Cocoa flavonoid-enriched diet modulates systemic and intestinal immunoglobulin synthesis in adult Lewis rats

Previous studies have reported that a diet containing 10% cocoa, a rich source of flavonoids, has immunomodulatory effects on rats and, among others effects, is able to attenuate the immunoglobulin (Ig) synthesis in both systemic and intestinal compartments. The purpose of the present study was focused on investigating whether these effects were attributed exclusively to the flavonoid content or to other compounds present in cocoa. To this end, eight-week-old Lewis rats were fed, for two weeks, either a standard diet or three isoenergetic diets containing increasing proportions of cocoa flavonoids from different sources: one with 0.2% polyphenols from conventional defatted cocoa, and two others with 0.4% and 0.8% polyphenols, respectively, from non-fermented cocoa. Diet intake and body weight were monitored and fecal samples were obtained throughout the study to determine fecal pH, IgA, bacteria proportions, and IgA-coated bacteria. Moreover, IgG and IgM concentrations in serum samples collected during the study were quantified. At the end of the dietary intervention no clear changes of serum IgG or IgM concentrations were quantified, showing few effects of cocoa polyphenol diets at the systemic level. However, in the intestine, all cocoa polyphenol-enriched diets attenuated the age-related increase of both fecal IgA and IgA-coated bacteria, as well as the proportion of bacteria in feces. As these effects were not dependent on the dose of polyphenol present in the diets, other compounds and/or the precise polyphenol composition present in cocoa raw material used for the diets could be key factors in this effect.

Evaporação da água na superfície do solo em sistemas de plantio direto e preparo convencional

Este trabalho teve por objetivos quantificar a evaporação da água na superfície do solo, em plantio direto e em preparo convencional, e avaliar o uso de microlisímetros de pesagem para medir esse processo. As medições foram feitas em campo, nos verões de 2001/2002, 2002/2003 e 2004/2005, em experimentos irrigados e não irrigados. Utilizaram-se delineamentos inteiramente casualizados, com quatro repetições, cujos tratamentos consistiram de sistemas de manejo do solo – plantio direto e preparo convencional –, e a presença ou ausência de cultivo de milho. Mediu-se a evaporação diária durante períodos de secagem do solo, entre precipitações ou irrigações consecutivas. Em experimentos sem irrigação, a evaporação acumulada foi maior sob plantio direto, na maioria dos períodos de medição, independentemente da presença da lavoura de milho. Nos experimentos com irrigação, a evaporação não apresentou diferenças regulares entre sistemas de manejo de solo. Normalmente, no início dos períodos de medição, com dois a cinco dias de secagem do solo, a evaporação foi maior em solo sob preparo convencional, tendo-se tornado maior em plantio direto, no restante do período de secagem. O emprego de microlisímetros de pesagem é eficiente para medir a evaporação na superfície do solo.

Protective effect of intravitreal injection of vasoactive intestinal peptide-loaded liposomes on experimental autoimmune uveoretinitis.

PURPOSE: The aim of this study was to investigate the effect of a single intravitreal (i.v.t.) injection of vasoactive intestinal peptide (VIP) loaded in rhodamine-conjugated liposomes (VIP-Rh-Lip) on experimental autoimmune uveoretinitis (EAU). METHODS: An i.v.t. injection of VIP-Rh-Lip, saline, VIP, or empty-(E)-Rh-Lip was performed simultaneously, either 6 or 12 days after footpad immunization with retinal S-antigen in Lewis rats. Clinical and histologic scores were determined. Immunohistochemistry and cytokine quantification by multiplex enzyme-linked immunosorbent assay were performed in ocular tissues. Systemic immune response was determined at day 20 postimmunization by measuring proliferation and cytokine secretion of cells from inguinal lymph nodes (ILNs) draining the immunization site, specific delayed-type hypersensitivity (DTH), and the serum concentration of cytokines. Ocular and systemic biodistribution of VIP-Rh-Lip was studied in normal and EAU rats by immunofluorescence. RESULTS: The i.v.t. injection of VIP-Rh-Lip performed during the afferent, but not the efferent, phase of the disease reduced clinical EAU and protected against retinal damage. No effect was observed after saline, E-Rh-Lip, or VIP injection. VIP-Rh-Lip and VIP were detected in intraocular macrophages and in lymphoid organs. In VIP-Rh-Lip-treated eyes, macrophages expressed transforming growth factor-beta2, low levels of major histocompatibility complex class II, and nitric oxide synthase-2. T-cells showed activated caspase-3 with the preservation of photoreceptors. Intraocular levels of interleukin (IL)-2, interferon-gamma (IFN-gamma), IL-17, IL-4, GRO/KC, and CCL5 were reduced with increased IL-13. At the systemic level, treatment reduced retinal soluble autoantigen lymphocyte proliferation, decreased IL-2, and increased IL-10 in ILN cells, and diminished specific DTH and serum concentration of IL-12 and IFN-gamma. CONCLUSIONS: An i.v.t. injection of VIP-Rh-Lip, performed during the afferent stage of immune response, reduced EAU pathology through the immunomodulation of intraocular macrophages and deviant stimulation of T-cells in ILN. Thus, the encapsulation of VIP within liposomes appears as an effective strategy to deliver VIP into the eye and is an efficient means of the prevention of EAU severity.

Calagem e desenvolvimento radicular, nutrição e produção de laranja 'Valência' sobre porta-enxertos e sistemas de preparo do solo

O objetivo deste trabalho foi avaliar o efeito da calagem e de porta-enxertos sobre a quantidade de raízes, a nutrição e a produção de laranjeira 'Valência', em diferentes sistemas de preparo do solo. O experimento foi instalado em blocos ao acaso, com parcelas subsubdivididas e três repetições. As parcelas consistiram de dois sistemas de preparo do solo: convencional e com preparo mínimo. As subparcelas consistiram de níveis de calagem: com ou sem. As subsubparcelas consitiram de três porta-enxertos: limoeiro 'Cravo' (Citrus limonia), tangerineira 'Cleópatra' (Citrus reshni) e Poncirus trifoliata. O tratamento com calagem recebeu 3 Mg ha-1 de calcário dolomítico, antes da implantação, e 1,65 Mg ha-1 quatro anos após o plantio. A produção foi avaliada durante 12 anos; o teor de Ca e Mg nas folhas foram avaliados 13 anos após a implantação do experimento; e as raízes e as características químicas do solo, 14 anos após a implantação. O preparo de solo, e sua interação com os demais fatores, não influenciou as variáveis avaliadas. O limoeiro 'Cravo' adaptou-se melhor à acidez, ao Al e a menores teores de Ca e Mg no solo, e não respondeu ao tratamento com calagem. Poncirus trifoliata apresentou a pior adaptação à acidez do solo, com aumentos de 126% na quantidade de raízes e de 26,4% na produção de frutos em razão da calagem.

Mortalidade de percevejo-do-colmo do arroz no preparo do solo para cultivo mínimo

O objetivo deste trabalho foi verificar a influência do manejo da resteva do arroz irrigado, durante o preparo do solo, sobre a redução populacional pré-hibernante do percevejo-do-colmo (Tibraca limbativentris). Os tratamentos consistiram dos seguintes manejos da resteva: uma passada de grade aradora; duas passadas de grade aradora; duas passadas de grade aradora mais uma passada de grade niveladora; e controle. Em cada unidade amostral, determinou-se o número de ninfas e adultos do percevejo-do-colmo. No preparo do solo, no mínimo duas passadas com grade aradora são necessárias para a redução populacional pré-hibernante do percevejo-do-colmo.

Recognition of Gram-positive Intestinal Bacteria by Hybridoma- and Colostrum-derived Secretory Immunoglobulin A Is Mediated by Carbohydrates.

Humans live in symbiosis with 10(14) commensal bacteria among which >99% resides in their gastrointestinal tract. The molecular bases pertaining to the interaction between mucosal secretory IgA (SIgA) and bacteria residing in the intestine are not known. Previous studies have demonstrated that commensals are naturally coated by SIgA in the gut lumen. Thus, understanding how natural SIgA interacts with commensal bacteria can provide new clues on its multiple functions at mucosal surfaces. Using fluorescently labeled, nonspecific SIgA or secretory component (SC), we visualized by confocal microscopy the interaction with various commensal bacteria, including Lactobacillus, Bifidobacteria, Escherichia coli, and Bacteroides strains. These experiments revealed that the interaction between SIgA and commensal bacteria involves Fab- and Fc-independent structural motifs, featuring SC as a crucial partner. Removal of glycans present on free SC or bound in SIgA resulted in a drastic drop in the interaction with Gram-positive bacteria, indicating the essential role of carbohydrates in the process. In contrast, poor binding of Gram-positive bacteria by control IgG was observed. The interaction with Gram-negative bacteria was preserved whatever the molecular form of protein partner used, suggesting the involvement of different binding motifs. Purified SIgA and SC from either mouse hybridoma cells or human colostrum exhibited identical patterns of recognition for Gram-positive bacteria, emphasizing conserved plasticity between species. Thus, sugar-mediated binding of commensals by SIgA highlights the currently underappreciated role of glycans in mediating the interaction between a highly diverse microbiota and the mucosal immune system.

Secretory IgA: an actor of the interplay between the commensal flora and the intestinal immune system?

In the gastro-intestinal tract,Peyers patches have been describedas a major inductive site for mucosalsecretory IgA (SIgA) responses directedagainst pathogens. The classicalview is that SIgAserves as the firstline of defense against microorganismsby agglutining potential invadersand faciliting their clearance byperistaltic and mucociliary movements,a mechanism called immuneexclusion. Our laboratory has shownthat SIgA is not only able to be"retrotransported" into Peyers patchesvia the associated M cells, but also todeliver sizeable cargos in the form ofSIgA-based immune complexes, resultingin the onset of non-inflammatorytype of responses. Such a novelfunction raises the question of thepossible role of mucosal SIgA in theinterplay with commensal bacteriaand the contribution of the antibody inbacterial homeostasis. To address thisquestion, Lactobacillus rhamnosus(LPR) was administered into a mouseligated loop comprising a Peyerspatch, in association or not with SIgA.The fate of fluorescently labelled bacteriawas followed by laser scanningconfocal microscopy at different incubationtimes. After 2 hours of incubationin the loop, LPR bacteria arefound more abundantly in thesubepithelial dome (SED) regionwhen they are coated with SIgA thanLPR administered alone despite theyare absent from neighboring villi.Herein, it is shown that this mechanismof entry involves M cells inPeyers pathes. After their sampling byM cells, bacteria are engulfed by thedendritic cells of the subjacent SEDregion. Interestingly, LPR bacteriaare found coated by the endogenousnatural SIgA present in mice intestinalsecretions, confirming the requirementof SIgA for this type of entry.The subsequent effect on the maturationof dendritic cells after interactionwith LPR was investigated in vitroin presence or not of SIgA by measuringthe expression of CD40, CD80and CD86 surface markers with flowcytometry analyses. Results show thatDCs respond in the same way in presenceof SIgA than with LPR bacteriaalone, indicating that SIgA does notmodulate the interaction betweenDCs and bacteria in this context. Thiswork gives new evidences about theinvolvement of SIgA in the mechanismby which the intestinal immunesystem permanently checks the contentof the intestine.

Adubação nitrogenada em milho e capim-xaraés sob plantio direto e preparo convencional em sistema agrossilvipastoril

O objetivo deste trabalho foi avaliar o desempenho agronômico das culturas do milho e do capim-xaraés (Urochloa brizantha 'Xaraés') em consórcio, cultivadas em plantio direto e convencional, em sistema agrossilvipastoril, com a aplicação de diferentes doses de nitrogênio em cobertura. O experimento foi conduzido em área de cultivo de milho com a espécie florestal mulateiro (Calycophyllum spruceanum). Utilizou-se o delineamento experimental de blocos ao acaso, com arranjo em parcelas subdivididas, com dois sistemas de manejo do solo (plantio direto e convencional com grade pesada) alocados nas parcelas e com cinco doses de adubação nitrogenada em cobertura (0, 50, 100, 150 e 200 kg ha-1 de N), nas subparcelas, o que totalizou dez tratamentos. A produtividade de grãos de milho respondeu linearmente à aplicação de N em área de plantio convencional. Em área de plantio direto, a dose de 165 kg ha-1 de N em cobertura foi necessária para a obtenção de produtividades satisfatórias. A adubação nitrogenada em cobertura na cultura do milho, até a dose de 200 kg ha-1 de N, não influencia o rendimento do capim-xaraés em consórcio nas entrelinhas, após a colheita do milho.

Desempenho agronômico de alface orgânica influenciado pelo sombreamento, época de plantio e preparo do solo no Acre

O objetivo deste trabalho foi avaliar o desempenho agronômico do cultivo de alface orgânica sob diferentes níveis de sombreamento, épocas de plantio e preparo do solo, no Acre. Para cada cultivar de alface avaliada, lisa (Baba de Verão) e crespa (Vera), foram instalados quatro experimentos em ambientes com níveis distintos de sombreamento (casa de vegetação, 35%; tela, 50%; latada de maracujazeiro, 52%; e a pleno sol), em duas épocas de plantio (estiagem e chuvosa). Utilizou-se o delineamento de blocos ao acaso, com três métodos de preparo do solo (plantio direto, cultivo mínimo e preparo convencional) e quatro repetições. O cultivo em casa de vegetação proporciona maior massa de matéria fresca e produtividade de alface 'Vera', e desempenho similar ao observado sob tela de sombreamento, com preparo mínimo do solo, para as duas épocas de plantio avaliadas. O cultivo a pleno sol, em plantio direto, proporciona maior massa de matéria fresca e produtividade de alface 'Vera', para o período de estiagem. A massa de matéria seca da parte aérea das cultivares Baba de Verão e Vera é maior em cultivo em casa de vegetação, seguida do cultivo sob tela de sombreamento.

Downregulation of endotoxin-induced uveitis by intravitreal injection of vasoactive intestinal Peptide encapsulated in liposomes.

PURPOSE: To reestablish the immunosuppressive microenvironment of the eye, disrupted by ocular inflammation during endotoxin-induced uveitis (EIU), by means of intravitreal injection of vasoactive intestinal peptide (VIP) in saline or encapsulated in liposomes, to increase its bioavailability and efficiency. METHODS: EIU was induced in Lewis rats by subcutaneous injection of lipopolysaccharide (LPS). Simultaneously, animals were intravitreally injected with saline, saline/VIP, VIP-loaded liposomes (VIP-Lip), or unloaded liposomes. EIU severity and cellular infiltration were assessed by clinical examination and specific immunostaining. VIP concentration was determined in ocular fluids by ELISA. Ocular expression of inflammatory cytokine and chemokine mRNAs was detected by semiquantitative RT-PCR. Biodistribution of rhodamine-conjugated liposomes (Rh-Lip) was analyzed by immunohistochemistry in eyes and regional cervical lymph nodes (LNs). RESULTS: Twenty-four hours after intravitreal injection of VIP-Lip, VIP concentration in ocular fluids was 15 times higher than after saline/VIP injection. At that time, EIU clinical severity, ocular infiltrating polymorphonuclear leukocytes (PMNs), and, to a lesser extent, ED1(+) macrophages, as well as inflammatory cytokine and chemokine mRNA expression, were significantly reduced in VIP-Lip-injected rats compared with rats injected with saline/VIP, unloaded liposomes, or saline. Rh-Lip was distributed in vitreous, ciliary body, conjunctiva, retina, and sclera. It was internalized by macrophages and PMNs, and VIP colocalized with liposomes at least up to 14 days after injection. In cervical LNs, resident macrophages internalized VIP-Rh-Lip, and some adjacent lymphocytes showed VIP expression. CONCLUSIONS: VIP was efficient at reducing EIU only when formulated in liposomes, which enhanced its immunosuppressive effect and controlled its delivery to all tissues affected by or involved in ocular inflammation.

Temperatura e tipo de preparo na conservação de produto minimamente processado de abacaxi-'Pérola'

O presente trabalho teve como objetivo avaliar o efeito do tipo de preparo ("rodelas" e "metades") e da temperatura de armazenamento (3ºC, 6ºC e 9ºC) na conservação de produto minimamente processado de abacaxi-'Pérola'. Os frutos, depois de selecionados, lavados e desinfectados com cloro, foram armazenados por 12 horas a 10ºC, antes de serem processados sob condições higiênicas, embalados em contentores de polietileno tereftalatado ("rodelas") ou bandeja de isopor recoberta com filme de cloreto de polivinila esticável ("metades") e armazenados por até 12 dias. Os produtos foram avaliados quanto à evolução da atmosfera interna na embalagem, respiração, quantidade de suco drenado e evolução da massa fresca e da aparência. Foram testadas, durante o período de armazenamento, a aceitabilidade pelos consumidores, no início do experimento e enquanto a aparência e a análise microbiológica permitiram. A presença de bactérias mesofílicas e coliformes totais e fecais foi avaliada a cada três dias. Durante o armazenamento, a porcentagem de O2 nas embalagens apresentou decréscimo, enquanto a de CO2 aumentou até 20% para as "metades" e até 1,86% para as "rodelas". A intensidade dos cortes no preparo teve influência direta na respiração, assim como nas perdas de suco e de massa fresca. A temperatura influenciou na respiração e foi fator limitante à vida de prateleira do produto, pois os produtos armazenados a 9ºC, conservaram-se por 6 dias, enquanto os mantidos a 3ºC e 6ºC, por até 9 dias.

Clostridium difficile toxin-induced inflammation and intestinal injury are mediated by the inflammasome.

BACKGROUND & AIMS: Clostridium difficile-associated disease (CDAD) is the leading cause of nosocomial diarrhea in the United States. C difficile toxins TcdA and TcdB breach the intestinal barrier and trigger mucosal inflammation and intestinal damage. The inflammasome is an intracellular danger sensor of the innate immune system. In the present study, we hypothesize that TcdA and TcdB trigger inflammasome-dependent interleukin (IL)-1beta production, which contributes to the pathogenesis of CDAD. METHODS: Macrophages exposed to TcdA and TcdB were assessed for IL-1beta production, an indication of inflammasome activation. Macrophages deficient in components of the inflammasome were also assessed. Truncated/mutated forms of TcdB were assessed for their ability to activate the inflammasome. The role of inflammasome signaling in vivo was assessed in ASC-deficient and IL-1 receptor antagonist-treated mice. RESULTS: TcdA and TcdB triggered inflammasome activation and IL-1beta secretion in macrophages and human mucosal biopsy specimens. Deletion of Nlrp3 decreased, whereas deletion of ASC completely abolished, toxin-induced IL-1beta release. TcdB-induced IL-1beta release required recognition of the full-length toxin but not its enzymatic function. In vivo, deletion of ASC significantly reduced toxin-induced inflammation and damage, an effect that was mimicked by pretreatment with the IL-1 receptor antagonist anakinra. CONCLUSIONS: TcdA and TcdB trigger IL-1beta release by activating an ASC-containing inflammasome, a response that contributes to toxin-induced inflammation and damage in vivo. Pretreating mice with the IL-1 receptor antagonist anakinra afforded the same level of protection that was observed in ASC-/- mice. These data suggest that targeting inflammasome or IL-1beta signaling may represent new therapeutic targets in the treatment of CDAD.

Preparo de amostras e métodos para a determinação do teor de óleo essencial de frutos de limoeiro

Objetivou-se avaliar alternativas de preparo da amostra e os métodos quantitativos para a determinação do teor de óleo em frutos de limão. Os tratamentos foram avaliados por meio do delineamento estatístico inteiramente casualizado, em esquema fatorial 2x2x6, com quatro repetições, sendo duas variedades-copa de limoeiro verdadeiro ('Siciliano' e 'Eureka'), dois métodos analíticos para a determinação do teor de óleo (Scott e Clevenger) e seis formas de preparo dos frutos para análise (análise do fruto inteiro; frutos cortados na longitudinal e análise de ¼; frutos cortados na longitudinal e análise de ¹/2; frutos cortados ao meio e análise da parte superior; frutos cortados ao meio e análise da parte inferior; e análise de ²/8 do fruto). O método denominado de Clevenger foi o mais eficiente na determinação do teor de óleo essencial, e as formas de preparo de amostras ½ inferior, ½ superior, ¼ longitudinal e ½ longitudinal dos frutos proporcionaram valores superiores de óleo essencial.

Immune Antibodies and Helminth Products Drive CXCR2-Dependent Macrophage-Myofibroblast Crosstalk to Promote Intestinal Repair.

Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/-) or activating Fc receptors (Fcrg-/-) displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb), whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3) by macrophages (MΦ) and myofibroblasts (MF) within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing.