Synergistic inhibition of influenza A virus replication by a plant polyphenol-rich extract and epsilon-aminocaproic acid in vitro and in vivo.

UNLABELLED: A combined antiviral effect of a polyphenol-rich extract of the medicinal plant Geranium sanguineum L. (PC) and a protease inhibitor, epsilon-aminocaproic acid (ACA) was examined in Influenza A virus (IAV)-infected MDCK cell cultures and mice. Synergistic, antagonistic, or indifferent antiviral effects were distinguished on the basis of virus yields, namely fractional yields of individual compounds and yields of both compounds in combination. Combinations of PC and ACA in particular concentrations proved synergistic in the inhibition of virus replication in MDCK cells and in protection of mice against virus infection as determined by virus titers, lung weight, mean survival time (MST), mortality rate, and protection rate (PR). Following the application of a combination of PC and ACA to the virus-infected mice, the levels of the lung protease and protease-inhibitory activity, which were increased due to the virus infection, were brought to normal. These results demonstrate the rationale for a combined application of viral inhibitors with different modes of action to the treatment of IAV infection, in particular PC as a natural inhibitor of early viral transcription and translation and ACA as a synthetic inhibitor of cellular proteases. KEYWORDS: Influenza A virus; antiviral effect; synergism; plant polyphenol extract; epsilon-aminocaproic acid; protease inhibitors.

Limbal explants from cryopreserved cadaver human corneas. Immunofluorescence and light microscopy of epithelial cells growing in culture.

The aim of the present study was to determine whether human cadaver corneas, that were subject to cryopreservation, would be a source of migrating epithelial cells in vitro and what kind of morphological features these cells possess. Limbal explant culture was used for expanding the epithelial cells. Non-quantitative light microscopical examinations of the cultures within a period of 28 days were carried out. The phenotype of cultured cells, particularly of the presumed adult stem cell population, was examined by indirect fluorescent immunostaining using antibodies against corneal stem cell associated markers p63 and vimentin. The effectiveness of the freezing-thawing protocol was confirmed by cultivation of limbal explants taken from non-cryopreserved cadaver corneoscleral rims. The result clearly showed that limbal tissue, subjected to cryopreservation and long lasting (up to 12 months) storage in liquid nitrogen, retains the capacity to be source of migrating and proliferating epithelial cells in vitro including the presumed adult stem cells and transient amplifying cells.

Reduction of nonlinear dynamic systems with an application to signal transduction pathways.

Mathematical modelling of kinetic processes with different time scales allows a reduction of the governing equations using quasi-steady-state approximations (QSSA). A QSSA theorem is applied to a mathematical model of the influence that Raf kinase inhibitor protein (RKIP) has on the ERK signalling pathway. On the basis of previously published parameter values, the system of 11 ordinary differential equations is rewritten in a form suitable for model reduction. In accordance with the terminology of the QSSA theorem, it is established that four of the protein and protein-complex concentrations are 'fast varying', such that the corresponding kinetic equations form an attached system. Another concentration is 'medium varying' such that the corresponding equation is reduced with respect to the four fast ones. The other six concentrations are 'slow varying', which means the corresponding kinetic equations also present a reduced system with respect to the others. Analytical solutions, relating the steady-state values of the fast varying protein concentrations and the slow varying ones, are derived and interpreted as restrictions on the regulatory role of RKIP on ERK-pathway.

Saponins from Tribulus terrestris L are less toxic for normal human fibroblasts than for many cancer lines: influence on apoptosis and proliferation.

The objective of the study was to explore the influence of saponins derived from Tribulus terrestris L. (TT) on normal human skin fibroblasts and to compare it with their anticancer properties. In this study, [3H]thymidine incorporation and MTT to assess cell proliferation and viability, respectively, and immunoblotting and HPLC analysis to explore intracellular signal transduction pathways have been used. We found that TT caused a dose-dependent decrease in [3H]thymidine incorporation into the DNA of treated fibroblast compared to the untreated controls. Viability of treated cells remained within the control levels with treatment of up to 5 micro g TT/ml medium. It was significantly depressed with incubation in > or =6 micro g TT/ml medium with an IC50 of 12.6 micro g TT/ml of cultivating media. ERK1/2 was significantly dephosphorylated at 5 mins of incubation with TT until the 48th hour, when phosphorylation slightly recovered, but was still below the control levels. In contrast, p38 and JNK phosphorylation was positively influenced, with peaks at 1 hr and 24 hrs of incubation respectively. Phosphorylation/dephosphorylation events of SAPK/MAPK clearly correlated with Mkp-1 induction. Procaspase 3 was activated after 5 mins of incubation and coincided with a rapid actin cleavage. There was a significant decrease of putrescine concentration and a concomitant increase of spermidine and spermine at 2 mins of treatment. According to our results, TT is less toxic for normal human skin fibroblasts in comparison to many cancer lines investigated in previous studies. The molecular mechanism of this cytotoxicity involves up- and downregulation of polyamines' homeostasis, suppression of proliferation, and induction of apoptosis. Further research in this field using animal models would help to explore and interpret the potential properties of TT as an anticancer supplement.

The penguin feathers as bioindicator of Antarctica environmental state.

Concentrations of biogenic and toxic elements (Na, K, Mg, Ca, P, S, Fe, Cu, Zn, Co, Mn, Se, Ni, Sr, Al, Cd, Pb, As) were determined for the first time in feathers of gentoo penguin (Pygoscelis papua) and chinstrap penguin (Pygoscelis antarctica) from Antarctica. A comparison of element levels was performed among these species in years 2002-2003. Penguins molt annually and this fact allows defining precisely the concentrations of accumulated toxic elements and heavy metals in plumage every year. A continual environmental biomonitoring could establish a possible trend to contamination of the Antarctica sea zones. The penguin feather is an excellent subject for monitoring because penguins have long life span, permanent ecological niche and dominate the aviafauna in Antarctica. Because of its remoteness, Antarctica is believed to be unpolluted. The relatively elevated levels of Cd established are due to the Cd-enrichment of the Antarctic marine food chain. Because of great bioaccumulation of lead in feathers, the concentration of Pb in penguin feather was higher (4-8 times) compared to that of Cd. In both penguin species the levels of Zn were 1.9 times higher than respective Fe levels. The concentrations of most of the investigated elements were significantly higher in P. papua than in P. antarctica and this probably could be explained by the different diet and feeding habit of these species.

Integrity of endothelium in cryopreserved human cornea.

The aim of the present study was to elaborate an optimal method for cryopreservation of human donor cornea for transplantation and to follow the morphological changes in the structure of the endothelial cell layer using scanning electron microscopy (SEM). Sixteen groups, with four donor cornea each, were cryopreserved at cooling rates of 1 degree C per min and 5 degree C per min. Four cryoprotectants (glycerol, dimethyl sulfoxide, 1,2-propanediol, polyethylene glycol-400) in two concentrations (5% and 10% v/v) were prepared on the bases of medium Optisol GS supplied with 20% v/v human serum albumin. Four additional human cornea were used as controls. Endothelial cell recovery of the cornea after thawing and 24 hours culture, was calculated as a percent of the preserved recovered cells. Sufficient recovery of the endothelial cell layer, making the cornea suitable for transplantation was obtained using the cryoprotectants dimethyl sulfoxide and especially polyethylene glycol-400.

Development of antibody-secreting cells and antigen-specific T cells in cervical lymph nodes after intranasal immunization.

Intranasal (i.n.) immunization with bacterial protein antigens coupled to cholera toxin B subunit (CTB) effectively induces mucosal, especially salivary immunoglobulin A (IgA), and nonmucosal antibody responses in mice. To examine the regional distribution of antigen-specific B and T cells after i.n. immunization, antibody-secreting cells and antigen-responsive T cells in cervical lymph nodes (CLN) were compared with those found after intraoral or subcutaneous (in the neck) administration of the same antigen and with T cells found in mesenteric lymph nodes (MLN) and spleen after intragastric immunization. The i.n. immunization induced predominantly IgA antibody-secreting cells in salivary glands and IgA and IgG antibody-secreting cells in the superficial and central CLN; these responses were quantitatively enhanced if the antigen was coupled to CTB. Intraoral immunization also induced IgA and IgG antibody-secreting cells in the superficial and central CLN, but only if intact cholera toxin was included as an adjuvant. In contrast, subcutaneous (neck) immunization induced IgG antibody-secreting cells mainly in the draining facial lymph nodes. CLN cell populations resembled those of MLN, except that CLN lymphocytes had higher proportions of T cells and lower proportions of B cells and a slightly higher CD4+/CD8+ ratio among T cells than the MLN lymphocytes did. T cells that proliferated in response to antigen in vitro were found especially in central CLN 2 days after i.n. immunization and persisted for up to 6 months, whereas after intragastric immunization, responsive T cells were not found in the MLN for up to 14 days. After culture with antigen in vitro, T cells from the superficial CLN of i.n. immunized mice secreted both gamma interferon and interleukin-4. Therefore, after i.n. immunization, superficial and central CLN represent sites of regional lymphocyte development, and the central CLN in particular appear to be sites where memory T cells persist.

Induction of antibody-secreting cells and T-helper and memory cells in murine nasal lymphoid tissue.

Intranasal (i.n.) immunization is an effective route for inducing mucosal immune responses especially in the upper respiratory tract and mouth. To characterize the cells involved in these responses, nasal lymphoid tissue (NALT; considered to be the equivalent of Waldeyer's ring in humans) of normal mice, and of mice immunized intranasally with a bacterial protein antigen conjugated to cholera toxin B subunit, was isolated and the lymphoid cells analysed according to surface phenotype, immunoglobulin and antibody secretion, and cytokine profile. Compared with cells obtained from Peyer's patches (PP), NALT cells contained a higher proportion of T cells, especially naive (CD45RB+hi) T-helper cells, and fewer surface (s)IgA+ cells. Both tissues contained high proportions of sIgM+ IgD+ unswitched B cells. After i.n. immunization, IgA antibody-secreting cells were increased, indicating that isotype switching and differentiation of B cells to IgA-secreting cells occurred in NALT, whereas smaller numbers of antibody-secreting cells were found in PP after intragastric (i.g.) immunization. Antigen-specific memory cells persisted in NALT for at least 8 months after initial immunization. The cytokine expression profiles of antigen-stimulated NALT and PP cells of immunized mice, revealed by reverse transcription polymerase chain reaction analysis of mRNA, were similar. Both NALT and PP cells tended to express type 2 earlier or for longer than type 1 cytokine mRNA, but NALT cells tended to express interleukin-4 (IL-4) earlier, and IL-5 for a longer period, than PP cells. Thus NALT shares with PP cell populations typical of a mucosal inductive site, including unswitched B cells and naive T-helper (Th) cells. After i.n. immunization, NALT has the capacity to provide help for B-cell maturation and differentiation, as well as to maintain immune memory.

Dual function of human IgA antibodies: inhibition of phagocytosis in circulating neutrophils and enhancement of responses in IL-8-stimulated cells.

We have sought to elucidate the responses of human peripheral blood neutrophils to antigenic surfaces complexed with human specific IgA antibodies obtained either as myeloma proteins that recognize staphylococcal alpha-toxin, or from the serum of patients with subacute bacterial endocarditis due to Streptococcus mutans, or from colostrum. In contrast to IgG, IgA antibodies bound to antigen-coated fluorescent microspheres, and subsequently exposed to complement (or not), did not promote phagocytosis, as measured by flow cytometric enumeration of cell-associated microspheres. Instead, IgA antibodies interfered with complement-dependent phagocytosis mediated by IgG antibodies. These properties were shown by different forms of IgA antibodies, including serum and secretory IgA, as well as by monoclonal or polyclonal antibodies. Neutrophils did not respond to the production of superoxide to IgA antibodies complexed with antigen-coated microspheres or with antigen deposited on a solid surface and IgA antibodies suppressed IgG antibody- and complement-mediated superoxide release. However, neutrophils pretreated with interleukin-8 ingested IgA-opsonized microspheres and released superoxide when exposed to IgA antibody-antigen complexes. IgG antibody-antigen complexes did not stimulate increased superoxide release in interleukin-8-treated neutrophils. These findings were consistent with a selective increase in the surface expression of Fc alpha R by interleukin-8-treated neutrophils. We conclude that IgA antibodies interfere with the phagocytic activities of normal circulating human neutrophils and may promote these activities in inflammatory neutrophils activated by interleukin-8 in which Fc alpha R is up-regulated.

The role of the carbohydrate chains in complement (C3) fixation by solid-phase-bound human IgA.

In contrast to antigen-antibody complexes containing native human IgA, solid-phase-deposited IgA activates the alternative complement pathway and binds C3b. To investigate the role of carbohydrate chains in this, various human IgA preparations were treated with neuraminidase alone or together with N-glycanase or O-glycanase, or with mixed glycosidases from the oral bacterium, Streptococcus mitis. Depletion of oligosaccharides was determined by carbohydrate analysis. Removal of sialic acid and N-linked glycan chains greatly increased the C3b-fixing properties of normal serum IgA1 and IgA2. Myeloma IgA1 and IgA2 proteins and secretory IgA had higher C3b-binding activity than normal serum IgA, and this was further increased by removal of sialic acid and N-linked glycans. Fc alpha and Fc alpha-SC fragments of myeloma and secretory IgA1, respectively, but not Fab alpha fragments, obtained by cleavage with bacterial IgA1 proteases and also free secretory component, fixed C3b by the alternative pathway.

All forms of human IgA antibodies bound to antigen interfere with complement (C3) fixation induced by IgG or by antigen alone.

Polyclonal human secretory IgA1 and IgA2 antibodies to a bacterial protein antigen Streptococcus mutans AgI/II, and polyclonal human serum IgA1 and IgA2 antibodies to staphylococcal alpha-toxin, were found to interfere with antigen-mediated C3b fixation. In fluid phase, immune complexes of antigen and IgA failed to fix C3b, whereas antigen-IgG complexes did fix C3b. Partial removal of glycan chains with Streptococcus mitis SK96 glycosidases diminished the capacity of IgA antibodies to interfere with antigen-mediated C3b fixation by the alternative complement pathway. The authors conclude that native serum or secretory IgA antibodies suppress C3b fixation, and that the glycan chains play a significant role in maintaining this property.

Inhibition of Streptococcus mutans adherence to saliva-coated hydroxyapatite by human secretory immunoglobulin A (S-IgA) antibodies to cell surface protein antigen I/II: reversal by IgA1 protease cleavage.

The effect of human secretory immunoglobulin A (S-IgA) and serum antibodies to surface protein antigen (Ag) I/II on the adherence of Ag I/II-bearing Streptococcus mutans and of free Ag I/II to saliva-coated hydroxyapatite (SHA) was investigated. The inhibition by S-IgA of binding of both S. mutans and free Ag I/II to SHA was dependent on antibody to Ag I/II. Essentially no difference was found between S-IgA1 and S-IgA2 with respect to antibody-dependent inhibition of Ag I/II binding to SHA, but S-IgA1 inhibited S. mutans adherence more effectively than did either serum immunoglobulin A1 (IgA1) or IgG antibodies. The antiadherence effect of S-IgA was abrogated after cleavage by IgA1 protease. Purified Fab alpha fragments containing Ag I/II-binding activity enhanced the binding of free Ag I/II to SHA and showed greater binding to SHA than did intact S-IgA1. Despite its relative inability to interact with precoated SHA, S-IgA1 containing antibody to Ag I/II was readily incorporated into the salivary pellicle during coating, but this did not promote Ag I/II binding. These data suggest that S-IgA antibodies can inhibit the initial adherence of S. mutans to salivary pellicle-coated tooth surfaces in an adhesin-specific fashion, but the presence in the oral cavity of bacterial IgA1 proteases would potentially interfere with this antiadherence mechanism.

Kinetics of MHC class II and transferrin receptor expression by colostral cells stimulated in vitro with concanavalin A and myelin basic protein.

The colostral cells, regarded generally as being protective, have been shown to differ in a number of membrane properties (rosetting, adherence, mobility) from the corresponding peripheral blood mononuclear cells. After in vitro stimulation with Con A or MBP 50% to 70% of the human colostral cells appeared HLA-DR positive at the first 24 h of culturing. The CD71 expression reached a maximum on culture days 2-3 coinciding with the maximal proliferative response. With regard to the phenotypic characteristics and their kinetics, the human colostral cells did not show significant differences from the peripheral blood mononuclear cells.

Analysis of suppressor activity in experimental allergic encephalomyelitis inhibited by graft-versus-host reaction.

The induction of local graft-versus-host reaction (GvH) prior to the encephalitogenic challenge resulted in the conversion of acute experimental allergic encephalomyelitis (EAE) to the chronic-like EAE. This inhibitory effect of GvH on EAE development was cyclophosphamide (CY) sensitive. Cell-free supernatants of peripheral blood lymphocytes (PBL) isolated from guinea pigs with chronic-like EAE and during recovery from EAE showed suppressor activity on the in vitro proliferative response of myelin basic protein (MBP) sensitized PBL. The appearance of anaphylactic anti-MBP antibodies and a change in the ratio complement fixing: haemagglutinating (CF/HA) antibodies was also registered.

Interleukin production by human colostral cells after in vitro mitogen stimulation.

Human colostral cells were pulsed with PHA, Con A, or LPS and cultivated in serum-free medium. The culture supernatants were tested for IL-1 activity in C3H/HeJ thymocyte assay and for IL-2 activity on human lymphoblasts. The IL-1 activity was the highest at the 24th h of cultivation and IL-2 activity at the 48th h of cultivation.

[Demonstration of specific VIA antibodies in foot-and-mouth disease]. / Dokazvane na spetsifichni VIA antitela pri shapa.

Experiments were carried out to obtain a specific VIA antigen of F.M.D. During the research, 7 series of VIA antigen were extracted with high purity and specificity. It was established that 90% of the infected animals build specific VIA antibodies against F.M.D.V. Up to 70% of the repeatedly vaccinated cattle against F.M.D. contain VIA antibodies. The percentage of the positive reagent depends on the age of the animals, on the number of the immunizations and on the sort of the used vaccine. The cause for the high percentage of the seropositive to VIA not vaccinated animals is not elucidated.

Examination of two anti-HLA-DR-like monoclonal antibodies.

The cellular distribution of two anti-HLA-DR-like monoclonal antibodies was examined. The reagents showed typical affinity to E- cells, adherent cells and activated T cells. Functionally, they affected the presentation of PPD antigen from APC to T cells in autologous and allogeneic mixed lymphocyte reaction. One of them significantly influenced the proliferation of T helper cells activated polyclonally with PHA.