Sites responsible for infectivity and antigenicity on nervous necrosis virus (NNV) appear to be distinct.

Nervous necrosis virus (NNV) is a pathogenic fish-virus belonging to the genus Betanodavirus (Nodaviridae). Surface protrusions on NNV particles play a crucial role in both antigenicity and infectivity. We exposed purified NNV particles to different physicochemical conditions to investigate the effects on antigenicity and infectivity, in order to reveal information regarding the conformational stability and spatial relationships of NNV neutralizing-antibody binding sites and cell receptor binding sites. Treatment with PBS at 37 °C, drastically reduced NNV antigenicity by 66-79% on day one, whereas its infectivity declined gradually from 107.6 to 105.8 TCID50/ml over 10 days. When NNV was treated with carbonate/bicarbonate buffers at different pHs, both antigenicity and infectivity of NNV declined due to higher pH. However, the rate of decline with respect to antigenicity was more moderate than for infectivity. NNV antigenicity declined 75-84% after treatment with 2.0 M urea, however, there was no reduction observed in infectivity. The antibodies used in antigenicity experiments have high NNV-neutralizing titers and recognize conformational epitopes on surface protrusions. The maintenance of NNV infectivity means that receptor binding sites are functionally preserved. Therefore, it seems highly likely that NNV neutralizing-antibody binding sites and receptor binding sites are independently located on surface protrusions.

Identification of viral SIM-SUMO2-interaction inhibitors for treating primary effusion lymphoma.

Primary effusion lymphoma (PEL) is an aggressive B-cell malignancy without effective treatment, and caused by the infection of Kaposi's sarcoma-associated herpesvirus (KSHV), predominantly in its latent form. Previously we showed that the SUMO2-interacting motif within the viral latency-associated nuclear antigen (LANASIM) is essential for establishment and maintenance of KSHV latency. Here, we developed a luciferase based live-cell reporter system to screen inhibitors selectively targeting the interaction between LANASIM and SUMO2. Cambogin, a bioactive natural product isolated from the Garcinia genus (a traditional herbal medicine used for cancer treatment), was obtained from the reporter system screening to efficiently inhibit the association of SUMO2 with LANASIM, in turn reducing the viral episome DNA copy number for establishment and maintenance of KSHV latent infection at a low concentration (nM). Importantly, Cambogin treatments not only specifically inhibited proliferation of KSHV-latently infected cells in vitro, but also induced regression of PEL tumors in a xenograft mouse model. This study has identified Cambogin as a novel therapeutic agent for treating PEL as well as eliminating persistent infection of oncogenic herpesvirus.

New adenine analogues and a pyrrole alkaloid from Selaginella delicatula.

Phytochemical study on the n-BuOH extract of Selaginella delicatula lead to the isolation, characterization and structure elucidation of two new adenine analogues, delicatulines A (1) and B (2), one new pyrrole alkaloid (4), and five known compounds (3, 5-8). These new substances all contain an aliphatic chain in their parent nucleus, which were unusual to find in plants. In the present study, they were identified from Selaginellaceae for the first time. The structures and absolute configurations of these new compounds were determined by a combination of NMR and CD spectroscopic analyses. Compounds 1, 3 and 4 were evaluated for their inhibitory activities on HBV surface antigen and HBV DNA in HepAD38 cells. The results showed that these compounds had only weak or no inhibitive effects on HBV.

Decrease in Dengue virus-2 infection and reduction of cytokine/chemokine production by Uncaria guianensis in human hepatocyte cell line Huh-7.

BACKGROUND: Dengue fever may present hemorrhages and cavitary effusions as result of exacerbated immune responses. We investigated hydro-alcoholic extracts from leaves (UGL) and bark (UGB) of the medicinal species Uncaria guinanensis with respect to antiviral effects in Dengue virus (DENV) infection and in immunological parameters associated with in vivo physiopathological features. METHODS: Chemical profiles from UGB or UGL were compared in thin layer chromatography and 1H nuclear magnetic resonance using flavonoid compounds and a pentacyclic oxindole alkaloid-enriched fraction as references. DENV-2-infected hepatocytes (Huh-7) were treated with extracts. Cell viability, DENV antigens and immunological factors were detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry. FINDINGS: The UGL mainly differed from UGB by selectively containing the flavonoid kaempferitrin. UGB and UGL improved hepatocyte viability. Both extracts reduced intracellular viral antigen and inhibited the secretion of viral non-structural protein (NS1), which is indicative of viral replication. Reduction in secretion of macrophage migration inhibitory factor was achieved by UGB, of interleukin-6 by UGL, and of interleukin-8 by both UGB and UGL. MAIN. CONCLUSIONS: The U. guianensis extracts presented, antiviral and immunomodulatory effects for DENV and possibly a hepatocyte-protective activity. Further studies may be performed to consider these products as potential candidates for the development of an herbal product for the future treatment of dengue.

Decrease in Dengue virus-2 infection and reduction of cytokine/chemokine production by Uncaria guianensis in human hepatocyte cell line Huh-7

ABSTRACT BACKGROUND Dengue fever may present hemorrhages and cavitary effusions as result of exacerbated immune responses. We investigated hydro-alcoholic extracts from leaves (UGL) and bark (UGB) of the medicinal species Uncaria guinanensis with respect to antiviral effects in Dengue virus (DENV) infection and in immunological parameters associated with in vivo physiopathological features. METHODS Chemical profiles from UGB or UGL were compared in thin layer chromatography and 1H nuclear magnetic resonance using flavonoid compounds and a pentacyclic oxindole alkaloid-enriched fraction as references. DENV-2-infected hepatocytes (Huh-7) were treated with extracts. Cell viability, DENV antigens and immunological factors were detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry. FINDINGS The UGL mainly differed from UGB by selectively containing the flavonoid kaempferitrin. UGB and UGL improved hepatocyte viability. Both extracts reduced intracellular viral antigen and inhibited the secretion of viral non-structural protein (NS1), which is indicative of viral replication. Reduction in secretion of macrophage migration inhibitory factor was achieved by UGB, of interleukin-6 by UGL, and of interleukin-8 by both UGB and UGL. MAIN CONCLUSIONS The U. guianensis extracts presented, antiviral and immunomodulatory effects for DENV and possibly a hepatocyte-protective activity. Further studies may be performed to consider these products as potential candidates for the development of an herbal product for the future treatment of dengue.

Total Synthesis and in Vitro Anti-Tumor-Promoting Activities of Racemic Acetophenone Monomers from Acronychia trifoliolata.

Six acetophenone derivatives, acronyculatins I (1), J (2), K (3), L (4), N (5), and O (6), were recently isolated from Acronychia trifoliolata, and the structure of the known acronyculatin B (7) was revised. Because of the limited quantities of isolated products as well as their structure similarity, racemic acronyculatins I-L, N, O, and B (1-7) were synthesized to confirm their structures and to obtain sufficient material for biological evaluation. Trihydroxyacetophenone was converted to the target compounds by various sequences of hydroxy group protection, allylation or prenylation, and epoxidation followed by cyclization. C-Prenylations were carried out by direct addition of a prenyl group or through 1,3- or 3,3-sigmatropic rearrangement. The synthesized racemic compounds were evaluated in an anti-tumor-promoting assay using the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. All tested compounds significantly inhibited EBV-EA activation. Especially, racemic acronyculatin I (1) displayed the most potent inhibitory effects, with an IC50 value of 7.3 µM.

Disinfection protocols for necropsy equipment in rabies laboratories: Safety of personnel and diagnostic outcome.

In the last decades, molecular techniques have gradually been adopted for the rapid confirmation of results obtained through gold standard methods. However, international organisations discourage their use in routine laboratory investigations for rabies post-mortem diagnosis, as they may lead to false positive results due to cross-contamination. Cleaning and disinfection are essential to prevent cross-contamination of samples in the laboratory environment. The present study evaluated the efficacy of selected disinfectants on rabies-contaminated necropsy equipment under organic challenge using a carrier-based test. The occurrence of detectable Rabies virus (RABV) antigen, viable virus and RNA was assessed through the gold standard Fluorescent Antibody Test, the Rabies Tissue Culture Infection Test and molecular techniques, respectively. None of the tested disinfectants proved to be effective under label conditions. Off label disinfection protocols were found effective for oxidizing agents and phenolic, only. Biguanide and quaternary ammonium compound were both ineffective under all tested conditions. Overall, discordant results were obtained when different diagnostic tests were compared, which means that in the presence of organic contamination common disinfectants may not be effective enough on viable RABV or RNA. Our results indicate that an effective disinfection protocol should be carefully validated to guarantee staff safety and reliability of results.

Triterpene glycosides and other polar constituents of shea (Vitellaria paradoxa) kernels and their bioactivities.

The MeOH extract of defatted shea (Vitellaria paradoxa; Sapotaceae) kernels was investigated for its constituents, and fifteen oleanane-type triterpene acids and glycosides, two steroid glucosides, two pentane-2,4-diol glucosides, seven phenolic compounds, and three sugars, were isolated. The structures of five triterpene glycosides were elucidated on the basis of spectroscopic and chemical methods. Upon evaluation of the bioactivity of the isolated compounds, it was found that some or most of the compounds have potent or moderate inhibitory activities against the following: melanogenesis in B16 melanoma cells induced by α-melanocyte-stimulating hormone (α-MSH); generation of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, against Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-teradecanoylphorbol 13-acetate (TPA) in Raji cells; t TPA-induced inflammation in mice, and proliferation of one or more of HL-60, A549, AZ521, and SK-BR-3 human cancer cell lines, respectively. Western blot analysis established that paradoxoside E inhibits melanogenesis by regulation of expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1) and TRP-2. In addition, tieghemelin A was demonstrated to exhibit cytotoxic activity against A549 cells (IC50 13.5 µM) mainly due to induction of apoptosis by flow cytometry. The extract of defatted shea kernels and its constituents may be, therefore, valuable as potential antioxidant, anti-inflammatory, skin-whitening, chemopreventive, and anticancer agents.

Hydrogen peroxide inactivation of influenza virus preserves antigenic structure and immunogenicity.

The use of live virus in the laboratory requires additional precautions, such as personnel training and special equipment, in order to limit the transmission risk. This is a requirement which not all laboratories can fulfill. In this study, a viral inactivation method is introduced using hydrogen peroxide (H2O2), which maintains antigenicity. Three strains of influenza viruses were inactivated and the ex vivo cellular and humoral immune responses were further analyzed, by comparing them to live viruses, in ELISpot, Multiplex and ELISA assays. In all assays, the H2O2 inactivated viruses displayed comparable responses to the live viruses, suggesting that the inactivated viruses still elicited immunogenic responses even though inactivation was confirmed by lack of viral replication in MDCK cells. Taken together, this study demonstrates that influenza viruses inactivated with H2O2 retain immunogenicity and are able to both detect humoral and elicit cellular immune responses in vitro, which could reduce the need to handle live viruses in the laboratory.

Synergistic action of compounds isolated from the hexane extract of Ardisia crispa root against tumour-promoting effect, in vitro.

An isomeric mixture of α,ß-amyrin (triterpene) and 2-methoxy-6-undecyl-1,4-benzoquinone (quinone) isolated from the Ardisia crispa root hexane (ACRH) extract was reported to possess anti-inflammatory properties in vivo. Considering the close association between inflammation and cancer, on top of the lack of antitumour study on those compounds, this study aimed to determine the potential of both compounds against tumour promotion in vitro, either as single agent or in combination. Triterpene and quinone compounds, as well as triterpene-quinone fraction (TQF) and ACRH were subjected to inhibition of Epstein-Barr virus-early antigen (EBV-EA) activation assay for that purpose. Compared with curcumin (positive control), inhibition against EBV-EA activation occurred in the order: ACRH>TQF ≥ curcumin>α,ß-amyrin ≥ 2-methoxy-6-undecyl-1,4-benzoquinone. These findings reported, for the first time, the antitumor-promoting effect of α,ß-amyrin and 2-methoxy-6-undecyl-1,4-benzoquinone from the roots of A. crispa, which was enhanced when both compounds act in synergy.

α-Helix peptides designed from EBV-gH protein display higher antigenicity and induction of monocyte apoptosis than the native peptide.

We tested the hypothesis that stabilizing α-helix of Epstein-Barr virus gH-derived peptide 11438 used for binding human cells will increase its biological activity. Non-stable α-helix of peptide 11438 was unfolded in an entropy-driven process, despite the opposing effect of the enthalpy factor. Adding and/or changing amino acids in peptide 11438 allowed the designing of peptides 33207, 33208 and 33210; peptides 33208 and 33210 displayed higher helical content due to a decreased unfolding entropy change as was determined by AGADIR, molecular dynamics and circular dichroism analysis. Peptides 33207, 33208 and 33210 inhibited EBV invasion of peripheral blood mononuclear cells and displayed epitopes more similar to native protein than peptide 11438; these peptides could be useful for detecting antibodies induced by native gH protein since they displayed high reactivity with anti-EBV antibodies. Anti-peptide 33207 antibodies showed higher reactivity with EBV than anti-peptide 11438 antibodies being useful for inducing antibodies against EBV. Anti-peptide 33210 antibodies inhibit EBV invasion of epithelial cells better than anti-peptide 11438 antibodies. Peptide 33210 bound to normal T lymphocytes and Raji cells stronger than peptide 11438 and also induced apoptosis of monocytes and Raji cells but not of normal T cells in a similar way to EBV-gH. Peptide 33210 inhibited the monocytes' development toward dendritic cells better than EBV and peptide 11438. In conclusion, stabilizing the α-helix in peptides 33208 and 33210 designed from peptide 11438 increased the antigenicity and the ability of the antibodies induced by peptides of inhibiting EBV invasion of host cells.

Cancer preventive agents 10. Prenylated dehydrozingerone analogs as potent chemopreventive agents.

Dehydrozingerone analogs and related compounds were screened as potential antitumor promoters by using the in vitro short-term 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus early antigen activation assay. Among the 40 synthesized compounds, the prenylated analogs 16 and 34-36 showed the most significant and promising activity (100% inhibition of activation at 1 x 10(3) mol ratio/TPA, and 82-80%, 37-35%, and 13-11% inhibition at 5 x 10(2), 1 x 10(2), and 1 x 10 mol ratio/TPA, respectively) in this screening. Their activity profiles were comparable to those of the reference standard curcumin. While a prenyl moiety conferred potent chemopreventive activity, an extended prenyl unit such as a farnesyl moiety did not improve activity. Because in vitro inhibitory effects in this assay generally correlate well with in vivo inhibitory effects on tumor promotion, our results strongly suggested that prenylated 16 and 34-36 are likely to be promising antitumor promoters.

Sesquiterpenes from Maytenus jelskii as potential cancer chemopreventive agents.

Seven new (1-4 and 7-9) sesquiterpenes with a dihydro-beta-agarofuran skeleton, along with four known compounds (5, 6, 10, and 11), have been isolated from the leaves of Maytenus jelskii. The structures of the new compounds were elucidated by means of spectroscopic data analysis, including 1D and 2D NMR techniques, and their absolute configurations were determined by circular dichroism and chemical correlations. The compounds have been tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Compound 10 was found to be an effective antitumor-promoting agent and also showed a potent chemopreventive effect in an in vivo two-stage carcinogenesis model.

The influence of the inactivating agent on the antigen content of inactivated Newcastle disease vaccines assessed by the in vitro potency test.

An in vitro potency test has recently been included in the European Pharmacopoeia (EP) monograph (01/2007:0870) to assess the potency of inactivated Newcastle disease (ND) vaccines. This enzyme linked immunosorbent assay (ELISA) is an attractive alternative for the existing in vivo potency tests especially with regard to the objective of the European Authorities to Replace, Reduce and Refine the use of laboratory animals for production and quality control of immunobiologicals. In the present study the influence of the inactivant on the antigen content established by ELISA was evaluated. Therefore, oil based vaccines containing similar concentrations of beta-propiolactone (BPL) or formaldehyde inactivated Newcastle disease virus (NDV) were examined by ELISA and in the in vivo potency tests outlined in the EP. The results obtained demonstrate that the use of formaldehyde as inactivant lowered the in vitro potency compared to BPL as inactivant. In contrast, the in vivo potency was not affected. Therefore, the ELISA should not be used to compare the potency of commercial ND vaccines containing formaldehyde inactivated NDV with those containing BPL inactivated NDV. However, the ELISA is considered an attractive alternative for the existing in vivo potency tests since it can be used by vaccine manufacturers for the release of inactivated ND vaccines.

Oxidative stress can alter the antigenicity of immunodominant peptides.

APCs operate frequently under oxidative stress induced by aging, tissue damage, pathogens, or inflammatory responses. Phagocytic cells produce peroxides and free-radical species that facilitate pathogen clearance and can in the case of APCs, also lead to oxidative modifications of antigenic proteins and peptides. Little information is available presently about the consequences of such modifications on the immune response. To model oxidative modification of an immunodominant antigenic peptide, we oxidized the methionine residue of the human CMV pp65(495-503) (NLVPMVATV) peptide. Such modifications of an antigenic peptide can affect MHC binding or TCR recognition. Using binding and dissociation assays, we demonstrate that oxidative modification of the CMVpp65(495-503) peptide leads to a decreased binding of the pMHC complex to the TCR, whereas binding of the peptide to the MHC class I molecule is not impaired. Additionally, we show that CD8(+) T cells have a decreased proliferation and IFN-gamma production when stimulated with oxidized CMVpp65(495-503) peptide. Spectratyping the antigen-binding site of the TCR of responding T cells demonstrates that the CMVpp65(495-503) and the CMVoxpp65(495-503) peptides preferentially stimulate BV8 T cells. Sequencing of this dominant BV family reveals a highly conserved CDR3 amino acid motif, independent of the mode of stimulation, demonstrating the recruitment of the same T cell clonotypes. Our results suggest that oxidative modification of antigenic peptides may affect T cell responses severely by binding T cell clones with different affinity. This may lead to an altered immune response against infectious agents as well as against tumor or autoantigens under oxidative stress conditions.

Posttransplant lymphoproliferative disorder in pediatric liver transplantation.

The success rate of pediatric liver transplantation has improved in recent years. Advances in immunosuppression have reduced the risk of rejection, but have enhanced the risk of posttransplant lymphoproliferative disorder (PTLD). Since 1994, we have performed 197 orthotopic liver transplantations in 157 recipients younger than 15 years. Herein we have performed a retrospective study to review the incidence and clinical characteristics, along with the treatment and outcomes of PTLD diagnosed over this 14-year experience. We documented 8 cases of PTLD (5%), half of which occurred during the first 2 years posttransplantation; 5 presented with abdominal involvement and 2 with thoracic masses. The histological findings showed lymphoma in 6 cases. All were treated with reduction of immunosuppression and 2 received Rituximab. Three patients died, a mortality rate of 37.5%. One subject experienced rejection, and the others responded to treatment. PTLD is a life-threatening condition that requires a high index of suspicion, appropriate imaging, biopsy diagnosis, and prompt treatment to achieve positive results. Quantitative monitoring of Epstein-Barr virus load may be useful to detect a high-risk population.

Anti-tumor-promoting effects of 25-methoxyporicoic acid A and other triterpene acids from Poria cocos.

Nine new (1, 3, 5, 8, 12, 13, 15, 17, and 18) and nine known (2, 4, 6, 7, 9-11, 14, and 16) lanostane-type triterpene acids and a known diterpene acid (19) were isolated from the epidermis of the sclerotia of Poria cocos. The structures of the new compounds were established as 16alpha,27-dihydroxydehyrotrametenoic acid (1), 25-hydroxy-3-epitumulosic acid (3), 16alpha,25-dihydroxyeburiconic acid (5), 25-methoxyporicoic acid A (8), 26-hydroxyporicoic acid DM (12), 25-hydroxyporicoic acid C (13), poricoic acid GM (15), poricoic acid HM (17), and 6,7-dehydroporicoic acid H (18), on the basis of spectroscopic methods. On evaluation of the nine new and two of the known compounds, 4 and 19, against the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, all of the compounds exhibited inhibitory effects, with IC(50) values in the range 187-348 mol ratio/32 pmol TPA. In addition, compound 8 exhibited an inhibitory effect on skin tumor promotion in an in vivo two-stage carcinogenesis test using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter. Further, 17 compounds, 1-14, 16, 18, and 19, were evaluated for their cytotoxic activity against two human tumor cell lines, HL60 (leukemia) and CRL1579 (melanoma).

Cancer chemopreventive activity of oleanane-type triterpenoids from the stem bark of Betula ermanii.

In search for cancer chemopreventive agents from natural sources, three oleanane- and four known lupane-type triterpenoids, and sitosterol from the stem bark of Betula ermanii were tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among them, 3beta-acetoxy-12alpha-hydroxyoleanan-13beta,28-olide (1) and 3beta-acetoxy-11alpha,12alpha-epoxyoleanan-13beta,28-olide (2) were investigated for the inhibitory effect in a two-stage carcinogenesis test on mouse skin using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter. 3beta-Acetoxy-11alpha,12alpha-epoxyoleanan-13beta,28-olide (2) was found to exhibit the potent antitumor promoting activity in the in vivo carcinogenesis test.

2-O-beta-d-glucopyranosyl-sn-glycerol based analogues of sulfoquinovosyldiacylglycerols (SQDG) and their role in inhibiting Epstein-Barr virus early antigen activation.

New sulfoquinovosyldiacylglycerols derived from 2-O-beta-d-glucopyranosyl-sn-glycerol, carrying acyl chains of various length on the glycerol moiety, were prepared through a convenient synthetic procedure in which a sulfonate is introduced at the C-6 position of glucose by oxidation of a thioacetate in the presence of the unprotected secondary hydroxyl groups, and tested for their anti-tumor-promoting activity using a short-term in vitro assay for Epstein-Barr virus early antigen (EBV-EA) activation. Our study has allowed to ascertain the role of the 6'-sulfonate group and the need of a free hydroxyl group on the glycerol moiety in inhibiting the EBV activation promoted by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).

Cancer preventive agents 9. Betulinic acid derivatives as potent cancer chemopreventive agents.

C-3 esterifications of betulinic acid (BA, 1) and its A-ring homolog, ceanothic acid (CA, 2), were carried out to provide sixteen terpenoids, 4-19, including nine new compounds (4-12). All synthesized compounds were evaluated in an in vitro antitumor-promoting assay using the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Among them, compounds 4-6, 11-14, 16, and 17 displayed remarkable inhibitory effects of EBV-EA activation. BA analog 6, which contains a prenyl-like group, showed the most potent inhibitory effect (100%, 76%, 37%, and 11% inhibition of EBA activation at 1000, 500, 100, and 10mol ratio/TPA, respectively, with IC(50) value of 285mol ratio/32pmol TPA). Compound 6 merits further development as a cancer preventive agent.