Punica granatum L. Leaf Extract Attenuates Lung Inflammation in Mice with Acute Lung Injury.

The hydroalcoholic extract of Punica granatum (pomegranate) leaves was previously demonstrated to be anti-inflammatory in a rat model of lipopolysaccharide- (LPS-) induced acute peritonitis. Here, we investigated the anti-inflammatory effects of the ethyl acetate fraction obtained from the pomegranate leaf hydroalcoholic extract (EAFPg) on the LPS-induced acute lung injury (ALI) mouse model. Male Swiss mice received either EAFPg at different doses or dexamethasone (per os) prior to LPS intranasal instillation. Vehicle-treated mice were used as controls. Animals were culled at 4 h after LPS challenge, and the bronchoalveolar lavage fluid (BALF) and lung samples were collected for analysis. EAFPg and kaempferol effects on NO and cytokine production by LPS-stimulated RAW 264.7 macrophages were also investigated. Pretreatment with EAFPg (100-300 mg/kg) markedly reduced cell accumulation (specially neutrophils) and collagen deposition in the lungs of ALI mice. The same animals presented with reduced lung and BALF TNF-α and IL-1ß expression in comparison with vehicle controls (p < 0.05). Additionally, incubation with either EAFPg or kaempferol (100 µg/ml) reduced NO production and cytokine gene expression in cultured LPS-treated RAW 264.7 macrophages. Overall, these results demonstrate that the prophylactic treatment with EAFPg attenuates acute lung inflammation. We suggest this fraction may be useful in treating ALI.

Xanthomonas axonopodis pv. punicae uses XopL effector to suppress pomegranate immunity.

Xanthomonas axonopodis pv. punicae (Xap) causing bacterial blight is an important pathogen that incurs significant losses to the exportability of pomegranate. Xap uses the Xop TTSS-effector, via the type three secretion system, to suppress pomegranate immunity. Here, we investigate the role of XopL during blight pathogenesis. We observed that XopL is essential for its in planta growth and full virulence. Leaves inoculated with Xap ΔxopL produced restricted water-soaked lesions compared to those inoculated with wild-type Xap. XopL supports Xap for its sustained multiplication in pomegranate by suppressing the plant cell death (PCD) event. We further demonstrated that XopL suppresses immune responses, such as callose deposition and production of reactive oxygen species (ROS). RT-qPCR analysis revealed that immune responsive genes were upregulated when challenged with Xap ΔxopL, whereas upregulation of such genes was compromised in the complemented strain containing the xopL gene. The transiently expressed XopL::EYFP fusion protein was localized to the plasma membrane, indicating the possible site of its action. Altogether, this study highlights that XopL is an important TTSS-effector of Xap that suppresses plant immune responses, including PCD, presumably to support the multiplication of Xap for a sufficient time-period during blight disease development.

Pomegranate Cultivars: Identification of the New IgE-Binding Protein Pommaclein and Analysis of Antioxidant Variability.

The consumption of pomegranate is increasing as it is considered a health-promoting food. Nevertheless, it can trigger allergic reactions, sometimes severe. The LTP Pun g 1 is the only pomegranate allergen so far reported. Based on preliminary clinical observations, the main aim of this study was the investigation of still unknown allergens contained in this fruit. Pommaclein, a homologue of peamaclein, the peach allergen Pru p 7, was isolated, identified by protein sequencing, and characterized as an IgE-binding protein by different test systems. RP-HPLC protein profiles revealed significant variations of LTP and pommaclein content in the red pulp of selected cultivars and accessions. Conversely, the mesocarp appeared free of proteins and much richer in antioxidants. In conclusion, a new allergen has been identified, and it could contribute to improving allergy diagnosis. The study highlights that pomegranate mesocarp could represent a rich and safe source of nutraceuticals also for allergic subjects.

Non-covalent pomegranate (Punica granatum) hydrolyzable tannin-protein complexes modulate antigen uptake, processing and presentation by a T-cell hybridoma line co-cultured with murine peritoneal macrophages.

In this work we characterize the interaction of pomegranate hydrolyzable tannins (HT) with hen egg-white lysozyme (HEL) and determine the effects of non-covalent tannin-protein complexes on macrophage endocytosis, processing and presentation of antigen. We isolated HT from pomegranate and complex to HEL, the resulting non-covalent tannin-protein complex was characterized by gel electrophoresis and MALDI-TOF MS. Finally, cell culture studies and confocal microscopy imaging were conducted on the non-covalent pomegranate HT-HEL protein complexes to evaluate its effect on macrophage antigen uptake, processing and presentation to T-cell hybridomas. Our results indicate that non-covalent pomegranate HT-HEL protein complexes modulate uptake, processing and antigen presentation by mouse peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas a non-covalent pomegranate HT-HEL complex had already reached maximum IL-2 expression. Pomegranate HT may increase rate of endocytose of HEL and subsequent expression of IL-2 by the T-cell hybridomas.

Pomegranate ( Punica granatum L.) expresses several nsLTP isoforms characterized by different immunoglobulin E-binding properties.

BACKGROUND: Pomegranate allergy is associated with sensitization to non-specific lipid transfer proteins (nsLTPs). Our aim was to identify and characterize the non-specific nsLTPs expressed in pomegranate at the molecular level and to study their allergenic properties in terms of immunoglobulin E (IgE)-binding and cross-reactivity with peach nsLTP (Pru p 3). METHODS: A non-equilibrium two-dimensional (2-D) electrophoretic approach based on acid-urea PAGE and sodium dodecyl sulfate PAGE was set up to separate pomegranate nsLTPs. Their immunoreactivity was tested by immunoblotting carried out with anti-Pru p 3 polyclonal antibodies and sera from pomegranate-allergic patients. For final identification, pomegranate nsLTPs were purified by chromatography and subjected to trypsin digestion and mass spectrometry (MS) analysis. For this purpose, the sequences obtained by cDNA cloning of three pomegranate nsLTPs were integrated in the database that was subsequently searched for MS data interpretation. RESULTS: Four nsLTPs were identified by 2-D immunoblotting. The detected proteins showed different IgE-binding capacity and partial cross-reactivity with Pru p 3. cDNA cloning and MS analyses led to the identification of three nsLTP isoforms with 66-68% amino acid sequence identity named Pun g 1.0101, Pun g 1.0201 and Pun g 1.0301. CONCLUSIONS: By 2-D electrophoresis, we could separate different nsLTP isoforms possessing different IgE-binding properties, which might reflect peculiar allergenic potencies. The contribution of Pru p 3 to prime sensitization is not central as in other plant nsLTPs.

Compostos fenólicos da polpa e sementes de romã ( Púnica granatum, L)atividade antioxidante e protetrora em células MDCK

Os radicais livres, dentre os quais as EROs e as ERNs, são espécies reativas envolvidas no início de reaçõescomo a peroxidação, que no sistema biológico pode dar início ao desenvolvimento de diversos processos patológicos. Para conter o avanço da cadeia oxidativa iniciada pelos radicais livres e minimizar os seus efeitos deletérios ao organismo, algumas substâncias antioxidantes como compostos fenólicos, dentre os quais os ácidos fenólicos, podem ser encontrados em diversas fontes vegetais. Asfrutas são uma boa fonte de compostos antioxidantes, e a romã (Punica granatum, L.) apresenta expressiva quantidade de compostos fenólicos. Os objetivos deste trabalho foram identificar os principais ácidos fenólicos, presentes na fruta (polpa e sementes), através de cromatografi a gasosa, a avaliação da capacidade antioxidante, utilizando dois métodos: o de co-oxidação de substratos (β- caroteno e ácido linoléico) e o da redução do radical 1,1- diphenyl- 2- picryl- hydrazyl (DPPH•). Ainda, foi verifi cado o efeito da fração de ácidos fenólicos livres da polpa da romã (AFLp) sobre a proliferação e viabilidade em cultura de células MDCK. Pôde-se verifi car que a fração AFLp apresentou o maior conteúdo de compostos redutores (1.399,02μg/mL) enquanto que a de ácidos fenólicos ligados a compostos solúveis apresentou o maior conteúdo (770,58 μg/mL) nas sementes(AFESs). Todas as frações de ácidos fenólicos apresentaram resultados signifi cativamente (p<0,05) superiores aos do antioxidante sintético butil-hidroxitolueno, utilizado como padrão. Os principais ácidos fenólicos encontrados foram o salicílico, o protocatequínico e o gálico. As célulastratadas com baixas concentrações da fração AFLp apresentaram aumento da proliferação celular e a viabilidade manteve-se alta.

Effect of traditional Korean medicinal (TKM) triherbal extract on the innate immune system and disease resistance in Paralichthys olivaceus against Uronema marinum.

We report the effect of aqueous-, ethanol- and methanol-solvent-derived extracts of three traditional Korean herbs, Punica granatum, Chrysanthemum cinerariaefolium and Zanthoxylum schinifolium, by monitoring the innate immune mechanisms, such as phagocytosis activity, respiratory burst activity, alternative complement activity and lysozyme activity and the functional immunity in terms of percentage mortality and relative percent survival (RPS) in olive flounder (Paralichthys olivaceus) against Uronema marinum (1 x 10(5)ciliates ml(-1)) for 30 days. Fish were intraperitoneally administered with 5, 50 and 100 mg kg(-1) body weight of each traditional Korean medicinal (TKM) solvent extract except the control and infected untreated groups. In all the treated groups at concentrations of 50 and 100 mg kg(-1) body weight, the chosen innate immune parameters were found significantly enhanced when compared to 0 mg kg(-1) dose. However, at 5 mg kg(-1) the tested immune parameters did not vary. Administration of TKM solvent extracts preceding the challenge with U. marinum for 30 days significantly reduced the percentage mortality with the consequent increase in RPS. Administration of 50 and 100 mg kg(-1) TKM solvent extracts clearly enhanced the innate immune responses and disease resistance in P. olivaceus against U. marinum.

Anaphylaxis to excipient mannitol: evidence for an immunoglobulin E-mediated mechanism.

BACKGROUND: Anaphylaxis to mannitol present naturally in pomegranate and cultivated mushroom in a sensitized subject has been described recently, and an IgE-mediated mechanism to this sugar alcohol has been proposed. The same subject also experienced severe allergic reactions to a chewable pharmaceutical (cisapride drug). OBJECTIVE: The purpose of the study was to identify allergenic component in the pharmaceutical preparation, and also, to understand the mechanism of immediate hypersensitivity to mannitol. METHODS: Methodology involved skin prick tests (SPTs), high-performance liquid chromatographic (HPLC) analysis of pharmaceutical preparations, separation of mannitol by Ca++-ion-moderated cation-exchange chromatography, preparation of alditol-protein conjugates by reductive amination, SPT using the conjugates, hapten affinity purification of the allergic serum on D-mannitol-keyhole limpet haemocyanin (KLH)-Sepharose CL-6B, and detection of serum mannitol-specific IgE by ELISA. RESULTS: Component testing by SPT, and HPLC analysis of various pharmaceuticals indicated that the excipient mannitol is the causative allergen. Mannitol separated from Cisapid MPS showed allergenic activity by SPT. Among the several conjugates tested by SPT, D-mannitol-bovine serum albumin and D-mannitol-KLH showed positive weal/flare reaction, demonstrating the presence of cell-bound mannitol-specific IgE in vivo. Negative results with D-glucitol, D-galactitol, meso-erythritol, and L-mannitol protein conjugates clearly showed that the mannitol-specific human IgE is very specific to the D-isomer of mannitol. ELISA using the hapten affinity-purified allergic serum was positive, demonstrating the presence of mannitol-specific serum IgE in the allergic subject. CONCLUSION: Mannitol, which is widely used as a food and drug additive (excipient), can rarely cause IgE-mediated anaphylaxis. This study is the first one to demonstrate the presence of mannitol-specific human IgE in a sensitized allergic subject to validate an IgE-mediated hypersensitivity mechanism for mannitol.