Investigation of immunomodulatory and anti-inflammatory effects of eriodictyol through its cellular anti-oxidant activity

Many studies have been performed to assess the potential utility of natural products as immunomodulatory agents to enhance host responses against infection or to ameliorate immune-based pathologies. To determine whether eriodictyol has immunomodulatory effects and clarify which types of immune effector cells are stimulated in vitro, we investigated the stimulatory effect of eriodictyol on spleen cells isolated from BALB/c mice. Eriodictyol significantly stimulated splenocyte proliferation. However, only B lymphocytes (not T lymphocytes) could be stimulated by eriodictyol in a dose-related manner. Studies assessing potential effect of eriodictyol on innate immunity reported that eriodictyol enhanced significantly the killing activity of natural killer (NK) cells, T lymphocytes, and macrophages. We also demonstrated that eriodictyol inhibited nitric oxide (NO) production and lysosomal enzyme activity in murine peritoneal macrophages cultured ex-vivo, suggesting a potential anti-inflammatory effect in situ. Eriodictyol revealed also a cellular anti-oxidant activity in splenocytes and macrophages. Furthermore, eriodictyol increased catalase activity in spleen cells. From this data, it can be concluded that eriodictyol exhibited an immunomodulatory effect that could be ascribed in part to a cytoprotective effect related to its anti-oxidant activity.     

The immunomodulatory Pseudomonas aeruginosa signalling molecule N-(3-oxododecanoyl)-L-homoserine lactone enters mammalian cells in an unregulated fashion

The Pseudomonas aeruginosa quorum-sensing signal molecule N-3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been reported to affect the function of a wide range of mammalian cell types, including cells of the immune system. In T cells, it has been reported to inhibit the production of most cytokines, and it has been reported to inhibit the function of antigen-presenting cells. The intracellular target of OdDHL in these cells remains to be identified, although the lipophilic nature of the molecule suggested that the target could be membrane associated. We explored the association of radiolabelled OdDHL with the membrane and cytoplasm of Jurkat T-cell lines and of primary murine T cells and dendritic cells. We found that not only did 3H-OdDHL enter the cytoplasm of Jurkat cells without disproportionate association with the cell membrane, it also reached maximum levels in the cytoplasm very quickly, and that the intracellular concentration was proportional to the extracellular concentration. Similar results were obtained when 3H-OdDHL was incubated with primary murine T cells or cultured dendritic cells. In addition, we show that the cellular distribution of OdDHL does not significantly alter after stimulation of Jurkat cells or primary murine CD4 T cells with immobilized anti-CD3, with little activity being associated with nuclear fractions. Together, these data strongly suggest that OdDHL enters mammalian cells by passive mechanisms, and that it does not preferentially associate with the membrane or nucleus upon T-cell receptor ligation.     

Activation of human leukocytes by lipid A from<Emphasis Type="Italic">E. coli</Emphasis> strains adapted to quaternary ammonium salt and amine oxide

The immunomodulatory activities of monophosphoryl lipid A (MLA) and diphosphoryl lipid A analogues obtained from the sensitive strain of E. coli and from the resistant strains adapted to a quaternary ammonium salt and an amine oxide were compared. All analogues considerably stimulated the activity of human leukocytes although the analogue from the sensitive strain at a higher concentration significantly suppressed phagocytosis. The MLA analogue exhibited a suppressive effect on the microbicidal activity of human leukocytes against E. coli and the peroxidase activity. Adaptation of bacteria to amphiphilic antimicrobial compounds, which is accompanied by chemical changes in their lipid A, only slightly reduced their immunomodulatory activity when compared with the analogue from the sensitive strain. On the other hand, the diphosphoryl analogues were less active than MLA.     

L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase: production of dipeptides containing valine residue at its C-terminus

L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase (ACVS) has been recently studied as a model enzyme for peptide synthetases. It was found that in the absence of alpha-aminoadipic acid but in the presence of several cysteine analogues it was incorporated into several analogue dipeptides upon incubation of the potential cysteine analogues with ACVS. [(14)C]Cysteine was incorporated into the[(14)C]cysteinyl-valine analogue dipeptides. Notably, [(14)C]valine incorporation in the presence of N-acylated cysteine analogues was observed. The alpha-aminoadipic acid activation site is influential, inhibitory or promotive, on the production of these putative dipeptide products. The production of dipeptide analogues, containing valine or analogues at the C-terminus, leads to the speculation that the biosynthetic direction of ACV could be from the C-terminus to the N-terminus.     

Anti‐inflammatory and immune‐modulatory impacts of berberine on activation of autoreactive T cells in autoimmune inflammation

Autoreactive inflammatory CD4⁺ T cells, such as T helper (Th)1 and Th17 subtypes, have been found to associate with the pathogenesis of autoimmune disorders. On the other hand, CD4⁺ Foxp3⁺ T regulatory (Treg) cells are crucial for the immune tolerance and have a critical role in the suppression of the excessive immune and inflammatory response promoted by these Th cells. In contrast, dendritic cells (DCs) and macrophages are immune cells that through their inflammatory functions promote autoreactive T‐cell responses in autoimmune conditions. In recent years, there has been increasing attention to exploring effective immunomodulatory or anti‐inflammatory agents from the herbal collection of traditional medicine. Berberine, an isoquinoline alkaloid, is one of the main active ingredients extracted from medicinal herbs and has been shown to exert various biological and pharmacological effects that are suggested to be mainly attributed to its anti‐inflammatory and immunomodulatory properties. Several lines of experimental study have recently investigated the therapeutic potential of berberine for treating autoimmune conditions in animal models of human autoimmune diseases. Here, we aimed to seek mechanisms underlying immunomodulatory and anti‐inflammatory effects of berberine on autoreactive inflammatory responses in autoimmune conditions. Reported data reveal that berberine can directly suppress functions and differentiation of pro‐inflammatory Th1 and Th17 cells, and indirectly decrease Th cell‐mediated inflammation through modulating or suppressing other cells assisting autoreactive inflammation, such as Tregs, DCs and macrophages.     

Vitamin E analogues as inducers of apoptosis: implications for their potential antineoplastic role

Recent evidence suggests that vitamin E and its analogues, which have been used for many years as antioxidants, may not only protect cells from free radical damage but also induce apoptotic cell death in various cell types. While alpha-tocopherol (alpha-TOH) is mainly known as an anti-apoptotic agent, its redox-silent analogues either have no influence on cell survival (alpha-tocopheryl acetate, alpha-TOA), or induce apoptosis (alpha-tocopheryl succinate, alpha-TOS). Although precise mechanisms of apoptosis induction by alpha-TOS remain to be elucidated, there is evidence that this process involves both the antiproliferative and membrane destabilising activities of the agent. Alpha-TOS has been shown to induce apoptosis in malignant cell lines but not, in general, in normal cells, and to inhibit tumorigenesis in vivo. These features suggest that this semi-synthetic analogue of vitamin E could be a promising antineoplastic agent.     

Pheophorbide a: a photosensitizer with immunostimulating activities on mouse macrophage RAW 264.7 cells in the absence of irradiation

Pheophorbide a (Pa) has been proposed to be a potential photosensitizer for the photodynamic therapy of human cancer. However, the immunomodulatory effect of Pa, in the absence of irradiation, has not yet been investigated. The present study revealed that Pa possessed immunostimulating effect on a murine macrophages cell line RAW 264.7. Pa could significantly stimulate the growth of RAW 264.7 cells with the maximum effect at 1.0 μM after 24, 48 and 72 h of treatment (all p < 0.05). Besides, intracellular mitogen activated protein kinases (MAPK) including extracellular signal-regulated kinase (ERK) and p38 MAPK were activated by Pa treatment in a dose-dependent manner. The activation of ERK and p38 MAPK was found to be related to the Pa-induced intracellular reactive oxygen species. Furthermore, Pa could significantly induce the release of interleukine-6 and tumour necrosis factor-α, and enhance the phagocytic activity of RAW 264.7 cells (all p < 0.05). The present work is the first report to demonstrate the potential immunomodulatory effect of Pa on macrophages, apart from its well-studied anti-tumour activity.     

New substituted C-19-andrographolide analogues with potent cytotoxic activities

Andrographolide, the major diterpenoid lactone from Andrographis paniculata, is toxic against cancer cells. In the present study, we investigated the structure-activity relationships (SARs) of 19 andrographolide analogues which were synthesized by modification at the three hydroxyl groups. A number of the andrographolide analogues showed much higher cytotoxic activities than that of the parent compound on cancer cells including P-388, KB, COL-2, MCF-7, LU-1 and ASK cells. SAR studies of the synthetic analogues indicated that the introduction of silyl ether or triphenylmethyl ether group into C-19 of the parent compound led to increase in toxicity against the cancer cells. The 19-O-triphenylmethyl ether analogue 18 showed higher cytotoxic activity than the potent anticancer drug ellipticine, and this analogue may serve as a potential structure lead for the development of new anticancer drugs.     

Synthesis of cryptolepine analogues as potential bioreducible anticancer agents

A series of 10 novel nitro-analogues of cryptolepine (1) has been synthesised and these compounds were evaluated for their in-vitro cytotoxic properties as well as their potential for reductive activation by the cytosolic reductase enzymes NQO1 and NQO2. Molecular modelling studies suggest that cryptolepine is able to fit into the active site of NQO2 and thus raising the possibility that nitro-analogues of 1 could act as bioreductive prodrugs and be selectively reduced by NQO1 and NQO2 to more toxic species in cancer cells in which these enzymes are over-expressed. Analogues were screened against the RT112 cell line (high in NQO2), in the presence and absence of the essential cofactor dihydronicotinamide riboside (NRH), whereby all analogues were shown to be cytotoxic (IC50<2microM) in the absence of NRH. With the addition of NRH, one analogue, 2-fluoro-7,9-dinitrocryptolepine (7), exhibited a 2.4-fold increase in cytotoxic activity. Several nitro-derivatives were also evaluated as substrates for purified human NQO1 and analogues that were found to be substrates were subsequently tested against the H460 (high NQO1) and BE (low NQO1) cell lines to detect in-vitro activation by NQO1. The analogue 8-chloro-9-nitrocryptolepine (9) was found to be the best substrate for NQO1 but it was not more toxic to H460 than to BE cells. Fluorescence laser confocal microscopy of 1 and several analogues showed that in contrast to 1 the analogues were not localised into the nucleus suggesting that their cytotoxic mode(s) of action are different. This study has identified novel substrates for both NQO1 and NQO2 and further work on nitrocryptolepine derivatives as a lead towards novel anticancer agents would be worthwhile.     

Vitamin E analogues as inducers of apoptosis: implications for their potential antineoplastic role

Abstract

Recent evidence suggests that vitamin E and its analogues, which have been used for many years as antioxidants, may not only protect cells from free radical damage but also induce apoptotic cell death in various cell types. While -tocopherol (-TOH) is mainly known as an anti-apoptotic agent, its redox-silent analogues either have no influence on cell survival (-tocopheryl acetate, -TOA), or induce apoptosis (-tocopheryl succinate, -TOS). Although precise mechanisms of apoptosis induction by -TOS remain to be elucidated, there is evidence that this process involves both the antiproliferative and membrane destabilising activities of the agent. -TOS has been shown to induce apoptosis in malignant cell lines but not, in general, in normal cells, and to inhibit tumorigenesis in vivo. These features suggest that this semi-synthetic analogue of vitamin E could be a promising antineoplastic agent.     

Virulence of Mycobacterium tuberculosis

Abstract Different strains of Candida albicans show varied sensitivities to the peptide analogues bacilysin, polyoxins and nikkomycins. From a sensitive strain, B2630, spontaneous mutants were selected for resistance to each analogue; certain mutants showed cross-resistance to other analogues and associated defects in peptide transport. A bacilysin-resistant mutant was cross resistant to the other analogues and to m -fluorophenylalanyl-Ala (FPA) but retained sensitivity to m -fluorophenylalanyl-Ala—Ala (FPAA). It showed defective dipeptide transport but normal oligopeptide transport. A revertant, selected for its ability to utilize Ala-Ala as sole nitrogen source, regained wild-type dipeptide transport activity and analogue sensitivity. Thus, C. albicans has distinguishable mechanisms for dipeptide and oligopeptide transport which can be exploited for uptake of peptide-drug adducts.     

Differential Top10 promoter regulation by six tetracycline analogues in plant cells

The effects of five tetracycline analogues, anhydrotetracycline, doxycycline, minocycline, oxytetracycline, and tetracycline, on Top10 promoter activity in NT1 tobacco tissue culture cells have been analysed. The concentration that repressed Top10 promoter activity, the level of transgene repression and the kinetics of transgene de-repression were determined for each analogue, and could not be predicted from in vitro binding affinity to the tetracycline repressor or from comparison with animal cells. Doxycycline had the most potent effect on the Top10 promoter and completely inhibited transgene expression at 4 nmol l(-1). Tetracycline was the most versatile of the analogues tested; tetracycline inhibited the Top10 promoter at 10 nmol l(-1) and was easily washed out to restore Top10-driven expression in 12-24 h. A study was also made of the suitability for plant research of a novel tetracycline analogue, GR33076X. In animal cells, GR33076X de-repressed Top10 promoter activity in the presence of inhibitory concentrations of anhydrotetracycline. In NT1, it is shown that GR 33076X can antagonize repression of the Top10 promoter in the presence of tetracycline, but not of anhydrotetracycline or of doxycycline. Different tetracycline analogues can therefore be used to regulate the Top10 promoter in plant cells and this property may be exploited in planning an optimum course of transgene regulation.     

Diversity of interferon gamma and granulocyte-macrophage colony-stimulating factor in restoring immune dysfunction of dendritic cells and macrophages during polymicrobial sepsis

The development of immunosuppression during polymicrobial sepsis is associated with the failure of dendritic cells (DC) to promote the polarization of T helper (Th) cells toward a protective Th1 type. The aim of the study was to test potential immunomodulatory approaches to restore the capacity of splenic DC to secrete interleukin (IL) 12 that represents the key cytokine in Th1 cell polarization. Murine polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Splenic DC were isolated at different time points after CLP or sham operation, and stimulated with bacterial components in the presence or absence of neutralizing anti-IL-10 antibodies, murine interferon (IFN) gamma, and/or granulocyte macrophage colony-stimulating factor (GM-CSF). DC from septic mice showed an impaired capacity to release the pro-inflammatory and Th1-promoting cytokines tumor necrosis factor alpha, IFN-gamma, and IL-12 in response to bacterial stimuli, but secreted IL-10. Endogenous IL-10 was not responsible for the impaired IL-12 secretion. Up to 6 h after CLP, the combined treatment of DC from septic mice with IFN-gamma and GM-CSF increased the secretion of IL-12. Later, DC from septic mice responded to IFN-gamma and GM-CSF with increased expression of the co-stimulatory molecule CD86, while IL-12 secretion was no more enhanced. In contrast, splenic macrophages from septic mice during late sepsis responded to GM-CSF with increased cytokine release. Thus, therapy of sepsis with IFN-gamma/GM-CSF might be sufficient to restore the activity of macrophages, but fails to restore DC function adequate for the development of a protective Th1-like immune response.     

Treatment with the Antipsychotic Agent,Risperidone, Reduces Disease Severity in Experimental Autoimmune Encephalomyelitis

Recent studies have demonstrated that atypical antipsychotic agents, which are known to antagonize dopamine D2 and serotonin 5-HT_2a receptors, have immunomodulatory properties. Given the potential of these drugs to modulate the immune system both peripherally and within the central nervous system, we investigated the ability of the atypical anti-psychotic agent, risperidone, to modify disease in the animal model of multiple sclerosis (MS)⁴, experimental autoimune encephalomyelitis (EAE). We found that chronic oral administration of risperidone dose-dependently reduced the severity of disease and decreased both the size and number of spinal cord lesions. Furthermore, risperidone treatment substantially reduced antigen-specific interleukin (IL)-17a, IL-2, and IL-4 but not interferon (IFN)-γ production by splenocytes at peak disease and using an in vitro model, we show that treatment of macrophages with risperidone alters their ability to bias naïve T cells. Another atypical antipsychotic agent, clozapine, showed a similar ability to modify macrophages in vitro and to reduce disease in the EAE model but this effect was not due to antagonism of the type 1 or type 2 dopamine receptors alone. Finally, we found that while risperidone treatment had little effect on the in vivo activation of splenic macrophages during EAE, it significantly reduced the activation of microglia and macrophages in the central nervous system. Together these studies indicate that atypical antipsychotic agents like risperidone are effective immunomodulatory agents with the potential to treat immune-mediated diseases such as MS.     

bis(benzyl)polyamine analogues are substrates for a mammalian cell-transport system which is distinct from the polyamine-transport system.

Bis(benzyl)polyamine analogues (e.g. NN'-bis(3-[(phenylmethyl)amino]propyl)-1,8-diamino-octane [C6H5CH2NH-(CH2)3NH(CH2)8NH(CH2)3NHCH2C6H5]) have previously been shown to regulate polyamine biosynthesis and growth of rat hepatoma (HTC) cells. Saturable uptake of the analogues, the ability of other bis(benzyl)polyamine analogues to compete for this uptake and the trans-acceleration of this uptake in pre-loaded cells indicate that these novel compounds are accumulated through the action of a transport system in HTC cells. A mutant Chinese-hamster-ovary (CHO) cell line, CHOMG, which lacks a functional polyamine-transport system, exhibited saturable bis(benzyl)polyamine uptake identical with that observed in the parental CHO cells, which have normal polyamine transport. The uptake of the analogue by both CHOMG and CHO cells was competitively inhibited by other bis(benzyl)polyamine analogues, but was insensitive to excess spermine. Treatment with alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis, resulted in the enhancement of spermine uptake in CHO cells but did not alter the uptake of a bis(benzyl)polyamine analogue by either CHO or CHOMG cells. Thus it appears that bis(benzyl)polyamine analogues are substrates for a mammalian-cell-transport system distinct from the polyamine-transport system.     

Protective effects of green tea catechins on alveolar macrophages against bacterial infections

Bacterial pneumonia in immunocompromised patients as well as elderly persons often becomes a life threatening disease, even when effective antibiotics are used extensively. In addition, the appearance of antibiotic-resistant bacteria in medical facilities as well as in patients requires another approach to treat such patients besides treatment with antibiotics. In this regard, green tea catechins, such as epigallocatechin gallate (EGCg), may be one of the potential agents for such purpose due to its possible potential immunomodulatory as well as antimicrobial activity. The studies by us showed that EGCg enhanced the in vitro resistance of alveolar macrophages to Legionella pneumophila infection by selective immunomodulatory effects on cytokine formation. Furthermore, the tobacco smoking-induced impairment of alveolar macrophages regarding antibacterial as well as immune activity was also recovered by EGCg treatment. These results indicate that EGCg may be a possible potential immunotherapeutic agent against respiratory infections in immunocompromised patients, such as heavy smokers.     

Bordetella pertussis adenylate cyclase toxin modulates innate and adaptive immune responses: distinct roles for acylation and enzymatic activity in immunomodulation and cell death

Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the repeat in toxin family of pore-forming toxins, which require posttranslational acylation to lyse eukaryotic cells. CyaA modulates dendritic cell (DC) and macrophage function upon stimulation with LPS. In this study, we examined the roles of acylation and enzymatic activity in the immunomodulatory and lytic effects of CyaA. The adenylate cyclase activity of CyaA was necessary for its modulatory effects on murine innate immune cells. In contrast, acylation was not essential for the immunomodulatory function of CyaA, but was required for maximal caspase-3 activation and cytotoxic activity. The wild-type acylated toxin (A-CyaA) and nonacylated CyaA (NA-CyaA), but not CyaA with an inactive adenylate cyclase domain (iAC-CyaA), enhanced TLR-ligand-induced IL-10 and inhibited IL-12, TNF-alpha, and CCL3 production by macrophages and DC. In addition, both A-CyaA and NA-CyaA, but not iAC-CyaA, enhanced surface expression of CD80 and decreased CpG-stimulated CD40 and ICAM-1 expression on immature DC. Furthermore, both A-CyaA and NA-CyaA promoted the induction of murine IgG1 Abs, Th2, and regulatory T cells against coadministered Ags in vivo, whereas iAC-CyaA had more limited adjuvant activity. In contrast, A-CyaA and iAC-CyaA induced caspase-3 activation and cell death in macrophages, but these effects were considerably reduced or absent with NA-CyaA. Our findings demonstrate that the enzymatic activity plays a critical role in the immunomodulatory effects of CyaA, whereas acylation facilitates the induction of apoptosis and cell lysis, and as such, NA-CyaA has considerable potential as a nontoxic therapeutic molecule with potent anti-inflammatory properties.     

Coupling of isoprenoid triflates with organoboron nucleophiles: synthesis and biological evaluation of geranylgeranyl diphosphate analogues

The Suzuki coupling reaction has been used to introduce a methyl group derived from commercially available methylboronic acid into a vinyl triflate. This has led to a concise synthesis of all-trans-geranylgeraniol, with the key step being the palladium-catalyzed, silver-mediated methylation of triflate to give ethyl geranylgeranoate. This coupling protocol has also been used to produce the novel geranylgeranyl diphosphate (GGPP) analogue 3-phenyl-3-desmethylgeranylgeranyl diphosphate (3-PhGGPP, ). Our previously developed organocuprate coupling protocol has been used to introduce the cyclopropyl and tert-butyl moieties into the 3-position of vinyl triflate. The four GGPP analogues 3-vinyl-3-desmethylgeranylgeranyl diphosphate (3-vGGPP, ), 3-cyclopropyl-3-desmethylgeranylgeranyl diphosphate (3-cpGGPP, ), 3-tert-butyl-3-desmethyl-geranylgeranyl diphosphate (3-tbGGPP, ), and were then evaluated as potential inhibitors of recombinant yeast protein-geranylgeranyl transferase I (PGGTase I). The potential mechanism-based inhibitors 3-vGGPP and 3-cpGGPP did not exhibit time-dependent inactivation of PGGTase I. Instead, both analogues were alternative substrates, in accord with the interaction of the corresponding farnesyl analogues 3-vFPP and 3-cpFPP with PFTase. The tert-butyl and phenyl analogues were not substrates, but were instead competitive inhibitors of PGGTase I. Note that all four of the GGPP analogues were bound less tightly by the enzyme than the natural substrate, in contrast to the behavior of the 3-substituted FPP analogues.     

Experience with insulin analogues in children

Current data on rapid and long-acting insulin analogues in the paediatric age group is limited. While several studies indicate a benefit in reducing hypoglycaemia, particularly at night, with rapid or long-acting insulin analogue treatment, the effect on long-term glycaemic control remains controversial. The continuous glucose monitoring system offers a new option for tailoring treatment with insulin analogues to achieve optimal glycaemia. In 29 adolescents with diabetes this approach confirmed the non-inferiority of postprandial rapid-acting analogue administration compared to preprandial regular insulin, but revealed significant mealtime differences, with increased analogue requirement at breakfast and dinner. Although rapid- and long-acting insulin analogues may offer potential benefits for problems frequently encountered in paediatric diabetology, their value for the individual child still has to be tested in long-term observations in daily clinical practice.