Mental Well-being InSciEd Out: Health Partnerships with the Boys & Girls Clubs of Puerto Rico.

BACKGROUND: Mental health care is a top clinical concern for modern Puerto Rico, especially given a dramatically changing economic landscape paired with recurrent natural disasters. Youth are particularly at-risk due to long-term impacts of toxic stress and adverse childhood experiences on health and development. OBJECTIVES: Here we present a novel clinician-community-educator-scientist partnership to address Puerto Rican youth mental well-being and wellness. We deployed pilot health workshops within the Boys & Girls Clubs of Puerto Rico to build youth mental health conceptual understanding and competencies in stress recognition and management. The work in progress herein evaluates acceptability and feasibility of our curricular model. METHODS: Dialogue with community stakeholders guided curricular design of workshops for youth ages 6 to 13 and older. Prior to implementation, educators and volunteers attended a 1-day training on educational strategies. Workshop success was evaluated using qualitative approaches (i.e., narrative feedback, educator and volunteer reflections, youth Talking Drawings) to assess youth engagement, youth conceptual health understanding, and educator/volunteer impressions of feasibility and impact. RESULTS: Initial findings indicate high acceptability and feasibility of our curricular model. Youth engagement and enthusiasm were noted in educator feedback and continue to be sustained post-workshop. Preliminary analysis shows accompanying increases in youth conceptual mental health understanding, particularly for 6- to 12-year-olds in recognition of stress and healthy coping mechanisms. Reciprocal gains were observed for volunteers. CONCLUSIONS: Activities have evolved into a formal partnership called Semilla, which features expanded analysis of mental well-being and wellness outcomes. Our collaborative model continues to engage Puerto Rican youth in the science of their well-being.

Raman Spectroscopy Study of Skin Biopsies from Patients with Parkinson's Disease: Trends in Alpha-Synuclein Aggregation from the Amide I Region.

Parkinson's disease (PD) is one of the most common neurological pathologies with a high prevalence worldwide. PD is characterized by Lewy bodies, whose major component is the aggregates of α-synuclein (αSyn) protein. Interestingly, recent works have demonstrated that skin biopsy studies are a promising diagnostic tool for evaluating α-synucleinopathies. In this sense, this work focuses on the detection of αSyn in skin biopsies employing Raman spectroscopy, using three different approaches: (i) the in vitro Raman spectrum of α-synuclein, (ii) the ex vivo Raman spectra of human skin biopsies from healthy and Parkinson's disease patients, and (iii) theoretical calculations of the Raman spectra obtained from different model αSyn fragments using density functional theory (DFT). Significant differences in the intensity and location of Raman active frequencies in the amide I region were found when comparing healthy and PD subjects related to α-synuclein conformational changes and variations in their aggregation behavior. In samples from healthy patients, we identified well-known Raman peaks at 1655, 1664, and 1680 cm-1 associated with the normal state of the protein. In PD subjects, shifted Raman bands and intensity variations were found at 1650, 1670, and 1687 cm-1 associated with aggregated forms of the protein. DFT calculations reveal that the shape of the amide I Raman peak in model αSyn fragments strongly depends on the degree of aggregation. Sizable frequency shifts and intensity variations are found within the highly relevant 1600-1700 cm-1 domain, revealing the sensitivity of the amide I Raman band to the changes in the local atomic environment. Interestingly, we obtain that the presence of surrounding waters also affects the structure of the amide I band, leading to the appearance of new peaks on the low-frequency side and a notable broadening of the Raman spectra. These results strongly suggest that, through Raman spectroscopy, it is possible to infer the presence of aggregated forms of αSyn in skin biopsies, a result that could have important implications for understanding α-synuclein related diseases.

Potential of Mean Force Calculations for an SN2 Fluorination Reaction in Five Different Imidazolium Ionic Liquid Solvents Using Quantum Mechanics/Molecular Mechanics Molecular Dynamics Simulations.

The use of ionic liquids (ILs) as both catalysts and solvents in a wide range of chemical reactions has received considerable attention over the last few years due to their positive effects in enhancing reaction rates and selectivities. In this work, hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations were carried out in conjunction with umbrella-sampling techniques to study the bimolecular nucleophilic substitution (SN2) fluorination reaction between propyl-mesylate and potassium fluoride using five ILs as solvents, specifically, 1-butyl-3-methylimidazolium mesylate ([C4mim][OMs]), 1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim][BF4]), 1-butyl-3-methylimidazolium trifluoroacetate ([C4mim][CF3COO]), 1-butyl-3-methylimidazolium bromide ([C4mim][Br]), and 1-butyl-3-methylimidazolium chloride ([C4mim][Cl]) at 373.15 K. The QM region (reactive part) in all QM/MM systems was simulated using the Parametric Method 6 (PM6) semiempirical methods, and for the MM region (IL solvent), classical force fields (FF) were employed, with the FF developed within the group. The calculated activation free energy barriers (ΔG‡) for the SN2 reaction in the presence of [C4mim][OMs] and [C4mim][BF4] ILs were in agreement with the experimental values reported in the literature. On the other hand, only predicted values were obtained for the activation energies for the [C4mim][CF3COO], [C4mim][Br], and [C4mim][Cl] ILs. These activation energies indicated that the SN2 reaction would be more facile to proceed using the [C4mim][Cl] and [C4mim][OMs] ILs, in contrast with the use of [C4mim][Br] IL, which presented the highest activation energy. Energy-pair distributions, radial distribution functions, and noncovalent interactions (NCI) were also calculated to elucidate the molecular interactions between the reactive QM region and the solvents or reaction media. From these calculations, it was found that not only the reactivity can be enhanced by selecting a specific anion to increase the K-F separation but also the cation plays a relevant role, producing a synergetic effect by forming hydrogen bonds with the fluorine atom from KF and with the oxygen atoms within the mesylate leaving group. Three interactions are significant for the IL catalytic behavior, FQM-HX, KQM-anion, and OQM-HX interactions, where the FQM and KQM labels correspond to fluorine and potassium atoms from the KF salt, OQM corresponds to oxygen atoms within the mesylate leaving group (reactant), and HX refers to hydrogen atoms within the IL cation. The NCI analysis revealed that KQM-anion interactions are of weak type, indicating the importance of hydrogen bond interactions from the cation such as FQM-HX and OQM-HX for the catalytic behavior of ILs.

Thermodynamic, structural and dynamic properties of ionic liquids [C4mim][CF3COO], [C4mim][Br] in the condensed phase, using molecular simulations.

In this work a series of thermodynamic, structural, and dynamical properties for the 1-butyl-3-methylimidazolium trifluoroacetate ([C4mim][CF3COO]) and 1-butyl-3-methylimidazolium bromide, ([C4mim][Br]) ionic liquids (ILs) were calculated using Non-polarizable Force Fields (FF), parameterized using a methodology developed previously within the research group, for condensed phase applications. Properties such as the Vapor-Liquid Equilibrium (VLE) curve, critical points (ρ c, T c), Radial, Spatial and Combined Distribution Functions and self-diffusion coefficients were calculated using Equilibrium Molecular Dynamics simulations (EMD); other properties such as shear viscosities and thermal conductivities were calculated using Non-Equilibrium Molecular Dynamics simulations (NEMD). The results obtained in this work indicated that the calculated critical points are comparable with those available in the literature. The calculated structural information for these two ILs indicated that the anions interact mainly with hydrogen atoms from both the imidazolium ring and the methyl chain; the bromide anion displays twice the hydrogen coordination number than the oxygen atoms from the trifluoroacetate anion. Furthermore, Non-Covalent interactions (NCI index), determined by DFT calculations, revealed that some hydrogen bonds in the [C4mim][Br] IL displayed similar strength to those in the [C4mim][CF3COO] IL, in spite of the shorter O--H distances found in the latter IL. The majority of the calculated transport properties presented reasonable agreement with the experimental available data. Nonetheless, the self-diffusion coefficients determined in this work are under-estimated with respect to experimental values; however, by escalating the electrostatic atomic charges for the anion and cation to ±0.8e, only for this property, a remarkable improvement was obtained. Experimental evidence was recovered for most of the calculated properties and to the best of our knowledge, some new predictions were done mainly in thermodynamic states where data are not available. To validate the FF, developed previously within the research group, dynamic properties were also evaluated for a series of ILs such as [C4mim][PF6], [C4mim][BF4], [C4mim][OMs], and [C4mim][NTf2] ILs.

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.

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.

Manual wheelchair use: bouts of mobility in everyday life.

Background. This study aimed to describe how people move about in manual wheelchairs (MWCs) during everyday life by evaluating bouts of mobility or continuous periods of movement. Methods. A convenience sample of 28 MWC users was recruited. Participants' everyday mobility was measured using a wheel-mounted accelerometer and seat occupancy switch for 1-2 weeks. Bouts of mobility were recorded and characterized. Results. Across 29,200 bouts, the median bout lasted 21 seconds and traveled 8.6 m at 0.43 m/s. 85% of recorded bouts lasted less than 1 minute and traveled less than 30 meters. Participants' daily wheelchair activity included 90 bouts and 1.6 km over 54 minutes. Average daily occupancy time was 11 hours during which participants wheeled 10 bouts/hour and spent 10% of their time wheeling. Spearman-Brown Prophecy analysis suggested that 7 days were sufficient to achieve a reliability of 0.8 for all bout variables. Conclusions. Short, slow bouts dominate wheelchair usage in a natural environment. Therefore, clinical evaluations and biomechanical research should reflect this by concentrating on initiating movement, maneuvering wheelchairs, and stopping. Bouts of mobility provide greater depth to our understanding of wheelchair use and are a more stable metric (day-to-day) than distance or time wheeled.

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. (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. (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.

Qué es la Epilepsia? Attitudes and knowledge of epilepsy by Spanish-speaking adults in the United States.

BACKGROUND: Spanish-speaking adults are the largest minority population group in the United States and are disproportionately afflicted by epilepsy. METHODS: A unique 78-item survey instrument conducted entirely in Spanish and devoted to the topic of epilepsy was administered to 760 Spanish-speaking adults in seven large U.S. Hispanic metropolitan areas representing a cross section of the U.S. Hispanic community. The answers were compared with those of 272 non-Hispanic controls administered the same survey in English in June 2004. RESULTS: The Hispanic sample correlated well with U.S. Census data. Spanish-speaking adults are mostly unaware about epilepsy, with 21% reporting no familiarity with the condition (P=0.0001). The vast majority of Hispanics use the term convulsiones or ataque to describe a seizure. Thirteen percent of Hispanics with less than high school education believe that epilepsy is contagious (P=0.0001); 8% see "sins" as a cause of seizures (P=0.0001); and 10% agree that "exorcism" would be a good remedy (P=0.002). CONCLUSIONS: There is considerable misinformation about epilepsy in the U.S. Hispanic community. Neurologists must be made aware of U.S. Hispanic attitudes and beliefs regarding epilepsy to provide culturally competent care.

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.