The novel immunotherapeutic oligodeoxynucleotide IMT504 protects neutropenic animals from fatal Pseudomonas aeruginosa bacteremia and sepsis.

IMT504 is a novel immunomodulatory oligonucleotide that has shown immunotherapeutic properties in early preclinical and clinical studies. IMT504 was tested in a neutropenic rat model of Pseudomonas aeruginosa bacteremia and sepsis. This animal system recapitulates many of the pathological processes found in neutropenic patients with Gram-negative, bacterial infections. The research was conducted in the setting of an academic research laboratory. The test subjects were Sprague-Dawley rats. Animals were rendered neutropenic by administration of cyclophosphamide, colonized with P. aeruginosa by oral feeding, and then randomized to receive IMT504 over a range of doses and treatment regimens representing early and late therapeutic interventions. IMT504 immunotherapy conferred a significant survival advantage over the 12-day study period compared with the results seen with placebo-treated animals when the therapy was administered at the onset of neutropenia and even in the absence of antibiotics and after the onset of fever and systemic infection. Notably, even late salvage IMT504 monotherapy was highly effective (13/14 surviving rats with IMT504 therapy versus 2/14 controls, P=<0.001). Moreover, late salvage IMT504 monotherapy was as effective as antibiotic therapy (13/14 surviving rats versus 21/21 rats, P=0.88). In addition, no antagonism or loss of therapeutic efficacy was noted with combination therapy of IMT504 plus antibiotics. IMT504 immunotherapy provides a remarkable survival advantage in bacteremia and sepsis in neutropenic animals and deserves further study as a new treatment option in patients with, or at risk for, severe Gram-negative bacterial infections and sepsis.

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.

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.

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.

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.

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.

Phylogenetic relationships of the genus Kluyvera: transfer of Enterobacter intermedius Izard et al. 1980 to the genus Kluyvera as Kluyvera intermedia comb. nov. and reclassification of Kluyvera cochleae as a later synonym of K. intermedia.

In order to assess the relationship between the genus Kluyvera and other members of the family Enterobacteriaceae, the 16S rRNA genes of type strains of the recognized Kluyvera species, Kluyvera georgiana, Kluyvera cochleae, Kluyvera ascorbata and Kluyvera cryocrescens, were sequenced. A comparative phylogenetic analysis based on these 16S rRNA gene sequences and those available for strains belonging to several genera of the family Enterobacteriaceae showed that members of the genus Kluyvera form a cluster that contains all the known Kluyvera species. However, the type strain of Enterobacter intermedius (ATCC 33110T) was included within this cluster in a very close relationship with the type strain of K. cochleae (ATCC 51609T). In addition to the phylogenetic evidence, biochemical and DNA-DNA hybridization analyses of species within this cluster indicated that the type strain of E. intermedius is in fact a member of the genus Kluyvera and, within it, of the species Kluyvera cochleae. Therefore, following the current rules for bacterial nomenclature and classification, the transfer of E. intermedius to the genus Kluyvera as Kluyvera intermedia comb. nov. is proposed (type strain, ATCC 33110T=CIP 79.27T=LMG 2785T=CCUG 14183T). Biochemical analysis of four E. intermedius strains and one K. cochleae strain independent of the respective type strains further indicated that E. intermedius and K. cochleae represent the same species and are therefore heterotypic synonyms. Nomenclatural priority goes to the oldest legitimate epithet. Consequently, Kluyvera cochleae Muller et al. 1996 is a later synonym of Kluyvera intermedia (Izard et al. 1980) Pavan et al. 2005.

PyNTTTTGT prototype oligonucleotide IMT504 is a potent adjuvant for the recombinant hepatitis B vaccine that enhances the Th1 response.

PyNTTTTGT oligodeoxinucleotides (ODNs) cause activation, proliferation and immunoglobulin secretion on B cells, and the expression of co-stimulatory molecules on plasmacytoid dendritic cells of primates. It has now been discovered that these ODNs are also active on rat cells. This fact allowed us to investigate the adjuvant properties of PyNTTTTGT ODNs in a human Hepatitis B vaccine using this animal model. A very significant increment, as compared with the antigen alone, was observed in the antibody production induced by vaccination with the recombinant Hepatitis B surface antigen adjuvated with the PyNTTTTGT prototype IMT504 ODN. Analysis of the IgG subclass distribution in the sera of vaccinated animals indicated that, although an increase was observed in the titer of all the IgG subclasses, the increase on the Th1-associated IgG2b subclass was clearly more pronounced. Remarkably, this effect on the IgG2b titer was observed even if alum, a Th2 promoting adjuvant, was present together with IMT504 in the vaccine formulation. The increase in the Th1 response induced by IMT504 was also suggested by in vitro gamma interferon secretion assays. Monkeys of the species Cebus apella immunized with the recombinant Hepatitis B surface antigen plus alum and IMT504 also showed titers of antibodies against the antigen several times superior to the titers observed in control animals immunized with the antigen plus alum without ODN. Since rat and monkey cells are significantly less immunostimulated "in vitro" by PyNTTTTGT ODNs than human cells, the present results reasonably predict a very good performance of these ODNs as adjuvants in human vaccination.

Strong cytosine-guanosine-independent immunostimulation in humans and other primates by synthetic oligodeoxynucleotides with PyNTTTTGT motifs.

Synthetic oligodeoxynucleotides (ODNs) containing cytosine-guanosine (CpG) motifs stimulate B and plasmacytoid dendritic cells of the vertebrate immune system. We found that in primates strong stimulation of these cells could also be achieved using certain non-CpG ODNs. The immunostimulatory motif in this case is a sequence with the general formula PyNTTTTGT in which Py is C or T, and N is A, T, C, or G. Assays performed on purified cells indicated that the immunostimulatory activity is direct. The use of a nuclease-resistant phosphorothioate backbone is not a necessary condition, since phosphodiester PyNTTTTGT ODNs are active. It was also demonstrated that ODN 2006, a widely used immunostimulant of human B cells, possess two kinds of immunostimulatory motifs: one of them mainly composed of two successive TCG trinucleotides located at the 5' end and another one (duplicated) of the PyNTTTTGT kind here described. Even though PyNTTTTGT ODNs are mainly active on primate cells, some of them, bearing the CATTTTGT motif, have a small effect on cells from other mammals. This suggests that the immunostimulatory mechanism activated by these ODNs was present before, but optimized during, evolution of primates. Significant differences in the frequency of PyNTTTTGT sequences between bacterial and human DNA were not found. Thus, the possibility that PyNTTTTGT ODNs represent a class of pathogen-associated molecular pattern is unlikely. They could, more reasonably, be included within the category of danger signals of cell injury.

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. (AU)

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.

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. (AU)

Construcción de sondas moleculares para el diagnóstico de micobacterias / Development of molecular probes for the diagnosis of mycobacteria

Se demostró la presencia de una secuencia repetitiva en el DNA del Mycobacterium bovis BCG. Esta secuencia se encontró también en el DNA de M. tuberculosis, pero no se detectó en M. Kansasii, M. flavescens, M. fortuitum, M. vaccae, M. leprae, M. phlei, M. smagmatis y M. marinum. Esta secuencia repetitiva fue muy polimorfa en M. tuberculosis pero no en BCG. El análisis de hidridización sugiere que la secuencia repetitiva fue muy polimorfa en M. tuberculosis pero no en BCG. El análisis de hibridización sugiere que la secuencia repetitiva podría ser útil en la identificación de Mycobacterium patógeno en muestras clínicas

Condiloma acuminado gigante de Buschke y Lowenstein HPV tipo 16-18 tratado con metotrexato / Giant condyloma of Buschke-Lowenstein; type 16-18: treatment with methotrexate

Se presenta un caso de Condiloma acuminado gigante de Buschke y Lowenstein positivo para HPV tipo 16-18, de localización perianal tratado con metotrexato como primer paso para reducir masa tumoral. Se recalca la importancia de efectuar controles médicos periódicos con biopsias para pesquisar la aparición de un epitelioma espinocelular, principalmente si el H.P.V. causal es de conocido potencial carcinogenético. Realizamos una breve revisión del tema