Taraxacum mongolicum extract inhibited malignant phenotype of triple-negative breast cancer cells in tumor-associated macrophages microenvironment through suppressing IL-10 / STAT3 / PD-L1 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Triple-negative breast cancer (TNBC) is the most aggressive and the worst prognosis breast cancer with limited treatment options. Taraxacum mongolicum (also called dandelion) is a traditional Chinese medicine has been used to treat mastitis, breast abscess, and hyperplasia of mammary glands since ancient times. In modern pharmacological research, dandelion has been proven with anti-breast cancer activities. We previously reported that dandelion extract could induce apoptosis in TNBC cells. However, its anti-tumor effects and mechanisms in the tumor microenvironment have not yet been elucidated. AIM OF THE STUDY: Tumor-associated macrophages (TAMs) play an important role in regulating the interaction between tumor cells and the immune system. The present study aimed to investigate the effects and mechanisms of dandelion extract on TNBC cells under the microenvironment of TAMs, as well as its influence on the polarization of M2 macrophages. MATERIALS AND METHODS: M2 macrophages were induced by phorbol-12-myristate 13-acetate (PMA) and interleukin 4 (IL-4), and verified by flow cytometry, quantitative RT-PCR (qRT-PCR), Western blotting, and ELISA. MDA-MB-231 and MDA-MB-468 TNBC cells were co-cultured with the supernatant of M2 macrophage which providing the TAMs microenvironment. The antitumor activity of dandelion extract in TNBC cells was evaluated by MTT assay. The invasive and migratory capacity of TNBC cells was measured by transwell assays. The expression of protein and gene was assessed by Western blotting and qRT-PCR, respectively. RESULTS: TAMs microenvironment promoted the proliferation, migration, and invasion of TNBC cells. However, dandelion extract inhibited the malignant property of MDA-MB-231 and MDA-MB-468 cells induced by TAMs. Both of TAMs and IL-10 caused STAT3 activation and PD-L1 higher expression, the immunosuppressive molecules in TNBC cells, and this effect can be attenuated by IL-10 neutralizing antibody. Dandelion extract exerted inhibition on STAT3 and PD-L1 in TNBC cells under TAMs microenvironment. Furthermore, in M2 macrophages, dandelion extract remarkably promoted the expression of M1-like marker TNF-α, IL-8, and iNOS, but reduced M2-like marker IL-10, CD206, Arginase-1, and TGF-ß. CONCLUSION: Dandelion extract inhibited the proliferation, migration and invasion of TNBC cells in TAMs microenvironment through suppressing IL-10/STAT3/PD-L1 immunosuppressive signaling pathway. Furthermore, dandelion extract promoted the polarization of macrophages from M2 to M1 phenotype. Thus, our results indicated that dandelion may serve as a promising therapeutic strategy for TNBC by modulating tumor immune microenvironment.

Evaluating the effects of vanadyl sulfate on biomarkers of oxidative stress and inflammation in renal tissue of rats with diabetes type 2

Vanadyl sulfate (VS) is an ingredient in some food supplements and experimental drugs. This study was designed to assay the effects of VS on biomarkers of oxidative stress and inflammation in renal tissue of rats with diabetes type 2. 30 male Wistar rats were divided into three equal groups as follow: non-diabetics, non-treated diabetics and VS-treated diabetics. Diabetes type 2 has been induced through high fat diet and fructose in the animals. Diabetic rats were treated with 25 mg/kgBW of VS in water for 12 weeks. At the end of study, glucose and insulin were measured using commercially available kits in serum and biomarkers of oxidative stress and inflammation in renal homogenates of animals were measured by related methods. Compared to controls, glucose and insulin were increased significantly in non-treated diabetic rats (p-value <0.05) that showed the induction of diabetes type 2 in rats. The results showed that in VS-treated diabetic rats compared to the non-treated diabetic group, vanadyl sulfate significantly reduced the glucose and insulin secretion and changed renal inflammatory and oxidative markers, except protein carbonyl so that we couldn't find any significant changes. Our study showed that vanadyl supplementation had positive effects on oxidative stress and inflammation biomarkers in kidney of diabetic rats

Synthesized natural peptides from amphibian skin secretions increase the efficacy of a therapeutic vaccine by recruiting more T cells to the tumour site.

BACKGROUND: Therapeutic vaccines against cervical cancer remain ineffective. Previously, we demonstrated that blocking the signalling of a cytokine, interleukin 10, at the time of immunisation elicited significantly higher numbers of antigen specific T cells and inhibited tumour growth in mice. RESULTS: In the current paper, we demonstrate, in a HPV16 E6/E7 transformed TC-1 tumour mouse model, that despite increased antigen specific T cell numbers, blocking IL-10 signalling at the time of immunisation does not increase the survival time of the TC-1 tumour bearing mice compared to mice receiving the same immunisation with no IL-10 signalling blockade. Moreover, the function of tumour infiltrating T cells isolated 3 weeks post TC-1 transplantation is more suppressed than those isolated 2 weeks after tumour inoculation. We demonstrate that synthesized caerin peptides, derived from amphibian skin secretions, 1) were able to inhibit TC-1 tumour growth both in vitro and in vivo; 2) are environmentally stable; and 3) promote the secretion of pro-inflammatory interlukine-6 by TC-1 cells. Notably caerin peptides were able to increase the survival time of TC-1 tumour bearing mice after therapeutic vaccination with a HPV16E7 peptide-based vaccine containing IL-10 inhibitor, via recruiting increased levels of T cells to the tumour site. CONCLUSION: Caerin peptides increase the efficacy of a therapeutic vaccine by recruiting more T cells to the tumour site.

Real-time monitoring of IL-6 and IL-10 reporter expression for anti-inflammation activity in live RAW 264.7 cells.

In previous study, we suggested that the interleukin (IL)-6 and IL-10 could serve as a good biomarker for anti-inflammation that related to chronic inflammatory disease. Recently, we are finding new anti-inflammation compounds from natural products by screening of IL-6 and IL-10 levels. Although, we could measure IL-6 and IL-10 levels by several methods. However, all methods could not measure continuous kinetic of IL-6 and IL-10 levels. Most methods have multiple steps and take a long time. Therefore, there is no a suitable method for screening. To this end, we established IL-6 and IL-10 promoter assay which can monitor with reference gene as Glyceraldehyde 3-phosphate dehydrogenase (gapdh) promoter in living single cell. It could determine IL-6 and IL-10 levels continuously in real-time within two steps. We evaluated IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells with well-known anti-inflammatory compounds such as quercetin, xanthones, ß-D-glucan and dexamethasone. As the results, the expression of IL-6 and IL-10 reporters were strongly induced by LPS. The expression of IL-6 reporter was inhibited by all anti-inflammation compounds in LPS-induced RAW 264.7 cells. The expression of IL-10 reporter was inhibited by quercetin, xanthones and dexamethasone in LPS-induced RAW 264.7 cells. While, expression of IL-10 reporter was induced by ß-D-glucan. These results indicated that this assay could use for determination of IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells for anti-inflammation activity. Moreover, the results showed that natural compounds have an effect on the time course of IL-6 and IL-10 expressions. Therefore, real-time monitoring has a merit for natural compounds screening. We suggested that this assay could serve as a compound screening assay for anti-inflammation activity.

Vitamin A Oral Supplementation Induces Oxidative Stress and Suppresses IL-10 and HSP70 in Skeletal Muscle of Trained Rats.

Exercise training intensity is the major variant that influences the relationship between exercise, redox balance, and immune response. Supplement intake is a common practice for oxidative stress prevention; the effects of vitamin A (VA) on exercise training are not yet described, even though this molecule exhibits antioxidant properties. We investigated the role of VA supplementation on redox and immune responses of adult Wistar rats subjected to swimming training. Animals were divided into four groups: sedentary, sedentary + VA, exercise training, and exercise training + VA. Over eight weeks, animals were submitted to intense swimming 5 times/week and a VA daily intake of 450 retinol equivalents/day. VA impaired the total serum antioxidant capacity acquired by exercise, with no change in interleukin-1ß and tumor necrosis factor-α levels. In skeletal muscle, VA caused lipid peroxidation and protein damage without differences in antioxidant enzyme activities; however, Western blot analysis showed that expression of superoxide dismutase-1 was downregulated, and upregulation of superoxide dismutase-2 induced by exercise was blunted by VA. Furthermore, VA supplementation decreased anti-inflammatory interleukin-10 and heat shock protein 70 expression, important factors for positive exercise adaptations and tissue damage prevention. Our data showed that VA supplementation did not confer any antioxidative and/or protective effects, attenuating exercise-acquired benefits in the skeletal muscle.

The content of docosahexaenoic acid in the suckling and the weaning diet beneficially modulates the ability of immune cells to response to stimuli.

The objective of the study was to isolate the effect of feeding a diet supplemented with docosahexaenoic acid (DHA) during the suckling and/or the weaning period on immune system development and function in offspring. Dams were randomized to one of two nutritionally adequate diets: control diet (N=12, 0% DHA) or DHA diet (N=8, 0.9% DHA). Diets were fed to dams throughout lactation, and then at weaning (21d), two pups per dam were randomly assigned to continue on the same diet as the dam or consume the other experimental diet for an additional 21d. At 6 weeks, splenocyte phenotypes and ex vivo cytokine production after stimulation with concanavalin A (ConA), lipopolysaccharide (LPS) or ovalbumin were assessed. Pups who received the control diet during both periods had the lowest production of IL-2 after ConA (P<.05 for interaction). Pups fed DHA during suckling had higher IL-10 production after all mitogens, regardless of the weaning diet (P<.05). Feeding DHA at weaning, regardless of the suckling diet, resulted in a lower production of IL-1ß and TNF-α in LPS-stimulated splenocytes and a higher proportion of total CD27+ cells (all P<.03). Our findings suggest that providing no DHA during critical periods of immune development resulted in a less efficient Th1 response upon challenge (IL-2 production). Feeding DHA during suckling had a programming effect on the ability of splenocytes to produce the regulatory cytokine IL-10. Feeding a DHA diet during weaning led to a lower TNF-α and IL-1ß response to a bacterial antigen.

Antileishmanial and immunomodulatory effects of the essential oil from Tetradenia riparia (Hochstetter) Codd.

Cutaneous leishmaniasis usually presents therapeutic resistance to antimonials, and the existing therapies for leishmaniasis have many adverse effects and toxicity. Natural products may be regarded as possible candidates for alternative leishmaniasis treatment. The plant Tetradenia riparia has shown promise for the treatment of infectious diseases in folk medicine. We evaluated the antileishmanial activity of an essential oil from T. riparia (TrEO) and the modulatory effects of TrEO on cytokine modulation by peritoneal fluid cells that were infected with L. (L.) amazonensis. Peritoneal fluid cells were infected with Leishmania and incubated with TrEO (30 ng/mL) for 3, 6, and 24 h. Cytokines were screened using semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and flow cytometry. Antileishmanial activity was evaluated at 24 h by microscopic counting and quantitative PCR (qPCR). TrEO treatment induced the death of 50% of Leishmania amastigotes (indicated by microscopic counting) and 91% of the parasite load (indicated by qPCR). TrEO inhibited some of the most critical cytokines for parasite growth and the establishment of infection, including granulocyte-macrophage colony-stimulating factor, interleukin-4 (IL-4), IL-10, and tumour necrosis factor. The parasite inhibited interferon-γ and IL-12, and TrEO blocked this inhibition, indicating that these cytokines are critical for activating mechanisms associated with the death and elimination of the parasite. These results suggest that TrEO may be an alternative leishmaniasis therapy when considering its antileishmanial and immunomodulatory activity.

Antiviral and Immunomodulatory Effects of Norantea brasiliensis Choisy on Dengue Virus-2.

BACKGROUND/AIMS: Severe dengue fever is a result of exacerbated immune responses and no specific treatments are available. We evaluated the antiviral and immunomodulatory effects of Norantea brasiliensis Choisy. METHODS: Human adherent monocytes infected in vitro with dengue virus (DENV)-2 were incubated with the crude ethanol extract from leaves (NB1) or 3 derived fractions: dichloromethane (NB3), ethyl acetate (NB5), and butanolic (NB6) partitions. The antiviral and immunomodulatory activities were determined by intracellular detection of DENV antigen within monocytes and by secreted NS1 viral protein and cytokines. RESULTS: The crude extract alone exhibited both antiviral activities (intracellular and secreted antigens) and all fractions derived from this extract modulated NS1 production. Regarding the immunomodulatory effect, among the secreted factors, TNF-α was inhibited by NB3 and NB6; IL-6 was inhibited by NB1, NB3, and NB6; IL-10 by NB1 and NB3; and IFN-α by NB6. The crude extract (NB1) presented the best antiviral effect, whereas the dichloromethane fraction (NB3) presented an immunomodulatory effect in the inflammatory and anti-inflammatory cytokines. CONCLUSION: During in vitro DENV infection, N. brasiliensis Choisy exerts both antiviral and immunomodulatory effects that are likely associated, considering that less viral load may lead to less immunostimulation.

Anti-Inflammatory Constituents from Bidens frondosa.

A new polyacetylene glucoside (3E,5E,11E)-tridecatriene-7,9-diyne-1,2,13-triol-2-O-ß-D-glucopyranoside (1), a new phenylpropanoid glucoside 2'-butoxyethylconiferin (2), and a new flavonoid glycoside 8,3',4'-trihydroxyflavone-7-O-(6''-O-p-coumaroyl)-ß-D-glucopyranoside (3), have been isolated from Bidens frondosa together with fifty-three known compounds 4-56. The structures of these compounds were established by spectroscopic methods. mainly ESIMS, 1D- and 2D-NMR spectroscopic data. and comparison with literature data. Compounds 1-34, 36, 39, 43, 47, 51, and 52 were tested for inhibition of nuclear factor kappa B (NF-κB) in 293-NF-κB-luciferase report cell line induced by lipopolysaccharide (LPS), and compounds 1, 2, 3, 9, 15, 21, 24 and 51 were tested for the production of TNF-α, IL-1ß, IL-6, IL-10 in RAW 264.7 macrophages induced by LPS. In conclusion, the isolated compounds 1, 2, 3, 9, 15, 21, 24 and 51 exhibited significant activity in anti-inflammatory activity assays.

Anti-inflammatory effects of naringin in chronic pulmonary neutrophilic inflammation in cigarette smoke-exposed rats.

Naringin, a well-known flavanone glycoside of grapefruit and citrus fruits, was found to be as an effective anti-inflammatory compound in our previous lipopolysaccharide-induced acute lung injury mouse model via blockading activity of nuclear factor κB. The current study sought to explore the anti-inflammatory effects of naringin on chronic pulmonary neutrophilic inflammation in cigarette smoke (CS)-induced rats. Seventy Sprague-Dawley rats were randomly divided into seven groups to study the effects of CS with or without various concentrations of naringin or saline for 8 weeks. The results revealed that naringin supplementation at 20, 40, and 80 mg/kg significantly increased body weight of CS-induced rats as compared to that in the CS group. Moreover, naringin of 20, 40, and 80 mg/kg prevented CS-induced infiltration of neutrophils and activation of myeloperoxidase and matrix metalloproteinase-9, in parallel with suppression of the release of cytokines, such as tumor necrosis factor-α and interleukin-8 (IL-8). IL-10 in bronchoalveolar lavage fluid was significantly suppressed after CS exposure, but dose dependently elevated by naringin. The results from hematoxylin and eosin staining revealed that naringin dose dependently reduced CS-induced infiltration of inflammatory cells, thickening of the bronchial wall, and expansion of average alveolar airspace. In conclusion, our data suggest that naringin is an effective anti-inflammatory compound for attenuating chronic pulmonary neutrophilic inflammation in CS-induced rats.

Scopoletin attenuates allergy by inhibiting Th2 cytokines production in EL-4 T cells.

Scopoletin is an antioxidant found in certain weedy plants. It exerts anti-inflammatory, anti-allergic, and anti-diabetic activities. It remains unknown whether scopoletin regulates T helper (Th) cells, including Th 1 and Th 2 cells. We found that scopoletin significantly inhibited phorbol myristate acetate (PMA)/ionomycin-induced interleukin-4 (IL-4), IL-5, and IL-10 production in EL-4 T cells. In addition, scopoletin significantly enhanced the inhibitory action of PMA/ionomycin on interferon-γ (IFN-γ) expression. In EL-4 T cells, PMA/ionomycin treatment markedly increased the expression of nuclear factor of activated T cells (NFAT) and GATA-3; in contrast, scopoletin significantly down-regulated expressions of these transcription factors. Furthermore, this downregulation depended on protein kinase C (PKC) attenuation. This leads us to suggest that the anti-allergic properties of scopoletin might be mediated by the downregulation of cytokine expression in Th 2 cells.

Scutellaria extract and wogonin inhibit tumor-mediated induction of T(reg) cells via inhibition of TGF-ß1 activity.

A number of studies have implicated tumor-induced T(reg) cell activity in the sub-optimal response to therapeutic vaccines. Development of neo-adjuvant strategies targeting T(reg) cells is therefore imperative. Scutellaria extracts or constituent flavonoids have shown encouraging efficacy against various tumors, including gliomas, in both pre-clinical and clinical studies. We report here, for the first time, that Scutellaria ocmulgee leaf extract (SocL) and flavonoid wogonin could inhibit TGF-ß1-induced T(reg) activity in malignant gliomas. F344 rats, subcutaneously transplanted with F98 gliomas, were treated with SocL. There was a significant inhibition of intra-tumoral TGF-ß1 and T(reg) cell frequency as well as peripheral blood TGF-ß1 levels in SocL-treated animals compared to the controls. SocL extract and wogonin also inhibited glioma-induced, TGF-ß1-mediated T(reg) activity in vitro. SocL extract and wogonin also inhibited the secretion of IL-10 in T(reg) culture; whereas the level of IL-2 was either unchanged or marginally enhanced. We also observed an inhibition of Smad-3, GSK-3ß and ERK1/2 signaling by SocL and wogonin in T(reg) cells, while phosphorylation of P38 MAPK was considerably enhanced, indicating that SocL or wogonin could inhibit the T cells' response to TGF-ß1 via modulation of both Smad and non-Smad signaling pathways. Overall, this study suggests that Scutellaria can potentially reverse tumor-mediated immune suppression via inhibition of TGF-ß1 secretion as well as via inhibition of T cells' response to TGF-ß1. This may provide an opportunity for developing a novel adjuvant therapeutic strategy for malignant gliomas, combining Scutellaria with immunotherapy and chemo/radio-therapeutic regimen, which could potentially improve the disease outcome.

Leishmanicidal effect of Spiranthera odoratíssima (Rutaceae) and its isolated alkaloid skimmianine occurs by a nitric oxide dependent mechanism.

Leishmaniasis is one of the neglected diseases. High cost, systemic toxicity, and diminished efficacy due to development of resistance by the parasites has a negative impact on the current treatment options. Thus, the search for a new, effective and safer anti-leishmanial drug becomes of paramount importance. Compounds derived from natural products may be a better and cheaper source in this regard. This study evaluated the in vitro anti-leishmanial activity of Spiranthera odoratíssima (Rutaceae) fractions and isolated compounds, using promastigote and amastigote forms of different Leishmania species. J774 A.1 macrophage was used as the parasite host cell for the in vitro assays. Evaluations of cytoxicity, nitric oxide (NO), interleukin-10 and in silico analysis were carried out. In vitro experiments showed that the fruit hexanic fraction (Fhf) and its alkaloid skimmianine (Skm) have a significant (P<0·001) effect against L. braziliensis. This anti-L. braziliensis activity of Fhf and Skm was due to increased production of NO and attenuation of IL-10 production in the macrophages at concentrations ranging from 1·6 to 40·0 µg/ml. The in silico assay demonstrated significant interaction between Skm and amino acid residues of NOS2. Skm is thus a promising drug candidate for L. braziliensis due to its potent immunomodulatory activity.

The effect of caffeic acid phenethyl ester on the functions of human monocyte-derived dendritic cells.

BACKGROUND: Propolis, an ancient herbal medicine, has been reported the beneficial effect both in asthma patients and murine model of asthma, but the mechanism was not clearly understood. In this study, the effect of caffeic acid phenethyl ester (CAPE), the most extensively studied components in propolis, on the functions of human monocyte-derived dendritic cells (MoDCs) was investigated. RESULTS: CAPE significantly inhibited IL-12 p40, IL-12 p70, IL-10 protein expression in mature healthy human MoDCs stimulated by lipopolysaccharides (LPS) and IL-12 p40, IL-10, IP-10 stimulated by crude mite extract. CAPE significantly inhibited IL-10 and IP-10 but not IL-12 expression in allergic patients' MoDCs stimulated by crude mite extract. In contrast, the upregulation of costimulatory molecules in mature MoDCs was not suppressed by CAPE. Further, the antigen presenting ability of DCs was not inhibited by CAPE. CAPE inhibited IkappaBalpha phosphorylation and NF-kappaB activation but not mitogen-activated protein kinase (MAPK) family phosphorylation in human MoDCs. CONCLUSION: These results indicated that CAPE inhibited cytokine and chemokine production by MoDCs which might be related to the NF-kappaB signaling pathway. This study provided a new insight into the mechanism of CAPE in immune response and the rationale for propolis in the treatment of asthma and other allergic disorders.

Nramp1-functionality increases iNOS expression via repression of IL-10 formation.

In mice, resistance to certain intracellular microbes depends on the expression of a late phagosomal protein termed natural-resistance associated macrophage protein 1 (Nramp1, Slc11a1). Nramp1-functionality is associated with alterations of cellular iron homeostasis and a sustained pro-inflammatory immune response, including the formation of the antimicrobial effector molecule NO. To investigate the underlying mechanism we used RAW-264.7 murine macrophage cells stably transfected with a functional Nramp1 allele (RAW-37) or Nramp1 non-functional controls (RAW-21). We found that the production of and signalling by the anti-inflammatory cytokine IL-10 was significantly enhanced in macrophages lacking functional Nramp1. Upon infection of macrophages with Salmonella typhimurium pathogen survival was significantly better in RAW-21 than in RAW-37, which inversely correlated to NO and TNF-alpha formation. Addition of a neutralising anti-IL-10 antibody to RAW-21 cells led to a significantly reduced survival of S. typhimurium within these cells and enhanced formation of NO and TNF-alpha reaching levels comparable to that observed in cells bearing functional Nramp1. Oppositely, supplementation of iron to RAW-21 cells further increased IL-10 formation.Thus, Nramp1 mediates effective host defence in part via suppression of excessive IL-10 production which may relate to Nramp1-mediated reduction of cellular iron pools, thus strengthening antimicrobial effector mechanisms.

Anti-inflammatory mechanism of Rungia pectinata (Linn.) Nees.

This study is to explore the inner anti-inflammatory mechanism of the ethanol extract of Rungia pectinata (Linn.) Nees. As a result, the ethanol extract of Rungia pectinata (Linn.) Nees could not only strongly reduce production of pro-inflammatory cytokines and mediators via blocking NF-kappaB activation but slightly promote release of anti-inflammatory mediator HO-1 and suppress IL-10 secretion. In conclusion, compared to Dexamethasone, Rungia pectinata (Linn.) Nees has not only similar effects on antagonizing pro-inflammatory mediators and cytokines but also mild effects on promoting production of anti-inflammatory mediators.

Fish oil supplementation modulates immune function in healthy infants.

(n-3) PUFA influence immune function in adults and may also affect immune maturation during development. This randomized trial is, to our knowledge, the first to investigate whether fish oil supplementation in late infancy modifies immune responses. The study was a 2 x 2 intervention in 64 healthy Danish infants, who received cow's milk or infant formula alone or with fish oil (FO) (3.4 +/- 1.1 mL/d) from 9 to 12 mo of age. Before and after the intervention, fatty acid composition of erythrocyte membranes, plasma IgE, C-reactive protein, and soluble IL-2 receptor concentrations were measured. TNF-alpha, INF-gamma, and IL-10 concentrations in whole-blood cultures, stimulated for 22 h with LPS+phytohemaglutinin (PHA) or Lactobacillus paracasei, were also determined. IgA was measured in feces when infants were 10 mo of age. FO supplementation effectively raised erythrocyte (n-3) PUFA (P < 0.001), increased L. paracasei-induced INF-gamma (P = 0.05) and tended to reduce LPS+PHA-induced IL-10 (P = 0.08). The FO intervention did not affect any of the other analyzed immune variables. The erythrocyte content of eicosapentanoic acid was negatively associated with LPS+PHA-induced IL-10 (r = -0.38, P = 0.02). Feeding milk rather than formula did not affect cytokine production, but plasma soluble IL-2 receptor concentration was greater in the formula group than in the cow's milk group (P = 0.03). Since the capacity to produce INF-gamma has been proposed as a maturation marker for the immune system in early life, this study suggests a faster immune maturation with FO supplementation with no apparent reduction in immune activation. The implications for later health need further investigation.

Bruton's tyrosine kinase is required for TLR-induced IL-10 production.

Bruton's tyrosine kinase (Btk) is a critical signaling mediator downstream of the B cell Ag receptor. X-linked agammaglobulinemia is caused by mutations in Btk resulting in multiple defects in B cell development and function, and recurrent bacterial infections. Recent evidence has also supported a role for Btk in TLR signaling. We demonstrate that Btk is activated by TLR4 in primary macrophages and is required for normal TLR-induced IL-10 production in multiple macrophage populations. Btk-deficient bone marrow-derived macrophages secrete decreased levels of IL-10 in response to multiple TLR ligands, compared with wild-type (WT) cells. Similarly, Btk-deficient peritoneal and splenic macrophages secrete decreased IL-10 levels compared with WT cultures. This phenotype correlates with Btk-dependent induction of NF-kappaB and AP-1 DNA binding activity, and altered commensal bacteria populations. Decreased IL-10 production may be responsible for increased IL-6 because blocking IL-10 in WT cultures increased IL-6 production, and supplementation of IL-10 to Btk-deficient cultures decreased IL-6 production. Similarly, injection of IL-10 in vivo with LPS decreases the elevated IL-6 serum levels during endotoxemia in Btk-deficient mice. These data further support a role for Btk in regulating TLR-induced cytokine production from APCs and provide downstream targets for analysis of Btk function.

Purine P1 receptor-dependent immunostimulatory effects of antiviral acyclic analogues of adenine and 2,6-diaminopurine.

Acyclic nucleoside phosphonates are widely recognised antivirals. The oral prodrugs of prototype compounds, e.g., 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; adefovir), and 9-(R)-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; tenofovir] were approved by FDA for treatment of hepatitis B (Hepsera), and acquired immunodeficiency syndrome (AIDS) (Viread), respectively. A number of acyclic nucleoside phosphonates possess immunostimulatory activity. The present experiments demonstrate that activation of cytokine and chemokine secretion is mediated by adenosine receptors. Included in the study were 9-(R)-[2-(phosphonomethoxy)propyl]adenine [tenofovir], N(6)-cyclopentyl-(R)-9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine, N(6)-cyclopropyl-(R)-9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine, and N(6)-isobutyl-9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine. All of them activate secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), "regulated on activation of normal T cell expressed and secreted" (RANTES/CCL5), and macrophage inflammatory protein-1alpha (MIP-1alpha/CCL3) in murine macrophages. With exception of MIP-1alpha, the effects were inhibited by antagonists of adenosine A(1), A(2B), and A(3) receptors (not by adenosine A(2A) receptor antagonist). The adenosine A(1) receptor antagonist inhibited TNF-alpha, IL-10, and RANTES, adenosine A(2B) receptor antagonist inhibited TNF-alpha and RANTES, and adenosine A(3) receptor antagonist inhibited IL-10 and RANTES. The suppression is due to decreased transcription of cytokine mRNA. It may be suggested that acyclic nucleoside phosphonates are nonspecific ligands for purine P(1) receptors.