery) or postoperative (6 hours after surgery) subcutaneous vehicle (saline) or IMT504. Various groups of rats were prepared for pain-like behavior analyses, including subgroups receiving morphine or naloxone, as well as for flow-cytometry or quantitative RT-PCR analyses of the spleen and hindpaws (for analysis of inflammatory phenotype). Compared to vehicle-treated rats, pre-emptive IMT504 significantly reduced mechanical allodynia by 6 hours after surgery, and accelerated recovery of basal responses from 72 hours after surgery and onwards. Cold allodynia was also reduced by IMT504. Postoperative administration of IMT504 resulted in similar positive effects on pain-like behavior. In IMT504-treated rats, 3 mg/kg morphine resulted in comparable blockade of mechanical allodynia as observed in vehicle-treated rats receiving 10 mg/kg morphine. IMT504 significantly increased hindpaw infiltration of mesenchymal stem cells, CD4+T and B cells, and caused upregulated or downregulated transcript expressions of interleukin-10 and interleukin-1ß, respectively. Also, IMT504 treatment targeted the spleen, with upregulated or downregulated transcript expressions, 6 hours after incision, of interleukin-10 and interleukin-1ß, respectively. Altogether, pre-emptive or postoperative IMT504 provides protection against postincisional pain, through participation of significant immunomodulatory actions, and exhibiting opioid-sparing effects. PERSPECTIVE: This preclinical study introduces the noncoding non-CpG oligodeoxynucleotide IMT504 as a novel modulator of postoperative pain and underlying inflammatory events. The opioid-sparing effects observed for IMT504 appear as a key feature that could contribute, in the future, to reducing opioid-related adverse events in patients undergoing surgical intervention.

IMT504 blocks allodynia in rats with spared nerve injury by promoting the migration of mesenchymal stem cells and by favoring an anti-inflammatory milieu at the injured nerve.

ABSTRACT: IMT504, a noncoding, non-CpG oligodeoxynucleotide, modulates pain-like behavior in rats undergoing peripheral nerve injury, through mechanisms that remain poorly characterized. Here, we chose the spared nerve injury model in rats to analyze the contribution of mesenchymal stem cells (MSCs) in the mechanisms of action of IMT504. We show that a single subcutaneous administration of IMT504 reverses mechanical and cold allodynia for at least 5 weeks posttreatment. This event correlated with long-lasting increases in the percentage of MSCs in peripheral blood and injured sciatic nerves, in a process seemingly influenced by modifications in the CXCL12-CXCR4 axis. Also, injured nerves presented with reduced tumor necrosis factor-α and interleukin-1ß and increased transforming growth factor-ß1 and interleukin-10 protein levels. In vitro analysis of IMT504-pretreated rat or human MSCs revealed internalized oligodeoxynucleotide and confirmed its promigratory effects. Moreover, IMT504-pretreatment induced transcript expression of Tgf-ß1 and Il-10 in MSCs; the increase in Il-10 becoming more robust after exposure to injured nerves. Ex vivo exposure of injured nerves to IMT504-pretreated MSCs confirmed the proinflammatory to anti-inflammatory switch observed in vivo. Interestingly, the sole exposure of injured nerves to IMT504 also resulted in downregulated Tnf-α and Il-1ß transcripts. Altogether, we reveal for the first time a direct association between the antiallodynic actions of IMT504, its promigratory and cytokine secretion modulating effects on MSCs, and further anti-inflammatory actions at injured nerves. The recapitulation of key outcomes in human MSCs supports the translational potential of IMT504 as a novel treatment for neuropathic pain with a unique mechanism of action involving the regulation of neuroimmune interactions.

Oligonucleotide IMT504 Improves Glucose Metabolism and Controls Immune Cell Mediators in Female Diabetic NOD Mice.

Type 1 diabetes occurs as a consequence of progressive autoimmune destruction of beta cells. A potential treatment for this disease should address the immune attack on beta cells and their preservation/regeneration. The objective of this study was to elucidate whether the immunomodulatory synthetic oligonucleotide IMT504 was able to ameliorate diabetes in NOD mice and to provide further understanding of its mechanism of action. We found that IMT504 restores glucose homeostasis in a diabetes mouse model similar to human type 1 diabetes, by regulating expression of immune modulatory factors and improving beta cell function. IMT504 treatment markedly improved fasting glycemia, insulinemia, and homeostatic model assessment of beta cell function (HOMA-Beta cell) index. Moreover, this treatment increased islet number and decreased apoptosis, insulitis, and CD45+ pancreas-infiltrating leukocytes. In a long-term treatment, we observed improvement of glucose metabolism up to 9 days after IMT504 cessation and increased survival after 15 days of the last IMT504 injection. We postulate that interleukin (IL)-12B (p40), possibly acting as a homodimer, and Galectin-3 (Gal-3) may function as mediators of this immunomodulatory action. Overall, these results validate the therapeutic activity of IMT504 as a promising drug for type 1 diabetes and suggest possible downstream mediators of its immunomodulatory effect.

IMT504 Provides Analgesia by Modulating Cell Infiltrate and Inflammatory Milieu in a Chronic Pain Model.

IMT504 is a non-CPG, non-coding synthetic oligodeoxinucleotide (ODN) with immunomodulatory properties and a novel inhibitory role in pain transmission, exerting long-lasting analgesic effects upon multiple systemic administrations. However, its mechanisms of anti-nociceptive action are still poorly understood. In the present study in male adult rats undergoing complete Freund's adjuvant-induced hindpaw inflammation, we focused in the analysis of the immunomodulatory role of IMT504 over the cellular infiltrate, the impact on the inflammatory milieu, and the correlation with its anti-allodynic role. By means of behavioral analysis, we determined that a single subcutaneous administration of 6 mg/kg of IMT504 is sufficient to exert a 6-week-long full reversal of mechanical and cold allodynia, compromising neither acute pain perception nor locomotor activity. Importantly, we found that the anti-nociceptive effects of systemic IMT504, plus quick reductions in hindpaw edema, were associated with a modulatory action upon cellular infiltrate of B-cells, macrophages and CD8+ T-cells populations. Accordingly, we observed a profound downregulation of several inflammatory leukocyte adhesion proteins, chemokines and cytokines, as well as of ß-endorphin and an increase in the anti-inflammatory cytokine, interleukin-10. Altogether, we demonstrate that at least part of the anti-nociceptive actions of IMT504 relate to the modulation of the peripheral immune system at the site of injury, favoring a switch from pro- to anti-inflammatory conditions, and provide further support to its use against chronic inflammatory pain. Graphical abstract GA short description - IMT504 systemic Administration. Systemic administration of the non-CpG ODN IMT504 results in a 6-week long blockade of pain-like behavior in association with anti-inflammatory responses at the site of injury. These include modulation of lymphoid and myeloid populations plus downregulated expression levels of multiple pro-inflammatory cytokines and ß-endorphin. Nocifensive responses and locomotion remain unaltered.

Contribution of neural crest and GLAST+ Wnt1+ bone marrow pericytes with liver fibrogenesis and/or regeneration.

BACKGROUND AND AIMS: Liver fibrosis results from cycles of liver damage and scar formation. We herein aimed at analysing neural crest cells and/or bone marrow stromal cells contribution to the liver. METHODS: Two liver fibrosis and one hepatectomy model were applied on double-transgenic loxP-Cre mouse lines. RESULTS: Increased numbers of glia with more complex processes were found in fibrotic livers. During embryonic development, only few cells were traced in the liver and bone marrow, in a minor fraction of mice of different neural crest reporter strains analysed: therefore, a neural crest origin of such cells is doubtful. In the fibrotic liver, a significantly higher incidence of endothelial cells and hepatocyte-like cells expressing the reporter gene Tomato were found in Wnt1-Cre-Tom and GLAST-CreERT2-Tom mice. Consistently, during early fibrogenesis stromal Wnt1-traced cells, with progenitor (CFU-F) properties, get likely mobilized to peripheral blood. Circulating adult Wnt1-traced cells are stromal cells and lack from the expression of other bone marrow and endothelial progenitor cells markers. Furthermore, in a 70% hepatectomy model GLAST+ Wnt1-traced pericytes were found to be mobilized from the bone marrow and the incidence of GLAST-traced hepatocyte-like cells was increased. Finally, GLAST-traced hepatocyte like-cells were found to maintain the expression of stromal markers. CONCLUSIONS: Our data suggest a gliosis process during liver fibrogenesis. While neural crest cells probably do not contribute with other liver cell types than glia, GLAST+ Wnt1-traced bone marrow pericytes are likely a source of endothelial and hepatocyte-like cells after liver injury and do not contribute to scarring.

Long-lasting ameliorating effects of the oligodeoxynucleotide IMT504 on mechanical allodynia and hindpaw edema in rats with chronic hindpaw inflammation.

PURPOSE: Previously we showed that systemic administration of IMT504 prevents or ameliorates mechanical and thermal allodynia in rats with sciatic nerve crush. Here we analyzed if IMT504 is also effective in reducing mechanical allodynia and inflammation in rats undergoing hindpaw inflammation. MATERIALS AND METHODS: Male Sprague-Dawley rats received unilateral intraplantar injection of complete Freund́s adjuvant (CFA), and were grouped into: 1) untreated CFA, 2) vehicle-treated CFA, 3) IMT504-treated CFA (5 daily (5*) doses of 20, 2 or 0.2 mg/kg, or 3*2 mg/kg). Naïve groups were also included. Finally, early (immediately after intraplantar CFA) and late (7 days after intraplantar CFA) IMT504 treatment protocols were also tested. Hindpaw mechanical allodynia, dorsoventral thickness, edema and cellular infiltration of ipsilateral hindpaws were evaluated in all groups. RESULTS: Untreated CFA rats exhibited mechanical allodynia of quick onset (day 1) and long duration (7 weeks inclusive). Early and late treatments with 5*20 mg/kg IMT504 to CFA rats resulted in both quick and long-lasting antiallodynic effects, as compared to untreated CFA rats. This was also the case in CFA rats undergoing late IMT504 treatment at lower doses (3* and 5*2 mg/kg). Very low doses of IMT504 (5*0.2 mg/kg) only showed a mild improvement in withdrawal threshold, never reaching basal levels. Finally, rats treated with 3* or 5*2 mg/kg or 5*0.2 mg/kg exhibited significant decreases in dorsoventral thickness, edema, and inflammatory cell infiltration of the inflamed hindpaw. CONCLUSION: Early and late administration of IMT504 results in quick and long-lasting reductions in mechanical allodynia and hindpaw edema. While the mechanisms behind these effects remain to be established, data suggests that IMT504 administration could be a promising strategy in the control of inflammatory pain.

Immunomodulatory oligonucleotide IMT504: Effects on mesenchymal stem cells as a first-in-class immunoprotective/immunoregenerative therapy.

The immune responses of humans and animals to insults (i.e., infections, traumas, tumoral transformation and radiation) are based on an intricate network of cells and chemical messengers. Abnormally high inflammation immediately after insult or abnormally prolonged pro-inflammatory stimuli bringing about chronic inflammation can lead to life-threatening or severely debilitating diseases. Mesenchymal stem cell (MSC) transplant has proved to be an effective therapy in preclinical studies which evaluated a vast diversity of inflammatory conditions. MSCs lead to resolution of inflammation, preparation for regeneration and actual regeneration, and then ultimate return to normal baseline or homeostasis. However, in clinical trials of transplanted MSCs, the expectations of great medical benefit have not yet been fulfilled. As a practical alternative to MSC transplant, a synthetic drug with the capacity to boost endogenous MSC expansion and/or activation may also be effective. Regarding this, IMT504, the prototype of a major class of immunomodulatory oligonucleotides, induces in vivo expansion of MSCs, resulting in a marked improvement in preclinical models of neuropathic pain, osteoporosis, diabetes and sepsis. IMT504 is easily manufactured and has an excellent preclinical safety record. In the small number of patients studied thus far, IMT504 has been well-tolerated, even at very high dosage. Further clinical investigation is necessary to demonstrate the utility of IMT504 for resolution of inflammation and regeneration in a broad array of human diseases that would likely benefit from an immunoprotective/immunoregenerative therapy.

Proposed mechanisms for oligonucleotide IMT504 induced diabetes reversion in a mouse model of immunodependent diabetes.

Type 1 diabetes (T1D) originates from autoimmune ß-cell destruction. IMT504 is an immunomodulatory oligonucleotide that increases mesenchymal stem cell cloning capacity and reverts toxic diabetes in rats. Here, we evaluated long-term (20 doses) and short-term (2-6 doses) effects of IMT504 (20 mg·kg(-1)·day(-1) sc) in an immunodependent diabetes model: multiple low-dose streptozotocin-injected BALB/c mice (40 mg·kg(-1)·day(-1) ip for 5 consecutive days). We determined blood glucose, glucose tolerance, serum insulin, islet morphology, islet infiltration, serum cytokines, progenitor cell markers, immunomodulatory proteins, proliferation, apoptosis, and islet gene expression. IMT504 reduced glycemia, induced ß-cell recovery, and impaired islet infiltration. IMT504 induced early blood glucose decrease and infiltration inhibition, increased ß-cell proliferation and decreased apoptosis, increased islet indoleamine 2,3-dioxygenase (IDO) expression, and increased serum tumor necrosis factor and interleukin-6 (IL-6). IMT504 affected islet gene expression; preproinsulin-2, proglucagon, somatostatin, nestin, regenerating gene-1, and C-X-C motif ligand-1 cytokine (Cxcl1) increased in islets from diabetic mice and were decreased by IMT504. IMT504 downregulated platelet endothelial cell adhesion molecule-1 (Pecam1) in islets from control and diabetic mice, whereas it increased regenerating gene-2 (Reg2) in islets of diabetic mice. The IMT504-induced increase in IL-6 and islet IDO expression and decreased islet Pecam1 and Cxcl1 mRNA expression could participate in keeping leukocyte infiltration at bay, whereas upregulation of Reg2 may mediate ß-cell regeneration. We conclude that IMT504 effectively reversed immunodependent diabetes in mice. Corroboration of these effects in a model of autoimmune diabetes more similar to human T1D could provide promising results for the treatment of this disease.

PyNTTTTGT and CpG immunostimulatory oligonucleotides: effect on granulocyte/monocyte colony-stimulating factor (GM-CSF) secretion by human CD56+ (NK and NKT) cells.

CD56+ cells have been recognized as being involved in bridging the innate and acquired immune systems. Herein, we assessed the effect of two major classes of immunostimulatory oligonucleotides (ODNs), PyNTTTTGT and CpG, on CD56+ cells. Incubation of human peripheral blood mononuclear cells (hPBMC) with some of these ODNs led to secretion of significant amounts of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and granulocyte/monocyte colony-stimulating factor (GM-CSF), but only if interleukin 2 (IL2) was present. IMT504, the prototype of the PyNTTTTGT ODN class, was the most active. GM-CSF secretion was very efficient when non-CpG ODNs with high T content and PyNTTTTGT motifs lacking CpGs were used. On the other hand, CpG ODNs and IFNα inhibited this GM-CSF secretion. Selective cell type removal from hPBMC indicated that CD56+ cells were responsible for GM-CSF secretion and that plasmacytoid dendritic cells (PDCs) regulate this process. In addition, PyNTTTTGT ODNs inhibited the IFNα secretion induced by CpG ODNs in PDCs by interference with the TLR9 signaling pathway. Since IFNα is essential for CD56+ stimulation by CpG ODNs, there is a reciprocal interference of CpG and PyNTTTTGT ODNs when acting on this cell population. This suggests that these synthetic ODNs mimic different natural alarm signals for activation of the immune system.

Non-clinical safety studies of IMT504, a unique non-CpG oligonucleotide.

IMT504 is a non-CpG 24-mer oligodeoxynucleotide (ODN) with immunomodulatory as well as tissue repair activity. IMT504 has been previously proven to be effective in animal models of vaccine potency, chronic lymphocytic leukemia, tissue regeneration, and sepsis. Here, we assessed the safety, including pharmacokinetics and toxicity studies in rats and monkeys, of IMT504 in a single- or repeated-dose administration by the subcutaneous (SC) or intravenous (IV) routes. In rats, the maximum tolerated dose was determined to be 50 mg/kg when administered SC. Adverse effects at 50 mg/kg were mild and reversible liver injury, revealed as lobular inflammation, focal necrosis, and small changes in the transaminase profile. Dose-dependent splenomegaly and lymphoid hyperplasia, most probably associated with immune stimulation, were commonly observed. Rats and monkeys were also IV injected with a single dose of 10 or 3.5 mg/kg, and no adverse effects were observed. Rats injected IV with 10 mg/kg showed a transient increase in spleen weight, together with a slight increase in the marginal zone of the white pulp and in leukocyte count 2 days post-administration. In monkeys, this dosage caused slight changes in total serum complement and leukocyte count on day 14. No adverse effects were observed at 3.5 mg/kg IV in rats or monkeys. Therefore, this dose was defined as the "no observed adverse effect level" for this route. Furthermore, repeated-dose toxicity studies were performed in these species using 3.5 or 0.35 mg/kg/day IV for 6 weeks. A transient increase in the spleen and liver weight was observed at 3.5 mg/kg/day only in female rats. No changes in clotting time and activation of the alternative complement pathway were observed. The toxicity profile of IMT504 herein reported suggests a dose range in which IMT504 can be used safely in clinical trials.

Addition of the immunostimulatory oligonucleotide IMT504 to a seasonal flu vaccine increases hemagglutinin antibody titers in young adult and elder rats, and expands the anti-hemagglutinin antibody repertoire.

Flu vaccines are partially protective in infants and elder people. New adjuvants such as immunostimulatory oligonucleotides (ODNs) are strong candidates to solve this problem, because a combination with several antigens has demonstrated effectiveness. Here, we report that IMT504, the prototype of a major class of immunostimulatory ODNs, is a potent adjuvant of the influenza vaccine in young adult and elderly rats. Flu vaccines that use virosomes or whole viral particles as antigens were combined with IMT504 and injected in rats. Young adult and elderly animals vaccinated with IMT504-adjuvated preparations reached antibody titers 20-fold and 15-fold higher than controls, respectively. Antibody titers remained high throughout a 120 day-period. Animals injected with the IMT504-adjuvated vaccine showed expansion of the anti-hemagglutinin antibody repertoire and a significant increase in the antibody titer with hemagglutination inhibition capacity when confronted to viral strains included or not in the vaccine. This indicates that the addition of IMT504 in flu vaccines may contribute to the development of significant cross-protective immune response against shifted or drifted flu strains.

A high dose of IMT504, the PyNTTTTGT prototype immunostimulatory oligonucleotide, does not alter embryonic development in rats.

Synthetic oligodeoxynucleotides (ODNs) are currently being evaluated as vaccine adjuvants for inducing protective immunity. As maternal vaccination is becoming increasingly common, the potential risk of vaccine formulation using ODN adjuvants should be warranted. A recent study performed in mice suggests that exposure to CpG motifs during pregnancy could result (although at very high doses as compared to the ones proposed for human vaccination) in fetal loss and morphological defects. PyNTTTTGT ODNs are immunostimulatory ODNs not bearing CpG motifs, which are very efficient vaccine adjuvants. In this report, we analyzed the potential teratogenic effect of its prototype IMT504 in rats. This animal model was chosen because PyNTTTTGT ODNs are barely active in mice. Intraperitoneal injection of IMT504 at a dose of 20 mg/kg (more than 1000 times higher than the one proposed for a vaccine dose in humans) at day 6 of pregnancy did not produce a significant decrease in the mean number of implanted fetuses or in the number of live pups delivered. Neither the fetuses nor the offspring presented malformations.

Oligonucleotide IMT504 reduces neuropathic pain after peripheral nerve injury.

We have recently shown that the administration of bone marrow stromal cells (MSCs) prevents the development of mechanical and thermal allodynia in animals subjected to a sciatic nerve injury. Furthermore, exogenously administered MSCs have been shown to participate in the repair and regeneration of damaged tissues in a variety of animal models. However, some limitations of this therapeutic approach, basically related to the ex vivo cell manipulation procedure, have arisen. IMT504, the prototype of the PyNTTTTGT class of immunostimulatory oligonucleotides, stimulates MSC expansion both in vitro and in vivo. In this study, we evaluated the effect of IMT504 systemic administration on the development of mechanical and thermal allodynia in rats subjected to a sciatic nerve crush. Animals were treated with IMT504, MSCs or saline either immediately after performing the lesion or 4 days after it, and were evaluated using the von Frey and Choi tests at different times after injury. Control animals developed both mechanical and thermal allodynia. Animals receiving either IMT504 or MSCs immediately after injury did not develop mechanical allodynia and presented a significantly lower number of nociceptive responses to cold stimulation as compared to controls. Moreover, injury-induced allodynia was significantly reduced after IMT504 delayed treatment. Our results show that the administration of IMT504 reduces neuropathic pain-associated behaviors, suggesting that IMT504 could represent a possible therapeutic approach for the treatment of neuropathic pain.

IMT504, the prototype of the immunostimulatory oligonucleotides of the PyNTTTTGT class, increases the number of progenitors of mesenchymal stem cells both in vitro and in vivo: potential use in tissue repair therapy.

Bone marrow (BM)-derived adult mesenchymal stem cells (MSCs) have the capacity to differentiate in vitro into different cell lines. This makes them a likely source for application in tissue repair therapies. Here, we report evidence indicating that, both in vivo and in vitro, IMT504, the prototype of the PyNTTTTGT class of immunostimulatory oligonucleotides, significantly increases the number of fibroblast colony-forming units (CFU-Fs) that originate MSCs. When rat BM cells were cultured with IMT504, the mean number of CFU-Fs increased about three times as compared with untreated controls (CFU-F: 19 +/- 6.3 vs. 6.8 +/- 2.0/2 x 10(6) seeded BM cells, p = .03). Furthermore, rats inoculated with IMT504 had a significantly higher number of CFU-Fs both in BM (CFU-F: 124 +/- 33 vs. 38 +/- 17/femur, p = .04) and in peripheral blood (animals with detectable CFU-Fs in circulation 8/12 vs. 2/12, p = .04) as compared with untreated animals. On the other hand, BM-derived adherent cells either treated in vitro with IMT504 or obtained from animals injected with IMT504 possess the capacity to differentiate to the osteogenic and adipogenic cell lineages as regular MSCs. Finally, we found that repair of a bone defect was accelerated in rats injected with IMT504 as compared with control animals (area with consolidated bone: 80% +/- 6.4% vs. 49% +/- 3.5%, p = .03, n = 10 rats per group). Importantly, when two human BM were cultured in the presence of IMT504, the mean number of fibroblastic adherent colonies also increased as compared with controls. These results suggest the possibility of clinical use of IMT504 in bone, and presumably other, tissue repair therapies.

Immunostimulatory PyNTTTTGT oligodeoxynucleotides: structural properties and refinement of the active motif.

It is well known that synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides within a given context stimulate B lymphocytes and plasmacytoid dendritic cells (PDCs) of the vertebrate immune system. We have reported that B lymphocyte and PDC stimulation in humans could also be efficiently achieved by using non-CpG ODNs bearing the immunostimulatory sequence (motif) PyNTTTTGT, wherein Py is C or T and N is any deoxyribonucleotide. We are now reporting a series of studies that gives further precision regarding the composition of this immunostimulatory motif. The analysis of hundreds of ODNs led us to the conclusion that the motif for optimal CpG-independent immune stimulation can be represented by a sequence of the following general formula: PyN(T/A)(T/C/G)(T/C/G)(T/G)GT, wherein Py is C or T and N is any deoxyribonucleotide and wherein at least two of the positions represented within parentheses are Ts. Requirements for optimal ODN activity are as follows: (1) at least one of the versions of the general motif must be located near the central portion of the ODN; and (2) the ODN must be 20 or more nucleotides long. PyN(T/A)(T/C/G)(T/C/G)(T/G)GT ODNs are active in a phosphorothioate or in a phosphodiester backbone. In a phosphodiester backbone a canonical motif is strongly required whereas in a phosphorothioate backbone specificity is, within certain limits, less strict. On the other hand, PyN(T/A)(T/C/G)(T/C/G)(T/G)GT oligonucleotides are inactive as a double chain or as a modified (phosphorothioate or hydroxyl-methyl modified) or unmodified RNA backbone.

Oligonucleotide IMT504 induces an immunogenic phenotype and apoptosis in chronic lymphocytic leukemia cells.

Oligonucleotides (ODNs) of the PyNTTTTGT class directly stimulate B lymphocytes and plasmacytoid dendritic cells of the immune system of primates. Here we investigated the ability of the PyNTTTTGT ODN prototype IMT504 to regulate the expression of surface molecules and apoptosis in human B-chronic lymphocytic leukemia (CLL) cells. The surface molecules CD25, CD40, CD80 and CD86 were up-regulated upon incubation of the B-CLL cells with IMT504. Co-stimulation with IL-2 resulted in further up-regulation. IMT504-activated B-CLL cells were also good stimulators of T cells in allogeneic mixed lymphocyte reactions and co-stimulation with IL-2 improved this stimulation capacity. Apoptosis of the B-CLL cells in vitro was also stimulated by incubation with IMT504. In this case, co-stimulation with IL-2 was not significant. Furthermore, B-CLL cells of all the patients studied developed an immunogenic phenotype and entered stimulated apoptosis upon in vitro incubation with IMT504 independently of the mutational status of their IgV(H) genes, becoming a good marker for tumor progression.

Ganglioside GM1-binding peptides as adjuvants of antigens inoculated by the intranasal route.

Forty-five GM1-binding peptides were identified using phage-displayed peptides libraries of random peptides. Most have a motif containing a hydrophobic amino acid followed by a serine (S). Based on a GM1-binding assays, two of these GM1-binding peptides (named 15 and 40) were chosen to investigate its immunostimulatory properties when chemically coupled to antigens. Mice intra-nasally (i.n.) vaccinated with some of these complexes developed a better local and systemic antibody response than mice i.n. vaccinated with the respective uncoupled antigens. The efficiency of the complex GM1-binding peptide-antigen strongly depends on the composition and structure of both of the components of the complex.

Oligonucleotide imt504 induces an immunogenic phenotype and apoptosis in chronic lymphocytic leukemia cells

Los oligonucleótidos (ODNs) de tipo PyNTTTTGT estimulan directamente las células B y las células dendríticas plasmacitoides del sistema inmune de primates. En este trabajo, investigamos la habilidad del IMT504, prototipo de los ODN tipo PyNTTTTGT, para regular la expresión demoléculas de superficie y la apoptosis en células B de leucemia linfocítica crónica (LLC). La expresión de lasmoléculas de superficie CD25, CD40, CD80 y CD86 fue aumentada al incubar las células B-LLC con IMT504. La co-estimulación con IL-2 provocó un aumento mayor. Las células B-LLC activadas fueron buenas estimuladorasde las células T en cultivo mixto de linfocitos alogeneicos y la co-estimulación con IL-2 mejoró esta capacidad. La apoptosis de las células B-LLC también fue estimulada por incubación con IMT504. En este caso, la coestimulación con IL-2 no fue significativa. Más aún, las células B-LLC de todos los pacientes estudiados,desarrollaron un fenotipo inmunogénico y entraron en apoptosis luego de la incubación in vitro con IMT504,independientemente del estado mutacional de sus genes IgVH , un indicador del pronóstico de la patología.