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.

Expression of osteoprotegerin, receptor activator of nuclear factor kappa-B ligand, tumor necrosis factor-related apoptosis-inducing ligand, stromal cell-derived factor-1 and their receptors in epithelial metastatic breast cancer cell lines.

BACKGROUND: While breast cancer (BC) is the major cause of death among women worldwide, there is no guarantee of better patient survival because many of these patients develop primarily metastases, despite efforts to detect it in its early stages. Bone metastasis is a common complication that occurs in 65-80 % of patients with disseminated disease, but the molecular basis underlying dormancy, dissemination and establishment of metastasis is not understood. Our objective has been to evaluate simultaneously osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), stromal cell-derived factor-1 (SDF-1), and their receptors (R) in 2 human BC cell lines, MDA-MB-231 and MCF-7. METHODS: OPG, RANKL, TRAIL and SDF-1 expression and release, in addition to the expression of their receptors has been investigated using immunofluorescence, immunocytochemistry and ELISA analyses. RESULTS: MCF-7 cells released higher levels of OPG in conditioned media (CM) than MDA-MB-231 cells; 100 % of both types of cell expressed OPG, RANKL, TRAIL and SDF-1. Moreover, 100 % in both lines expressed membrane RANKL and RANK, whereas only 50 % expressed CXCR4. Furthermore, 100 % expressed TRAIL-R1 and R4, 30-50 % TRAIL-R2, and 40-55 % TRAIL-R3. CONCLUSIONS: MCF-7 and MDA-MB-231 cells not only released OPG, but expressed RANKL, TRAIL and SDF-1. The majority of the cells also expressed RANK, CXCR4 and TRAIL-R. Since these ligands and their receptors are implicated in the regulation of proliferation, survival, migration and future bone metastasis during breast tumor progression, assessment of these molecules in tumor biopsies of BC patients could be useful in identifying patients with more aggressive tumors that are also at risk of bone metastasis, which may thus improve the available options for therapeutic intervention.

Megakaryocyte-stromal cell interactions: Effect on megakaryocyte proliferation, proplatelet production, and survival.

Bone marrow stromal cells provide a proper environment for the development of hematologic lineages. The incorporation of different stromal cells into in vitro culture systems would be an attractive model to study megakaryopoiesis and thrombopoiesis. Our objective was to evaluate the participation of different types of stromal cells in in vitro megakaryopoiesis, thrombopoiesis, and megakaryocyte (MK) survival. CD34-positive progenitors from umbilical cord blood were differentiated into MK precursors and then cocultured with umbilical vein endothelial cells (HUVECs), bone marrow mesenchymal stem cells (MSCs), skin fibroblasts (SFs) (all human), or the mouse fibroblast cell line L929. The number of MKs (CD61-positive cells) was increased in the presence of HUVECs and SFs, whereas L929 cells decreased total and mature MK counts. With respect to thrombopoiesis, HUVECs increased proplatelet (PP)-producing MKs, while MSCs, L929 cells, and SFs had the opposite effect (immunofluorescence staining and microscopic analysis). MK survival was enhanced in MSC and SF co-cultures, as assessed by evaluation of pyknotic nuclei. However, HUVECs and L929 did not prevent apoptosis of MKs. Reciprocally, the cloning efficiency of MSCs was decreased in the presence of MKs, while the ability of stromal cells (either MSCs or SFs) to produce the extracellular matrix proteins type III collagen, fibronectin, dermatan sulfate, heparan sulfate, and prolyl 4-hydroxylase subunit ß was preserved. These data indicate that each stromal cell type performs distinctive functions that differentially modulate MK growth and platelet production and, at the same time, that MKs also modify stromal cell behavior. Overall, our results highlight the relevance of considering the influence of stromal cells in MK research as well as the close interplay of different cell types within the bone marrow milieu.

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.

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.

Behaviour of mesenchymal stem cells from bone marrow of untreated advanced breast and lung cancer patients without bone osteolytic metastasis.

Tumour cells can find in bone marrow (BM) a niche rich in growth factors and cytokines that promote their self-renewal, proliferation and survival. In turn, tumour cells affect the homeostasis of the BM and bone, as well as the balance among haematopoiesis, osteogenesis, osteoclastogenesis and bone-resorption. As a result, growth and survival factors normally sequestered in the bone matrix are released, favouring tumour development. Mesenchymal stem cells (MSCs) from BM can become tumour-associated fibroblasts, have immunosuppressive function, and facilitate metastasis by epithelial-to-mesenchymal transition. Moreover, MSCs generate osteoblasts and osteocytes and regulate osteoclastogenesis. Therefore, MSCs can play an important pro-tumorigenic role in the formation of a microenvironment that promotes BM and bone metastasis. In this study we showed that BM MSCs from untreated advanced breast and lung cancer patients, without bone metastasis, had low osteogenic and adipogenic differentiation capacity compared to that of healthy volunteers. In contrast, chondrogenic differentiation was increased. Moreover, MSCs from patients had lower expression of CD146. Finally, our data showed higher levels of Dkk-1 in peripheral blood plasma from patients compared with healthy volunteers. Because no patient had any bone disorder by the time of the study we propose that the primary tumour altered the plasticity of MSCs. As over 70 % of advanced breast cancer patients and 30-40 % of lung cancer patients will develop osteolytic bone metastasis for which there is no total cure, our findings could possibly be used as predictive tools indicating the first signs of future bone disease. In addition, as the MSCs present in the BM of these patients may not be able to regenerate bone after the tumour cells invasion into BM/bone, it is possible that they promote the cycle between tumour cell growth and bone destruction.

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.

Higher oxidation and lower antioxidant levels in peripheral blood plasma and bone marrow plasma from advanced cancer patients.

BACKGROUND: Bone marrow (BM) is an important tissue in the generation of immunocompetent and peripheral blood cells. The precursors of hematopoietic cells in BM undergo continuous proliferation and differentiation and are highly vulnerable to acute and chronic oxidative stress. Little is known about the oxidant and antioxidant status in the BM of untreated patients with nonhematologic tumors. In this study, oxidative stress was evaluated in peripheral blood plasma (PBP) and BM plasma (BMP) from lung carcinoma (LC) and breast carcinoma (BC) patients. METHODS: The sample included 13 consecutive untreated LC patients, 15 BC patients, and 11 healthy controls. Luminol-dependent chemiluminescence was used to evaluate oxygen radical generation by peripheral blood neutrophils. Lipid oxidation, assessed by 2-thiobarbituric acid-reactive substances (TBARS), and alpha-tocopherol, beta-carotene, and total ubiquinol-10 levels were determined in PBP and BMP. RESULTS: In LC and BC patients, neutrophil chemiluminescence was higher (128% and 264%, respectively) than in controls (P < 0.05). In cancer patients, TBARS levels were higher in both PBP (51% and 243% for LC and BC patients, respectively) and BMP (66% and 305% for LC and BC patients, respectively) than in plasma from controls (P < 0.01). alpha-Tocopherol and total ubiquinol-10 levels were significantly lower in BMP from BC patients compared with controls. In BC patients, alpha-tocopherol content in PBP was significantly lower than in controls. CONCLUSIONS: Untreated cancer patients presented an imbalance between oxidant generation and lipid-soluble antioxidant levels in favor of the former.

Alteraciones leucocitarias en ratones portadores de sarcoma 180 / Leukocyte changes in mice bearing sarcoma 180

Se determinaron los valores avsolutos de la sdistintas poblaciones leucocitarias periféricas en ratones BALB/c normales y esplenectomizados (XB), desafiados con Sarcoma 180 sólido (S180s) y ascítico (S180a), inoculados por vías subcutánea e intraperitoneal, respectivamente. Las evaluaciones fueron realizadas al 8-, 15- y 25- día de crecimiento tumoral. Se observó: 1) leucocitosis en todos los grupos; 2) disminución del número de linfocitos al 15- y 25- día y disminución de monocitos al día 25-, en el grupo con S180s; 3) en el grupo XB, un incremento de todas las poblaciones leucocitarias, máximo entre el 8- y 15- día, valores que tendieron a normalizarse en el día 25-, en el grupo XB inoculado con S18s, neutrofilia, linfocitosis y monocitosis hasta el día 25-; 4) los valores máximos de neutrofilia correpsondieron al grupo S180a con 1905% de incremento en el día 15-, mientras que las cifras mayores de linfocitos y monocitos fueron observadas en el brupo XB inoculado con S180s en el día 15- con incrementos de 387% y 589% respectivamente, y 5) el aumento de neutrófilos fue constante en todos los grupos, observándose además altereaciones particulares en cada grupo

Antitumoral activity of polysaccharide isolated from Cyttaria johowii on the growth of sarcoma 180 in normal and splenectomized mice

Se estudió la actividad antitumoral del polisacárido PCj3 aislado del hongo Cyttaria johowii sobre el crecimiento de Sarcoma 180 (S180) sólido en ratones BALB/c normales y esolenectomizados, observando que este tratamiento inhibió el desarrollo del tumor tanto en ratones normales como esplenectomizados. Simultáneamente se evaluó el efecto del PCj3 sobre las poblaciones leucocitarias de sangre periférica al 8§, 15§ y 25§ día del experimento. Los resultados obtenidos al 8§ día muestran que todos los grupos inoculados con S180 sufrieron un incremento del número de linfocitos y neutrófilos, correspondiendo la cifra superior a los animales esplenectomizados desafiados con S180 y tratados con PCj3. El incremento de neutrófilos continuó en todos los animales con o sin tratamiento con PCj3 hasta el final del experimento, mientras que la linfocitosis sólo se mantuvo en los grupos esplenectomizados. El tratamiento con PCj3 causó un incremento del número de monocitos respecto al valor normal al 8§ día. Concluimos de acuerdo con estas observaciones que la actividad antitumoral del PCj3 fue más significativa en los ratones esplenectomizados, incrementando además este tratamiento los días de sobrevida, así como los valores de linfocitos, neutrófilos y monocitos en el 8§ día del crecimiento tumoral con respecto a los otros grupos

Accion de un polisacarido aislado del hongo Cyttaria johowii sobre el crecimiento del sarcoma 180 en ratones. / Effect of a polysaccharide isolated from the fungus Cyttaria johowii on the growth of sarcoma 180 in mice

Se estudio la actividad antitumoral de un polisacarido homogeneo obtenido del hongo Cyttaria johowii, a traves del efecto inhibitorio que produjo sobre el crecimiento del sarcoma 180 ascitico, inoculado por via intradermica o intraperitoneal en ratones endocriados de la cepa BALB/c. El polisacarido fue inoculado por via intraperitoneal durante 5 dias consecutivos comenzando 24 horas despues del inoculo tumoral. La dosis utilizada fue de 5 y 10 mg/kg raton/dia, para los grupos desafiados con sarcoma 180 intradermico y con sarcoma 180 intraperitoneal, respectivamente. Pudo observarse que el tratamiento con polisacarido produjo disminucion del crecimiento tumoral, maximo al 8o. dia tanto en los ratones inoculados con tumor por via id como ip. Ademas, se comprobo aumento del porcentaje de sobrevivientes y prolongacion de la media de dias de sobrevida, comparado con los animales tratados solo con tumor. Simultaneamente, fue analizada la accion del polisacarido sobre las poblaciones celulares de sangre periferica, observandose una disminucion significativa de linfocitos, maximo en el dia 15 en los ratones inoculados con sarcoma 180 ip, estas variaciones hematologicas no fueron observadas en los ratones desafiados con sarcoma 180 id. Ademas, se comprobo disminucion de la agregacion plaquetaria en los ratones con tumor tratados con polisacarido, respecto a los no tratados

Producción de interleuquina 1 durante el crecimiento tumoral / Production of interleukin 1 during tumor development

La producción de IL-1 por células esplénicas mononucleares adherentes (CMA) de ratones BALB/c inoculados con Sarcoma 180(S180) fue estudiada como uno de los posibles mecanismos responsables de la inmunosupresión observada durante el crecimiento tumoral. Como agentes estimulantes se utilizó un polisacárido químicamente definido, PCj3, y un lipopolisacárido de E. coli (LPS). La actividad de IL-1 se valoró en base al efecto estimulante de los sobrenadantes de cultivos en CMA sobre la respuesta proliferativa de timocitos murinos frente a la fitohemaglutina (PHA). Ambos esdtimulantes indujeron niveles comparables de IL-1 tanto en ratones normales como en portadores de S180 de 10 días. A los 20 y 30 días de desarrollo tumoral, las CMA disminuyeron significativamente la producción de IL-1 en respuesta a ambos estimulantes. Esos resultados permiten suponer que la inmunosupresión observada en los ultimos estadíos del crecimientos tumoral podría ser consecuencia de la inhibición de la producción de IL-1

Intracytoplasmatic and extracellular interleukin-1 production by monocytes of lung and colorectal cancer patients

We studied the production of interleukin-1 (IL-1) by peripheral blood monocytes (Mo) from twelve normal subjects (NS) and eight and nine untreated lung and colorectal cancer patients (CP), respectively. No significant changes of extracellular IL-1 biological activity was observed between CP and NS by thymocyte proliferation assay. This result was independent that the cells were treated or not with lipopolisaccharide from E. coli (LPS, 10 Mug/ml). Moreover, CP present normal amount of antigenic IL-1 Beta in LPS treated Mo culture supernatants by enzyme-linked immunosorbent asay (ELISA). The biological activity of IL-1 released was not significant modified after indomethacin (Indo, 10-6M) and LPS+Indo treatments. Furthermore, patients showed a low percentage of LPS activated Mo with intracytoplasmatic IL-1 (alpha + beta) compared to normal values. These results were obtained by immuno-alkaline phosphatase staining using monoclonal antibody anti IL-1 (alpha + beta). In conclusion, CP had a reduced number of Mo with intracytoplasmatic IL-1 (alpha + beta) and the difference observed may depend on degradation or in the rate of synthesis of this cytokine.