First report of in vitro selection of RNA aptamers targeted to recombinant Loxosceles laeta spider toxins

BACKGROUND: Loxoscelism is the envenomation caused by the bite of Loxosceles spp. spiders. It entails severe necrotizing skin lesions, sometimes accompanied by systemic reactions and even death. There are no diagnostic means and treatment is mostly palliative. The main toxin, found in several isoforms in the venom, is sphingomyelinase D (SMD), a phospholipase that has been used to generate antibodies intended for medical applications. Nucleic acid aptamers are a promising alternative to antibodies. Aptamers may be isolated from a combinatorial mixture of oligonucleotides by iterative selection of those that bind to the target. In this work, two Loxosceles laeta SMD isoforms, Ll1 and Ll2, were produced in bacteria and used as targets with the aim of identifying RNA aptamers that inhibit sphingomyelinase activity. RESULTS: Six RNA aptamers capable of eliciting partial but statistically significant inhibitions of the sphingomyelinase activity of recombinant SMD-Ll1 and SMD-Ll2 were obtained: four aptamers exert ~17% inhibition of SMD-Ll1, while two aptamers result in ~25% inhibition of SMD-Ll2 and ~18% cross inhibition of SMD-Ll1. CONCLUSIONS: This work is the first attempt to obtain aptamers with therapeutic and diagnostic potential for loxoscelism and provides an initial platform to undertake the development of novel anti Loxoscelesvenom agents.

Avaliação do método da imunofenotipagem por citometria de fluxo no diagnóstico das doenças displásicas (SMD, LMA-relacionada à mielodisplasia, LMMC e LMMJ) em adultos e crianças / Evaluation of the method of immunophenotyping by flow cytometry in the diagnosis of dysplastic disease (MDS, AML-related to myelodysplasia,CMML and JMML) in adults and children

As síndromes mielodisplásicas (SMD) se caracterizam por terem uma hematopoese displásica, citopenias e pelo risco de progressão para leucemia mielóide aguda. O diagnóstico baseia-se na clínica e nos achados citomorfológicos da medula óssea (MO) e citogenéticos. Na fase inicial ou quando a MO é hipocelular o diagnóstico é difícil e a citogenética frequentemente é normal. A imunofenotipagem (IMF) tem sido cada vez mais utilizada nos casos de SMD em adultos e pouco explorada na SMD pediátrica. Os nossos objetivos foram: estudar os casos de SMD e doenças correlatas (LMA relacioanda à SMD: LMA-rMD; leucemia mielomonocítica crônica: LMMC e leucemia mielomonocítica juvenil: LMMJ) em adultos e crianças, associando os dados clínicos e laboratoriais aos obtidos pela IMF, que utilizou um painel de anticorpos monoclonais para as várias linhagens medulares. No período compreendido entre 2000 e 2010 foram estudados 87 pacientes (64 adultos e 23 crianças) oriundos do HUPE/UERJ e IPPMG/UFRJ e 46 controles (23 adultos e 20 crianças). Todos os doentes realizaram mielograma, biópsia óssea, citogenética, citoquímica e estudo imunofenotípico. Segundo os critérios da OMS 50 adultos foram classificados como SMD, 11 como LMA-rMD e 3 LMMC. Entre as crianças 18 eram SMD, 2 LMA e 3 LMMJ. Os pacientes adultos com SMD foram divididos em alto risco (n=9; AREB-1 e AREB-2) e baixo risco (n=41; CRDU, CRDM, CRDM-SA, SMD-N e SMD-5q). As crianças com SMD em CR (n=16) e AREB (n=2). Anormalidades clonais recorrentes foram encontradas em 22 pacientes adultos e em 7 crianças. Na análise de IMF foi utilizada a metodologia da curva ROC para a determinação dos valores de ponto de corte a fim de identificar os resultados anormais dos anticorpos monoclonais nos pacientes e nos controles, permitindo determinar a sensibilidade e especificidade desses em cada linhagem. A IMF foi adequada para a análise em todos os pacientes e 3 ou mais anormalidades foram encontradas. A associação da IMF...

Myelodysplastic syndrome (MDS) is characterized by having a dysplastic hematopoiesis, cytopenias and risk of progression to acute myeloid leukemia. The diagnosis is based on clinical and cytomorphologic findings in bone marrow (BM) and cytogenetics. In the initial phase of when the BM is hypocellular, diagnosis is difficult and often with normal karyotype. The flow cytometry immunophenotyping (FCI) analysis has been broadly used in adult MDS cases but is rarely in pediatric MDS. The objectives of this work were: to study MDS cases and correlated diseases (AML with myelodysplasia-related changes; chronic myelomonocytic leukemia - CMML and juvenite myelomonocytic leukemia - JMML) and to correlate laboratorial data to FCI using a panel of monoclonal antibodies for the various marrow lineages in both adult and children. In the period between 2000 and 2010, 87 patients were studied (64 adults and 23 children) coming from HUPE/UERJ and IPPMG/UFRJ and 46 controls (26 adults and 20 children). All patients were submitted to myelogram, bone marrow biopsy, cytogenetic, cytochemistry and immunophenotypic study. According to WHO criteria 50 adults were classified MDS, 12 AML and 3 CMML. Among the children there were 18 MDS, 2 AML, and 3 JMML. MDS adult patients were subdivided into high risk (n=9; RAEB-1 and RAEB-2) and low risk (n=41; RCUD, RCMD-RS, MDS-U and MDS-5q). MDS children were classified as RCC (n=16) and RAEB (n=2). Clonal abnormalities were found in 22 (35%) adult patients and 7 (30%) children. In the analysis of FCI methodology ROC curve was used for determination of cut off abnormalities at monoclonal antibodies in patients and controls which allowed to estimate the sensitivity and specificity of each strain. The FCI was suitable for analysis in all patients and 3 or more abnormalities were found. The association of the FCI increased the sensitivity of morphological analysis in the erythroid lineage from 70 to 97% in adults and from 59 to 86% in children...

Alterações das células natural killer (NK) na síndrome mielodisplásica (SMD) / [Changes in natural killer (NK) cells in syndrome myelodysplastic syndrome (MDS)]

A síndrome mielodisplásica (SMD) compreende um grupo heterogêneo de doenças clonais da medula óssea (MO), caracterizadas por vários graus de citopenias periféricas, que pode evoluir para leucemia mielóide aguda (LMA). Tem sido discutida a possibilidade de a SMD ser mediada por mecanismos autoimunes. As células natural killer (NK) poderiam ter um papel na fisiopatologia e progressão da SMD, mas até o momento pouco se sabe sobre o assunto. Estas células são importantes componentes do sistema imune inato e possuem funções realizadas por dois subtipos distintos: as CD56dim são responsáveis pela atividade citotóxica natural contra células tumorais e infectadas por vírus e as CD56bright pela produção de citocinas imunoregulatórias. As funções destas células são controladas por um conjunto de receptores ativadores e inibidores que reconhecem as células alvo e pertencem a três famílias principais: receptores de citotoxicidade natural (NCR), lectina tipo C e receptores de células assassinas semelhantes a imunoglobulinas (KIR). O objetivo foi estudar alterações do comportamento das células NK em pacientes com SMD. Como a SMD é mais frequente em idosos e relativamente rara na infância, primeiramente foram avaliados os parâmetros de interesse em crianças, adultos e idosos saudáveis. Foi observada nos idosos saudáveis uma redução de expressão de receptores ativadores da família NCR em células NK e aumento da expressão de receptores da família KIR no subtipo CD56bright. As crianças apresentaram alterações semelhantes. Houve redução da expressão de NKG2D nos linfócitos T de idosos. A análise do genótipo KIR revelou que KIR2DL5 e KIR2DS3 estavam associados aos idosos...

Myelodysplastic syndrome (MDS) comprise a heterogeneous group of clonal disorders of bone marrow (BM), characterized by varying degrees of peripheral cytopenias that can develop into acute myeloid leukemia (AML). Recently, has been discussed the possibility that MDS may be mediated by autoimmune mechanisms. Natural killer cells (NK) cells could play a role in the pathophysiology and progression of MDS, but so far little is known about the subject. These cells are important components of the innate immune system and its functions are performed by two distinct subtypes: the CD56dim cells are responsible for natural cytotoxicity against tumor and cells virally infected while CD56bright cells are in charge by production of immunoregulatory cytokines. The functions of these cells are controlled by a set of inhibitory and activating receptors that recognize the target cells. There are three main families of NK cells receptors: natural cytotoxicity receptors (NCR), C-type lectin receptors and killer cell immunoglobulin-like receptors (KIR). The objective was to study changes in the performance of NK cells in MDS patients. As MDS is more common in the elderly and relatively rare in childhood, firstly the parameters of interest were evaluated in healthy children, adults and elderly. Healthy elderly presented a decrease of expression of activating receptors of NCR family in NK cells and increased expression of KIR receptors in CD56bright subset. Children showed similar results. NKG2D expression was decreased on T cells of elderly. Analysis of KIR genotype revealed that KIR2DL5 and KIR2DS3 were significantly associated with old age. Cytotoxic activity was preserved from childhood through old age, suggesting that the increase of the absolute number of CD56dim , observed in elderly, may represent a compensatory mechanism for the receptor expression alterations. In MDS patients, for the first time, was observed an increase of CD56dim and reduction of CD56bright subsets...

Perfil sorológico para circovírus suíno tipo 2 em granjas comerciais de suínos no Brasil / Serological profiles to porcine circovirus type 2 antibodies in Brazil

A técnica de imunoperoxidase em monocamada de células (IPMC) para demonstração de anticorpos contra o circovírus suíno tipo 2 (CVS2) foi empregada para a investigação sorológica em oito granjas de suínos destinadas à produção comercial. Das 240 amostras de soros testadas, 229 (95,4%) foram reagentes com títulos que variaram de 20 até 10.240. Títulos de anticorpos foram detectados nas faixas etárias de duas a três semanas até animais acima de 24 semanas e encontrados em granjas com e sem a síndrome multissistêmica do definhamento (SMD). A média dos títulos de anticorpos revelou diferenças estatísticas (P<0,05) nas faixas etárias de 11 a 13 e 14 a 22 semanas nos animais oriundos de granjas com e sem a SMD. Os resultados refletem a importância de conhecer o perfil sorológico do rebanho e assegurar a implantação de um efetivo cronograma de vacinação contra o CVS2.

The immunoperoxidase in monolayer cells (IPMC) technique for the demonstration of antibodies against type 2 porcine circovirus (PCV2) was used for the serological investigation in eight industrial swine farms. Out of the 240 tested sera, 229 (95,4%) were reactive with titers which varied from 20 up to 10.240. Titers of antibodies were detected in pigs from two to three weeks of age up to above 24 weeks of age, and were observed in farms with and without clinical signs indicative of the post-weaning multisystemic wasting syndrome (PMWS). Analysing the mean titers, statistical differences (P<0.05) were evidentiated in two age intervals, of 11 to 13 and 14 to 22 weeks of age. The results indicate the importance of determining the serological profiles of commercial swine herds, in order to enable the implantation of effective hygiene and vaccination protocols for PCV2 prevention.

Abscessos dentários periapicais em leitões com síndrome multissistêmica do definhamento / Dental periapical abscesses in piglets affected by postweaning multisystemic wasting syndrome PMWS

Duzentos e oitenta leitões, entre 40 e 70 dias de idade, afetados pela sindrome multissistêmica do definhamento (SMD) foram examinados após eutanásia para pesquisa de lesões dentárias. Pelo menos um abscesso periapical foi observado em 58 leitões (20,7 por cento), dos quais 12 apresentaram abscessos múltiplos. Terceiros incisivos superiores, 3os incisivos inferiores, caninos superiores, caninos inferiores e outros dentes apresentaram respectivamente 22 (31,4 por cento), 16 (22,9 por cento), 4 (5,7 por cento), 23 (32,9 por cento) e 5 (7,1 por cento) abscessos periapicais. A maior prevalência de abscessos observada nos dentes 3os incisivos e caninos inferiores está provavelmente associada com a maior área de corte ou desgaste que sofrem esses dentes. Entre as bactérias isoladas de amostras de 65 abscessos, Streptococcus sp. foi a mais prevalente e esteve presente em 21,48 por cento e 27,7 por cento dos isolados em aerobiose e anaerobiose, respectivamente. Na segunda posição em prevalência estiveram as bactérias corineformes, as quais foram mais freqüentemente isoladas em atmosfera anaeróbica do que aeróbica. Houve preponderância de isolamentos de bactérias Gram-positivas. Não foi possível determinar se as co-infecções bacterianas predispuseram às lesões características de SMD ou foram conseqüentes à imunossupressão causada pela infecção com PCV2.

Swine producers have historically resected needle teeth of newborn pigs to prevent potential injuries to the sow mammary gland and faces of littermates. However, the possible impact of this practice on pig performance has been questioned. As part of a study, 280 PMWS affected piglets, with 40-70 days of age, were examined for the presence of dental lesions immediately after euthanasia. Most pigs were confirmed as PMWS by the detection of typical microscopic lesions and immunohistochemical pattern. At least one periapical abscess was observed in 58 piglets (20.7 percent), of which 12 had multiple abscesses. There were 22 (31.4 percent), 16 (22.9 percent), 4 (5.7 percent), 23 (32.9 percent), and 5 (7.1 percent) abscesses in 3rd upper incisives, 3rd lower incisives, upper canines, lower canines, and other teeth, respectively. The higher prevalence of dental abscesses observed in 3rd incisives and lower canines is probably associated with the wider area of grinding or clipping to which these teeth were subjected. Among bacteria isolated from samples collected from 65 abscesses, Streptococcus sp. was the most prevalent bacteria and was present in 21.48 percent and 27.7 percent of the aerobic and anaerobic isolates, respectively. In the second most frequent group were coryneform microorganisms, which showed higher rate of isolation under anaerobic atmosphere than in aerobic culture. There was preponderance of Gram-positive isolates. It could not be determined whether these bacterial co-infections predisposed pigs to development of PMWS-type lesions or if were subsequent to the PCV2 infection.

A pilot study of molecular mechanism of salmon milt DNA (SMD) retards mouse insenescence / 中国组织工程研究

Aim To iuvestigate the effect and the mechanism of salmonmilt DNA (SMD) on age-related involutions in mouse thymus. MethodsFemale BALB/c of 10 months were divided randomly into three groupsaccording to their weights: high dosage group 333.33 mg/(kg @ d), lowdosage group 166. 67 mg/(kg @ d) and control group 0 mg/(kg @ d) .After five weeks, with Image-Pro Plus (version. 4.0) software, the thymusindexes and the thymoctytes in the thymus section were measured, as wellas the thymus cortex thickness. All the data were analyzed by SAS statisticsoftware. Mieroarray technique was applied to screen the gene fragments,which were differently expressed between the high dosage group and thecontrol group, together with RT-PCR to further confirm some of them.Results No significant differences of the variables including bodyweight, thymus weight and thymus indexes among the three groups werefound (F ＜ 3.0 and P ＞ 0.05, respectively). The thymocytes quanti-ties of thymus cortex and medulla in the high dosage group were significantlyhigher than those of the control group [cortex D(H, C) = 9.46, P ＜ 0.01;medulla t( H.C) = 2.53, P ＜ 0.05]. The thymus cortex thicknesses of bothSMD supplement groups were significantly higher than that of the control group[cortex D(L,C)=3.65, P＞ 0.05; medulla t(L, C)=0.8, P＞ 0.05] .112differently expressed gene fragments were isolated. Furthermore, we foundthe fragments with the logged number of U23789, X80232 and Aw209102were highly expressed in the high dosage group when RT-PCR techniquewas used. Conclusion SMD may reverse the age-related involutions inmouse thymus via up-regulation the expression of proliferation related genesand development and differentiation related genes simultaneously.