Hemolymph of triatomines presents fungistatic activity against Cryptococcus neoformans and improves macrophage function through MCP-I/TNF-α increase.

Background: Triatomines are blood-feeding arthropods belonging to the subfamily Triatominae (Hemiptera; Reduviidae), capable of producing immunomodulatory and water-soluble molecules in their hemolymph, such as antimicrobial peptides (AMPs). In this work, we evaluated the antifungal and immunomodulatory activity of the hemolymph of Meccus pallidipennis (MPH) and Rhodnius prolixus (RPH) against Cryptococcus neoformans. Methods: We assessed the activity of the hemolymph of both insects on fungal growth by a minimum inhibitory concentration (MIC) assay. Further, RAW 264.7 macrophages were cultivated with hemolymph and challenged with C. neoformans. Then, their phagocytic and killing activities were assessed. The cytokines MCP-1, IFN-γ, TNF-α, IL-10, IL-12, and IL-6 were measured in culture supernatants 4- and 48-hours post-infection. Results: Both hemolymph samples directly affected the growth rate of the fungus in a dose-dependent manner. Either MPH or RPH was capable of inhibiting fungal growth by at least 70%, using the lowest dilution (1:20). Treatment of RAW 264.7 macrophages with hemolymph of both insects was capable of increasing the production of MCP-I and TNF-α. In addition, when these cells were stimulated with hemolymph in the presence of C. neoformans, a 2- and a 4-fold increase in phagocytic rate was observed with MPH and RPH, respectively, when compared to untreated cells. For the macrophage killing activity, MPH decreased in approximately 30% the number of viable yeasts inside the cells compared to untreated control; however, treatment with RPH could not reduce the total number of viable yeasts. MPH was also capable of increasing MHC-II expression on macrophages. Regarding the cytokine production, MCP-I and TNF-α, were increased in the supernatant of macrophages treated with both hemolymphs, 4 and 48 hours after stimulation. Conclusion: These results suggested that hemolymph of triatomines may represent a source of molecules capable of presenting antifungal and immunomodulatory activity in macrophages during fungal infection.

Hemolymph of triatomines presents fungistatic activity against Cryptococcus neoformans and improves macrophage function through MCP-I/TNF-α increase

Triatomines are blood-feeding arthropods belonging to the subfamily Triatominae (Hemiptera; Reduviidae), capable of producing immunomodulatory and water-soluble molecules in their hemolymph, such as antimicrobial peptides (AMPs). In this work, we evaluated the antifungal and immunomodulatory activity of the hemolymph of Meccus pallidipennis (MPH) and Rhodnius prolixus (RPH) against Cryptococcus neoformans. Methods: We assessed the activity of the hemolymph of both insects on fungal growth by a minimum inhibitory concentration (MIC) assay. Further, RAW 264.7 macrophages were cultivated with hemolymph and challenged with C. neoformans. Then, their phagocytic and killing activities were assessed. The cytokines MCP-1, IFN-γ, TNF-α, IL-10, IL-12, and IL-6 were measured in culture supernatants 4- and 48-hours post-infection. Results: Both hemolymph samples directly affected the growth rate of the fungus in a dose-dependent manner. Either MPH or RPH was capable of inhibiting fungal growth by at least 70%, using the lowest dilution (1:20). Treatment of RAW 264.7 macrophages with hemolymph of both insects was capable of increasing the production of MCP-I and TNF-α. In addition, when these cells were stimulated with hemolymph in the presence of C. neoformans, a 2- and a 4-fold increase in phagocytic rate was observed with MPH and RPH, respectively, when compared to untreated cells. For the macrophage killing activity, MPH decreased in approximately 30% the number of viable yeasts inside the cells compared to untreated control; however, treatment with RPH could not reduce the total number of viable yeasts. MPH was also capable of increasing MHC-II expression on macrophages. Regarding the cytokine production, MCP-I and TNF-α, were increased in the supernatant of macrophages treated with both hemolymphs, 4 and 48 hours after stimulation. Conclusion: These results suggested that hemolymph of triatomines may represent a source of molecules capable of presenting antifungal and immunomodulatory activity in macrophages during fungal infection.(AU)

The Recombinant Form of Trypanosoma cruzi P21 Controls Infection by Modulating Host Immune Response.

Trypanosoma cruzi P21 protein (P21) is a putative secreted and immunomodulatory molecule with potent bioactive properties such as induction of phagocytosis and actin cytoskeleton polymerization. Despite the bioactive properties described so far, the action of P21 on parasite replication in muscle cell lineage or T. cruzi parasitism during acute experimental infection is unclear. We observed that recombinant P21 (rP21) decreased the multiplication of T. cruzi in C2C12 myoblasts, phenomenon associated with greater actin polymerization and IFN-γ and IL-4 higher expression. During experimental infection, lower cardiac nests, inflammatory infiltrate and fibrosis were observed in mice infected and treated with rP21. These results were correlated with large expression of IFN-γ counterbalanced by high levels of IL-10, which was consistent with the lower cardiac tissue injury found in these mice. We have also observed that upon stress, such as that induced by the presence of the IFN-γ cytokine, T. cruzi produced more P21. The effect of P21 in controlling the replication of T. cruzi, may indicate an evolutionary mechanism of survival developed by the parasite. Thus, when subjected to different stress conditions, the protozoan produces more P21, which induces T. cruzi latency in the host organism, enabling the protozoan to evade the host's immune system.

Seroprevalence of Toxoplasma gondii and Toxocara canis in a human rural population of Southern Rio Grande do Sul.

Due to the growing population of pets, especially homeless dogs and cats, zoonoses still represent a significant public health problem. Toxoplasma gondii and Toxocara spp. are epidemiologically important zoonotic agents as they are etiological factors of human toxoplasmosis and toxocariasis, respectively. These parasites remain neglected even though they are substantially prevalent in rural areas. The aim of this study was to investigate T. gondii and T. canis seroprevalence and risk factors of seropositivity in a rural population in Pelotas municipality, Brazil. The study participants (n=344) were patients of a Basic Healthcare Unit (BHU) located in Cerrito Alegre. Blood samples were collected and tested for T. gondii antibodies by indirect immunofluorescence and T. canis antibodies by an indirect ELISA that targets an excreted-secreted antigen (TES). T. gondii seropositivity was 53.2%, with higher titers (1:256 - 1:1,024) in individuals who habitually eat pork, beef, or chicken, while T. canis seropositivity was 71.8% and concomitant T. gondii and T. canis seropositivity was 38.3%. Among the seropositivity risk factors assessed, only habitual undercooked meat consumption was significant (p = 0.046; OR = 3.7) for T. gondii and none of them were associated with T. canis seropositivity. Both parasites have a high prevalence in rural areas, which reinforces the need to invest in rural community education and health.

Evolution of tonoplast P-ATPase transporters involved in vacuolar acidification.

Petunia mutants (Petunia hybrida) with blue flowers defined a novel vacuolar proton pump consisting of two interacting P-ATPases, PH1 and PH5, that hyper-acidify the vacuoles of petal cells. PH5 is similar to plasma membrane H(+) P3A -ATPase, whereas PH1 is the only known eukaryoticP3B -ATPase. As there were no indications that this tonoplast pump is widespread in plants, we investigated the distribution and evolution of PH1 and PH5. We combined database mining and phylogenetic and synteny analyses of PH1- and PH5-like proteins from all kingdoms with functional analyses (mutant complementation and intracellular localization) of homologs from diverse angiosperms. We identified functional PH1 and PH5 homologs in divergent angiosperms. PH5 homologs evolved from plasma membrane P3A -ATPases, acquiring an N-terminal tonoplast-sorting sequence and new cellular function before angiosperms appeared. PH1 is widespread among seed plants and related proteins are found in some groups of bacteria and fungi and in one moss, but is absent in most algae, suggesting that its evolution involved several cases of gene loss and possibly horizontal transfer events. The distribution of PH1 and PH5 in the plant kingdom suggests that vacuolar acidification by P-ATPases appeared in gymnosperms before flowers. This implies that, next to flower color determination, vacuolar hyper-acidification is required for yet unknown processes.

Aspectos radiográficos do sulco intertroclear de coelhos submetidos ao enxerto osteocondral alógeno associado à terapia com células mononucleares autógenas e dexametasona / Radiographic appearance of intertrochlear groove of rabbits submitted to osteochondral graft allographs associated to the therapy with mononuclear cell autographs and dexamethasone / Aspectos radiológicos de la ranura intertroclear de conejos sometidos a injerto osteocondral alógeno asociado a la terapia con células mononucleares autógenas y dexametasona

Vinte e quatro coelhos adultos foram separados em dois grupos (n=12) - controle (GI) e tratado (GII) e submetidos ao enxerto osteocondral alógeno para reparo ósseo e cartilaginoso do sulco troclear, conservado em glicerina a 98%. Os animais do GII receberam ainda injeção intra-articular de 2,0 x 106 células mononucleares autólogas e dexametasona intramuscular. Foram realizadas avalições radiográficas aos 45 e 90 dias de pós-operatório. Nos coelhos do grupo tratado e controle não foi notado sinais de reação características de enxerto-contra-hospedeiro e aos 45 e 90 dias de pós-operatório ocorre osteólise devido aos micromovimentos na interface implante-osso e pressão efetiva nos espaços articular. A implantação de enxerto alógeno conservado em glicerina, associado à inoculação de células mononucleares autógenas e dexametasona intramuscular promove intensa neoformação óssea e com bom reparo do defeito ósseo em coelhos.

Twenty-four adult rabbits were divided two groups (n = 12), control (GI) and treated (GII), and submitted to allogenic osteochondral graft for bone and cartilage repair of the trochlear groove, preserved in 98% glycerin. The GII yet received intra-articular injection of 2,0 x 106 autologous mononuclear cells and intramuscular dexamethasone. Radiographs at 45 and 90 days postoperatively avaliations were performed. In rabbits treated and control group was not skimmed signals characteristic reaction of graft-versus-host and at 45 and 90 days postoperatively osteolysis occurs due to micro motion at the implant-bone interface and effective pressure in the joint spaces. The implantation of allograft preserved in glycerol, associated with inoculation of autologous mononuclear cells and intramuscular dexamethasone intense bone and good repair of bone defects in rabbits neogenesis occurs.

Veinticuatro conejos adultos fueron divididos en dos grupos (n = 12) - control (GI) y tratado (GII) y sometidos a injerto osteocondral alógeno para reparo óseo y cartilaginoso de ranura tróclea, conservado en glicerina a 98%. Los animales del GII recibieron inyección interarticular de 2,0 x 106 células mononucleares autólogas y dexametasona intramuscular. Se realizó evaluaciones radiográficas a los 45 y 90 días posoperatorio. En los conejos tratados y del grupo control no se ha notado señales de reacciones características de injerto contra hospedero, y a los 45 y 90 días de posoperatorio ocurrió osteólisis debido a los micro movimientos en la interfaz, implante óseo y presión efectiva en los espacios articular. La implantación de injerto alógeno conservado en glicerina, asociado a la inoculación de células mononucleares autógenas y dexametasona intramuscular, promueve intensa neo formación ósea y con buen reparo del defecto óseo en conejos.

Patogenicidade e virulência de Toxoplasma gondii isolado de suínos de criação artesanal no sul do Brasil / Patogenicity and virulence of Toxoplasmagondii isolated from rustic farm pigs in Southern Brazil

Studies of Toxoplasma gondii infection in pigs are important because they are part of the human food chain. The main routes of transmission of this agent are: carnivorism, fecal-oral and congenital. Six isolates of T. gondii from pigs of rustic farms were evaluated for virulence and pathogenicity. Tachyzoites suspension used in the tests was obtained by aspiration or by washing the peritoneal cavity of mice that had developed ascites. Each sample of living tachyzoites was inoculated into groups of five mice with inoculum of 10¹, 10², 10³, 10(4), 10(5) and 10(6) intraperitoneally. Half of the isolates (3/6) were lethal and caused clinical signs in Swiss albino mice. The minimum lethal dose was 10³ tachyzoites by inoculum. The death of mice that had acute infection occurred between 12 and 26 days post-inoculation. The other three isolates were not pathogenic or virulent for mice. All isolates of the area studied had a high ability to form cysts, what could increase the risk for infection through ingestion of infected animal tissues.

Estudos com Toxoplasma gondii em suínos são relevantes porque seus produtos e subprodutos fazem parte da cadeia alimentar do ser humano. As principais vias de transmissão deste agente são o carnivorismo, fecal-oral e congênita. Seis isolados de Toxoplasma gondii de suínos de criação artesanal foram avaliados quanto à patogenicidade e virulência em camundongos suíços albinos. A suspensão de taquizoítos utilizada nos testes foi obtida através da punção ou lavagem da cavidade peritoneal de camundongos que apresentaram ascite. Cada amostra foi inoculada em grupos de cinco camundongos, com inóculo de 10¹, 10², 10³, 10(4), 10(5) e 10(6) taquizoítos vivos, via intraperitoneal. Dos isolados, 50% (3/6) foram letais e causaram sinais clínicos nos camundongos. A dose mínima letal foi de 10³ taquizoítos. A morte dos animais que apresentaram infecção aguda ocorreu entre 12 e 26 dias após a inoculação. Todos os isolados da região estudada apresentam alta capacidade de formar cistos, o que pode aumentar o risco de infecção pela ingestão de tecidos dos animais infectados pelos mesmos.

Uso da técnica TightRope modificada em cães com ruptura do ligamento cruzado cranial / Use of modification TightRope technique in dogs with rupture of cranial crucial ligament

A ruptura do ligamento cruzado cranial (RLCCr) figura entre as principais enfermidades ortopédicas na espécie canina, sendo responsável por instabilidade da articulação do joelho, o que resulta em claudicação e desenvolvimento de doença articular degenerativa (DAD). O objetivo deste trabalho foi avaliar os resultados da técnica de TightRope modificada (TRM) na estabilização do joelho de cães com ruptura do ligamento cruzado cranial (RLCCr). O procedimento cirúrgico foi realizado em seis animais, os quais foram avaliados por 120 dias de pós-operatório. A modificação da técnica para estabilização extra capsular da RLCCr, consiste na utilização de fio de fluorcarbono, e fio de aço inoxidável de 1 mm como forma de fixação óssea. A TRM promoveu a estabilidade do joelho dos pacientes com ruptura do ligamento cruzado cranial no pós-operatório imediato e durante o acompanhamento clínico pós-cirúrgico demonstrou, aos 30 dias, progressiva melhora da marcha e suporte de peso à deambulação, estando todos os animais recuperados clinicamente aos 120 dias de pós-operatório.

Rupture of the cranial cruciate ligament is one of the leading orthopedic diseases in dogs. Thus aimed to evaluate the techniqueof “Tightrope” modified (TRM) in the knees of dogs with the purpose of providing less invasive and cost-effective surgery. Thesurgical procedure was performed in six animals, and these were evaluated up to 120 days postoperatively. The modification of thetechnique for stabilization of extra capsular CCL consists in using fluorocarbon and the stainless steel wire of 1 mm in order bonefixation. The “TRM” promoted the stability of the knee of patients diagnosed with cranial cruciate ligament rupture in the immediatepostoperative period. Clinical follow-up after surgery showed after 30 days postoperatively, improved gait and progressive weightsupport for ambulation, being all animals recovered clinically at 120 days postoperatively.

Genetic machinery for RNA silencing and defense against viruses in Citrus

RNA silencing mechanisms are conserved throughout eukaryotic evolution, possibly due to their importance in viral resistance and other aspects of cell biology. Here, we explored the Citrus EST (CitEST) database in search of sequences related to the most important known genes involved in RNA silencing. Transcripts strongly matching Argonaute (AGO), Dicer-like (DCL), Hua enhancer (HEN), and RNA-dependent RNA Polymerase (RdRP) were found in many of the citrus libraries. The reads were clustered and quantified. This shows that post-transcriptional gene silencing apparatus is active in citrus. It seems plausible that a better understanding of the players of RNA silencing in Citrus spp. and related genera may help create new tools to defeat the viral diseases that affect the citrus industry. Functional analyses of these citrus genes would enable the pursuit of this hypothesis.

Treatment effects of methylphenidate on cognitive functioning in children with mental retardation and ADHD.

OBJECTIVE: Cognitive effects of stimulant medication were investigated in children with mental retardation (MR) and attention-deficit/hyperactivity disorder (ADHD). METHOD: Performance on tasks tapping sustained attention, visual and auditory selective attention, inhibition, and immediate memory was assessed for 24 children (mean age 10.9 years) during a placebo-controlled, double-blind, crossover treatment trial with 0.15, 0.30, and 0.60 mg/kg b.i.d. dosages of methylphenidate (MPH). RESULTS: Successively higher MPH doses were associated with consistent gains in cognitive task performance, with optimal performance noted at the highest dose. Analysis of dose-response curves revealed significant linear components of trend on measures tapping sustained attention, visual selective attention, auditory selective attention, as well as two tasks tapping inhibition/impulsivity: delay of gratification and match-to-sample. No evidence of a curvilinear dose-response relationship emerged for any measure. CONCLUSIONS: Inattention and disinhibition/impulsivity decline with MPH treatment in children with ADHD/MR, and consistent with the Multimodal Treatment Study of ADHD, higher MPH doses are most effective. These findings also suggest that cognitive testing, together with behavioral and medical assessment, can be an effective tool in assessing stimulant response in children with ADHD/MR.

Effects of methylphenidate treatment in children with mental retardation and ADHD: individual variation in medication response.

OBJECTIVE: Individual variation in cognitive and behavioral response to methylphenidate (MPH) was investigated in children with mental retardation and attention-deficit/hyperactivity disorder. METHOD: Twenty-four children (mean age 10.9 years, SD = 2.4) participated in a placebo-controlled, double-blind, crossover trial with 0.15-, 0.30-, and 0.60-mg/kg b.i.d. doses of MPH. Parent and teacher behavioral ratings, as well as cognitive task performance, were assessed at each dose. RESULTS: Relative to placebo, most children with attention-deficit/hyperactivity disorder and mental retardation showed some degree of behavioral and cognitive improvement with MPH treatment. However, fewer of these children made substantial gains (>30% improvement, relative to placebo) with MPH treatment. At the highest dose, 55% of the children showed substantial behavioral gains and 46% made substantial gains in cognitive task performance. However, there was substantial independence between changes in behavior and changes in cognitive performance. CONCLUSIONS: At the 0.60-mg/kg MPH dose, more children showed substantial cognitive and behavioral gains than those who showed substantial declines in a ratio of more than 5:1. However, it may be prudent to assess cognitive change as well as behavioral effects because improvements in the former do not necessarily forecast improvements in the latter in children with attention-deficit/hyperactivity disorder and mental retardation.

Treatment effects of methylphenidate on behavioral adjustment in children with mental retardation and ADHD.

OBJECTIVE: The effects of stimulant medication treatment were investigated in children with mental retardation (MR) and attention-deficit/hyperactivity disorder (ADHD). METHOD: Parent and teacher behavioral ratings and reports of side effects were obtained for children (N = 24, mean age = 10.9 years, SD = 2.4) during a placebo-controlled, double-blind, crossover treatment trial with 0.15 mg/kg, 0.30 mg/kg, and 0.60 mg/kg b.i.d. dosages of methylphenidate. RESULTS: The most significant improvements occurred at the 0.60 mg/kg methylphenidate dose for teacher ratings of inattention (p =.024), hyperactivity (p <.001), aggression (p <.001), and asocial behavior (p =.009). No significant improvements, relative to placebo, occurred at the 0.15 mg/kg dosage. Of interest, nearly all significant medication-related behavioral improvements were detected by teachers. However, parents were sensitive raters of side effects, noting more sleeping problems and loss of appetite at the 0.60 mg/kg dose compared with placebo. CONCLUSIONS: These results suggest that symptoms of ADHD can be treated successfully in children with ADHD/MR, and consistent with MTA study results, higher doses were most effective. Furthermore, these improvements were not accompanied by increases in symptoms such as staring, social withdrawal, or anxiety.