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.

Histochemical study of the oviducal gland and analysis of the sperm storage tubules of Mustelus schmitti Springer, 1939 (Chondrichthyes, Triakidae)

The paired oviducal glands of immature and mature females of Mustelus schmitti were examined macro and microscopically. Findings indicate that these glands possessed the same zonation as in most chondrichthyans from anterior to posterior: club, papillary, baffle and terminal zones. The whole gland is composed by simple tubular glands that connect with transverse grooves all along the organ. The club zone presents a typical indian club shape with a simple columnar and ciliated epithelium including secretory cells PAS (+) and AB (+). The papillary zone is characterized by lamella forming small and long cones in numbers of three. The epithelium of this zone contains ciliated cells with apical nuclei and secretory cells with basal nuclei that stain AB (+)The baffle zone consists of apically flattened lamellae alternating with spinnerets which are small projections disposed by both sides of the plateau. This whole structure is present in number of 8 or 9 units. A simple columnar ciliated epithelium covers the plateau and spinnerets and no AB or PAS staining is observed. The epithelium of the terminal zone is PAS (-) and AB (+), and elongated tubules, that run adjacent to the baffle zone are the site where groups of spermatozoa are clearly observed in the lumen. The epithelium of the sperm storage tubules do not stain with any of the dyes tested. Sperm was also observed in the baffle zone, presumably in its way to the fecundation in the oviduct because it displays no aggregation pattern and was between the folds of the epithelium. By scanning electron microscopy sperm was observed in the club and baffle zones in a gland which belonged to a pregnant female...

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.

Análise da associação da fadiga com variáveis clínicas e psicológicas em uma série de 371 pacientes brasileiros com artrite reumatoide / Analysis of the association of fatigue with clinical and psychological variables in a series of 371 Brazilian patients with rheumatoid arthritis

Objetivos: A fadiga é um sintoma altamente subjetivo e extremamente comum em pacientes com artrite reumatoide, embora seja difícil de caracterizar e definir. O objetivo desse estudo foi avaliar a fadiga em uma coorte de pacientes brasileiros e analisar a relação entre fadiga e variáveis específicas da doença. Métodos: Foram prospectivamente investigados 371 pacientes brasileiros diagnosticados com artrite reumatoide, de acordo com os critérios de classificação do Colégio Americano de Reumatologia de 1987. Dados demográficos, clínicos e laboratoriais foram obtidos dos registros clínicos. Foram registrados o número de articulações dolorosas, índice de massa corporal, duração da doença, qualidade de vida, capacidade funcional, ansiedade e depressão. A fadiga foi avaliada com o uso da subescala específica da escala Fatigue Assessment of Chronic Illness Therapy (FACIT-FATIGUE). Resultados: O escore mediano para fadiga foi 42 (10), negativamente correlacionado com a capacidade funcional (-0,507; p < 0,001), ansiedade e depressão (-0,542 e -0,545; p < 0,001, respectivamente) e predominantemente com o domínio físico do questionário Short Form-36 para qualidade de vida (SF-36P: 0,584; p < 0,001). Não houve correlação entre os escores e a velocidade de sedimentação das hemácias (-0,118; p <0,05), proteína C reativa (-0,089; p < 0,05), atividade da doença (-0,250;p < 0,001) ou número de articulações dolorosas (-0,135; p < 0,01). Para todas as medidas foi aplicado um intervalo de confiança de 95%. Conclusões: Nesta série de pacientes brasileiros com artrite reumatoide, sugerimos um novo significado para as queixas de fadiga como um parâmetro independente não relacionado com o número de articulações ...

Objectives: Fatigue is a highly subjective and extremely common symptom in patients with rheumatoid arthritis although it is difficult to characterize and define. The aim of this study was to assess fatigue in a cohort of Brazilian patients, and to analyze the relationship between fatigue and disease-specific variables. Methods: 371 Brazilian patients diagnosed with rheumatoid arthritis according to the 1987 American College of Rheumatology classification criteria were prospectively investigated. Demographic, clinical and laboratorial data were obtained from hospitals records. The number of painful joints, bone mass index, disease duration, quality of life, functional capacity, anxiety and depression were recorded. Fatigue was evaluated using the subscale of Fatigue Assessment of Chronic Illness Therapy (FACIT-FATIGUE scale). Results: The median fatigue score was 42.0 (10.0), negatively correlated with functional capacity (-0.507; P < 0.001), anxiety and depression (-0.542 and -0.545; P < 0.001 respectively), and predominantly with physical domain of Short Form 36-item quality of life questionnaire (SF-36P: 0.584; P < 0.001). The scores were not associated with the erythrocyte sedimentation rate (-0.118; P < 0.05), C-reactive protein (-0.089; P < 0.05), disease activity (-0.250; P < 0.001) or the number of painful joints (-0.135; P < 0.01). Confidence interval of 95% was applied for all measures. Conclusions: In this series of Brazilian patients with rheumatoid arthritis, we suggest a new significance for fatigue complains as an independent parameter not related with number of painful joints, disease or inflammatory activity scores. Psychological and functional impairments appear to be more related to fatigue. Additional studies and inclusion of standard measures for monitoring fatigue complains are required. .

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.

Perfil lipídico e excesso de peso em escolares / Lipid profile and weight excess among school children

Objetivo: avaliar o perfil lipídico de 603 escolares de instituições de ensino fundamental e médio no município de Barbacena-MG e a associação com o índice de massa corporal (IMC) e circunferência de cintura (CC). Metodologia: foram determinados valores séricos de colesterol total, de lipoproteínas de baixa densidade (LD L-c), alta densidade (H D L-c), triacilgliceróis (TG), IMC e CC. A avaliação do perfil lipídico seguiu critérios propostos pela III Diretrizes Brasileiras sobre Dislipidemias. A condição de sobrepeso e obesidade foi estabelecida entre os escolares por meio do IMC e este foi classificado segundo critérios de Cole et al.14. Os dados foram analisados por correlação bivariada utilizando-se o teste t de "Student" para as variáveis quantitativas e o de qui-quadrado para analisar as associações entre as variáveis qualitativas. Resultados: a hipercolesterolemia esteve presente em 40,29 por cento da amostra, a hipertrigliceridemia em 12,43 por cento, o LD L-c elevado em 6,62 por cento e os níveis de H D L-c foram adequados para todos os escolares. O percentual de excesso de peso encontrado foi de 13,59 por cento , sendo 11,94 por cento de sobrepeso e 1,65 por cento de obesidade. A circunferência da cintura (CC) e o IMC não tiveram associação estatisticamente significativa (p<0,05) com dislipidemia. Conclusão: os resultados revelaram elevada prevalência de dislipidemia nos escolares, cujos índice de massa corporal e circunferência de cintura não foram associados a tal problema.

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.

Satisfação do paciente com o atendimento interdisciplinar numambulatório de prevenção da doença renal crônica / Patient’s satisfaction with interdisciplinary care in a clinic of prevention of chronic kidney disease

Introdução: Semelhantemente ao observado em outras doenças, a satisfação do paciente com o seu tratamento pode determinar melhor evolução da doença renal crônica (DRC). Objetivo: Avaliar a satisfação dos pacientes com o atendimento interdisciplinar recebido num ambulatório de prevenção da DRC. Métodos: A pesquisa de satisfação foi aplicada a pacientes do PREVENRIM – Programa Interdisciplinar de Prevenção da DRC. O diagnóstico e o estagiamento da DRC se fundamentaram nas diretrizes do KDOQI da NKF americana. A pesquisa de satisfação baseou-se em perguntas relativas à logística do atendimento, à importância dos membros da equipe no manejo da doença, à dinâmica do atendimento e à compreensão das informações sobrea DRC. Resultados: Foram avaliados 101 pacientes, 52,6% eram do sexo masculino, com idade média de 56±13,9 anos acompanhados em média por 32,66±18,17 meses. Do total de pacientes, 43,8% possuíam ensino fundamental incompleto. A principal causa de DRC foi nefroesclerose hipertensiva. A média da FG estimada foi de 31,59±15,02mL/min/1,73m2. A maioria absoluta (95,6%) dos pacientes relatou estar satisfeita com as explicações sobre as suas doenças. O atendimento foi considerado importante ou muito importante pelos pacientes com relação à atuação do nefrologista e da nutricionista (100%), assistente social e enfermeiro (98,8%), bem como da psicóloga (97,7%). Todos recomendariam o PREVENRIM a outros pacientes com DRC. Conclusão: Nesta amostra de pacientes com DRC pré-dialítica acompanhados por uma equipe interdisciplinar, observamos um alto nível de satisfação com os cuidados de saúde ofertados. Estudos futuros avaliarão se a satisfação do paciente com o seu tratamento determinará melhores desfechos biopsicossociais na evolução da DRC.

Introduction: Similarly to the observed in other diseases, the patient’s satisfaction with his or her treatment may determine better outcomes of the chronic kidney disease (CKD). Aim: To assess the satisfaction of patients with the interdisciplinary care delivered in a Clinic of secondary prevention of CKD. Methods: The satisfaction assessment was applied to patients of the PREVENRIM – Interdisciplinary Program of Prevention of CKD. The diagnosis and staging of CKD were done as proposed by the KDOQI of the American NKF. The satisfaction assessment was made through questions covering the logistic of the Clinic, the importance of each members of the team in the management of the disease, the dynamic of the interdisciplinary care, and the understanding of the information on CKD. Results: The questionnaire were applied to 101 patients, followed for 32,66 ± 18,17 months, 52,6% were male and the mean age was 56±13,9 years. Incomplete basic education was observed in 43,8% of the patients. The main cause of CKD was hypertensive nephroesclerosis. The mean estimated GFR was 31,59±15,02mL/min/1,73m2. The great majority (95,6%) of the patients reported to be satisfied with theexplanation given about their disease. Regarding the management of their disease, the patients considered important or very important the role played by nephrologists and dietician (100%), social worker and nurse (95,6%), and psychologist (97,7%). All the patients would refer patients with CKD to the PREVENRIM. Conclusion: In this group of patients with CKD not yet on dialysis, followed by an interdisciplinary team, we observed a high satisfaction level with the health care received. Future studies should assess if the level of patients’ satisfaction with their treatment will determine better outcomes in CKD.

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.

A função da obra na estabilização psicótica: análise do caso do Profeta Gentileza / Art function in psychotic stabilization: analysis of Profeta Gentileza’s case

Partindo da Reforma Psiquiátrica, investigamos a possibilidade de construção de enlaçamento social e simbólico por psicóticos, via criação artística, a fim de contribuir na elaboração de estratégias clínicas inclusivas e de reinserção social. Estudamos o caso do Profeta Gentileza, que pintou as pilastras do Viaduto do Caju-RJ com símbolos e grafias inéditas. Concluímos que a construção de seu processo de estabilização se deu via metáfora delirante, tendo sua obra funcionado enquanto contorno estético ao excesso de gozo que restou insignificantizável do trabalho com o delírio, permitindo uma saída que prescindiu do ato. Esse trabalho sustentou sua inserção social, tendo sua obra se tornado patrimônio cultural do Rio de Janeiro. Como efeito de aprendizado para os trabalhadores da saúde mental, se destacou a importância de acompanhar o estilo do sujeito na sua tentativa de cura, sustentando os movimentos deste diante do campo do Outro junto ao trabalho de reinserção social.

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.

AEB-removível como opção de tratamento da má-oclusão de classe II / Removable extra-oral appliance as an option to class II treatment

A classe II é uma má olusão comumente observada na clínica ortodôntica e apresenta inúmeras opções de tratamentos que incluem a ortopedia funcional dos maxilares e aparelhos extrabucais, entre outros. De acordo com o diagnóstico, em relação à localização do problema, maxila ou mandíbula, é determinado o tipo de aparatologia mais adequado para cada caso. O objetivo deste artigo é descrever o caso de um paciente com Classe II, divisão 1, apresentando protrusão maxilar. Ele foi tratado com sucesso utilizando-se o aparelho AEB-removível.

Fragilidade osmótica eritrocitária em gatos acometidos por hepatopatias e gatos com insuficiência renal / Erythrocyte osmotic fragility in cats with hepatic disease and cats with renal failure

A fragilidade osmótica eritrocitária (FOE) avalia a resistência osmótica das células vermelhas em concentrações decrescentes de solução salina. A resistência depende da forma, volume, tamanho, conteúdo de hemoglobina e meia vida dos eritrócitos, e pode ser alterada por vários fatores fisiológicos ou patológicos. Existem poucos relatos sobre a FOE em gatos. O objetivo deste estudo foi avaliar a FOE em gatos hígidos, com enfermidades hepáticas e naqueles com insuficiência renal. Foram estudados 27 animais divididos em três grupos. A hemólise 50 por cento foi notada de forma similar em todos os grupos. Em 100 por cento de hemólise, os eritrócitos de gatos com insuficiência renal e doença hepática mostraram maior fragilidade osmótica, embora esses animais apresentassem maiores níveis de colesterol sérico quando comparados aos gatos sadios do grupo controle. O achado de maior FOE mesmo com níveis elevados de colesterol pode estar associado a características metabólicas da espécie.

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.

Determinaçäo sérica de haptoglobina, ceruloplasmina e Ó-glicoproteína ácida em cäes com gastrenterite hemorrágica / Determination of serum haptoglobin, ceruloplasmin and acid Ó-glycoprotein in dogs with haemorrhagic gastroenteritis

As proteínas de fase aguda (PFA) säo proteínas plasmáticas, cujo estímulo à síntese ocorre de forma rápida e marcante em resposta à injúria tecidual. Estas proteínas permitem o diagnóstico de processos inflamatórios em animais com supressäo ou depressäo medular. Além disso, säo úteis na monitorizaçäo da resoluçäo tecidual de traumas ou inflamaçäo e também na avaliaçäo da resposta orgânica ao tratamento. Uma vez que a leucopenia é observada nos estádios iniciais da parvovirose canina, a dosagem das PFA pode permitir a avaliaçäo do processo inflamatório sob estas condiçöes. Considerando-se estas hipóteses, foram determinados os níveis séricos das PFA (haptoglobina, ceruloplasmina e Ó-glicoproteína ácida) em 11 cäes saudáveis e 11 cäes leucopênicos com gastrenterite hemorrágica, com suspeita clínica de parvovirose canina. A avaliaçäo estatística mostrou diferença significativa, com intervalo de confiabilidade de 99 por cento (P<0,01) para a haptoglobina (p<0,0064) e para a glicoproteína ácida (p<0,0042) e 95 por cento de confiança (P<0,05) para a ceruloplasmina (p<0,0478) quando comparada com o grupo controle. Em conclusäo, cäes com gastrenterite hemorrágica durante a fase leucopênica apresentaram níveis elevados de haptoglobina, ceruloplasmina e Ó-glicoproteína ácida.

Immunostimulatory effects of plasmid DNA and synthetic oligodeoxynucleotides.

Nucleic acid immunization is a new vaccination technology. DNA vaccines do not only carry the genetic information for the antigen of interest but also deliver an adjuvant effect due to the presence of immunostimulatory sequences within the plasmid backbone. It is generally assumed that the adjuvant properties of plasmid DNA are equal to those described for oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. To challenge this hypothesis we have carried out a series of experiments comparing the ability of single- and double-stranded ODN containing CpG motifs to induce the activation of mouse spleen cells. Moreover, we compared the immunostimulatory properties of plasmids that were modified by the addition of two to four CpG motifs. Our results establish that plasmid DNA express their adjuvanticity as either double or single strands, and no differences were observed between modified and unmodified plasmids. On the other hand, the strongest stimulatory ODN sequences lost their adjuvant properties when administered as double-strand DNA. Furthermore, the profile of cytokines induced on spleen cells by plasmid DNA and ODN is different. Strikingly, plasmid DNA induces a moderate synthesis of IL-6 and a strong synthesis of IFN-gamma, whereas stimulation with ODN showed an inverse profile with a higher increase in the synthesis of IL-6 but a moderate increase in IFN-gamma. Finally, in vivo studies were consistent with the results obtained in vitro. Mice immunized with modified or unmodified plasmids encoding the glycoprotein D of HSV showed similar levels of cellular and humoral immune responses.

Modulation of the immune response mediated by oral transgene administration of IL-10.

In the current study, we analyze the immunomodulatory effect of oral transgene administration of IL-10 using a mice model of viral inflammation. Salmonella harboring a plasmid encoding the IL-10 gene (SLIL10) was administered by the oral route together with Salmonella carrying a plasmid encoding the glycoprotein D or B (SLgD, SLgB) of Herpes simplex virus type 2 (HSV-2). This resulted in a high inhibition of the cellular and human immune response against the viral proteins. When mice immunized against the HSV proteins were challenged with 10 lethal doses of HSV-2 by the intravaginal route, only those that had also received SLIL10 showed severe lesions and died. When Salmonella harboring pIL10 was administered orally to mice immunized by the intramuscular route with a plasmid encoding gD, inhibition of cellular and humoral immune responses were also observed but to a lesser extent than with oral immunization. By means of adoptive transfer experiments and in vitro experiments, we have subsequently determined that the mechanism possibly involved in the inhibition of the immune response could be a reduced antigenic presentation when mice receive SLIL10 that induced a state of anergy on specific T lymphocytes.