[Buzhong Yiqi Decoction ameliorates spleen deficiency syndrome by regulating gut microbiota].

This study aims to decipher the mechanism of Buzhong Yiqi Decoction(BZYQD) in the treatment of spleen deficiency syndrome via gut microbiota. The mouse models of spleen deficiency syndrome were established by fecal microbiota transplantation(FMT, from patients with spleen deficiency syndrome) and administration of Sennae Folium(SF, 10 g·kg~(-1)), respectively, and treated with BZYQD for 5 d. The pseudosterile mice(administrated with large doses of antibiotics) and the mice transplanted with fecal bacteria from healthy human were taken as the controls. The levels of IgA, interleukin(IL)-2, IL-1ß, interferon(IFN)-γ, tumor necrosis factor-alpha(TNF-α), and 5-hydroxytryptamine(5-HT) in the intestinal tissue of two models were measured by enzyme-linked immunosorbent assay, and the CD8~+/CD3~+ ratio was determined by flow cytometry. The composition and changes of the gut microbiota were determined by 16S rRNA high-throughput sequencing and qPCR. Furthermore, the correlation analysis was performed to study the mediating role of gut microbiota in the treatment. The results showed that BZYQD elevated the IgA level, lowered the IL-1ß, TNF-α, and 5-HT levels, and decreased the CD8~+/CD3~+ ratio in the intestinal tissue of the two models. Moreover, BZYQD had two-way regulatory effects on the levels of IL-2 and IFN-γ. BZYQD inhibited the overgrowth and reduced the richness of gut microbiota in the SF model, and improved the gut microbiota structure in the two models. Algoriphagus, Mycobacterium, and CL500＿29＿marine＿group were the common differential genera in the two models compared with the control. Acinetobacter, Parabacteroides, and Ruminococcus were the differential genera unique to the FMT model, and Sphingorhabdus, Lactobacillus, and Anaeroplasma were the unique differential genera in the SF model. BZYQD was capable of regulating all these genera. The qPCR results showed that BZYQD increased the relative abundance of Akkermansia muciniphila and decreased that of Bacteroides uniformis in the two models. The correlation analysis revealed that the levels of above intestinal cytokines were significantly correlated with characteristic gut microorganisms in different mo-dels. The IL-1ß level had a significantly positive correlation with Acinetobacter and CL500＿29＿marine＿group in the two models, while the different levels of IL-2 and IFN-γ in the two models may be related to its different gut microbiota structures. In conclusion, BZYQD could regulate the disordered gut microbiota structure in different animal models of spleen deficiency syndrome to improve the intestinal immune status, which might be one of the mechanisms of BZYQD in treating spleen deficiency syndrome.

Astragalus root increases Treg and Th17 involvement in embryo implantation and pregnancy maintenance by decreasing CTLA-4+ Tregs.

Maintenance of pregnancy is highly dependent on the maternal immune system. High levels of regulatory T cells (Tregs) accumulate in the maternal placenta to suppress immunoreactivity against fetal antigens. We assessed whether Astragalus root (AsR) and AsR-containing Kampo medicines modulate immunoreactivity and thereby increase mouse litter size. AsR-exposed murine splenocytes exhibited significantly increased IL-2 secretion. In AsR-exposed mice, total Tregs were significantly increased, whereas cytotoxic T lymphocyte antigen 4 (CTLA-4)-positive Tregs were decreased in AsR-exposed mice. Tregs express IL-2 receptor subunit alpha and are activated by IL-2. CTLA-4 interacts with B7 expressed in antigen-presenting cells (APCs) with high affinity, and CTLA-4/B7 signaling plays a critical role in inhibiting APC activity, thereby suppressing CD4+ T cell proliferation and activation. The decrease in CTLA-4+ Tregs in AsR-exposed mice is thought to induce an increase in CD4+ T cells, leading to increased IL-2 secretion from CD4+ T cells followed by Treg activation. Th17 cells prevent trophoblast apoptosis, resulting in trophoblast invasion into the decidua. AsR increases Th17 cells, thereby inducing dose-dependent increases in litter size. Although Keishikaogito (KO)- and Ogikenchuto (OK)-exposed mice exhibited increased IL-2 secretion and splenic Tregs, KO also increased CTLA-4+ Tregs. Therefore, KO promoted immunosuppression by increasing CTLA-4+ Tregs, which induced a decrease in Th17 and exerted little effect on litter size. Therefore, an increase in both Tregs and Th17 cells can be considered necessary for embryo implantation and pregnancy maintenance.

Effect of Huangdisan grain on improving cognitive impairment in VD rats and its mechanism in immune inflammatory response.

BACKGROUND: Vascular dementia (VD) is the second most common type of dementia after Alzheimer's disease (AD). Although the incidence rate is very high, there is no definitive treatment for VD. And it has serious impact on the quality of life of VD patients. In recent years, more and more studies about the clinical efficacy and pharmacological effects of traditional Chinese medicine (TCM) in the treatment of VD have been conducted. And Huangdisan grain has been used to treat VD patients with a good curative effect in clinic. OBJECTIVE: This study was designed to investigate the effect of Huangdisan grain on the inflammatory response and cognitive function of VD rats modeled by bilateral common carotid artery occlusion (BCCAO), that aimed to improve the treatment methods for VD. METHODS: 8-week-old healthy SPF male Wistar rats (280 ± 20 g) were randomly divided into the normal group (Gn, n = 10), sham operated group (Gs, n = 10), and operated group (Go, n = 35). The VD rat models in Go group were established by BCCAO. 8 weeks after surgery, the operated rats were screened by the hidden platform trail of Morris Water Maze (MWM), and the rats with cognitive dysfunction were further randomly divided into the impaired group (Gi, n = 10) and TCM group (Gm, n = 10). The VD rats in Gm group were given the intragastric administration of Huangdisan grain decoction once a day for 8 weeks, and the other groups were given intragastric administration of normal saline. Then the cognitive ability of rats in each group was detected by the MWM Test. The lymphocyte subsets in peripheral blood and hippocampus of rats were measured by flow cytometry. The levels of cytokines (IL-1ß, IL-2, IL-4, IL-10, TNF-α, INF-γ, MIP-2, COX-2, iNOS) in peripheral blood and hippocampus were measured by ELISA (enzyme linked immunosorbent assay). The number of Iba-1+ CD68+ co-positive cells in the CA1 region of hippocampus was measured by immunofluorescence. RESULTS: Compared with the Gn group, the escape latencies of the Gi group were prolonged (P < 0.01), the time spent in the former platform quadrant was shortened (P < 0.01), and the number of times of crossing over the former platform location was reduced (P < 0.05). But compared with the Gi group, the escape latencies of Gm group were shortened (P < 0.01), the time spent in the former platform quadrant was prolonged (P < 0.05), and the number of times of crossing over the former platform location was increased (P < 0.05). The number of Iba-1+ CD68+ co-positive cells in the CA1 region of hippocampus of VD rats in Gi group was increased (P < 0.01) compared with the Gn group. And the proportions of T Cells, CD4+ T Cells, CD8+ T Cells in the hippocampus were increased (P < 0.01). The level of pro-inflammatory cytokines in the hippocampus was increased significantly, such as IL-1ß (P < 0.01), IL-2 (P < 0.01), TNF-α (P < 0.05), IFN-γ (P < 0.01), COX-2 (P < 0.01), MIP-2 (P < 0.01) and iNOS (P < 0.05). And the level of IL-10 (P < 0.01), a kind of anti-inflammatory cytokine, was decreased. The proportions of T Cells (P < 0.05), CD4+ T Cells (P < 0.01) and NK Cells (P < 0.05) in the peripheral blood of the VD rats in Gi group were decreased, and the level of IL-1ß, IL-2, TNF-α, IFN-γ, COX-2, MIP-2 and iNOS was increased significantly (P < 0.01) compared with the Gn group. Meanwhile, the level of IL-4 and IL-10 was decreased (P < 0.01). Huangdisan grain could reduce the number of Iba-1+ CD68+ co-positive cells in the CA1 region of hippocampus (P < 0.01), decrease the proportions of T Cells, CD4+ T Cells, CD8+ T Cells and the level of IL-1ß, MIP-2 in hippocampus (P < 0.01) of VD rats. Moreover, it could rise the proportion of NK Cells (P < 0.01) and the level of IL-4 (P < 0.05), IL-10 (P < 0.05), and decrease the level of IL-1ß (P < 0.01), IL-2 (P < 0.05), TNF-α (P < 0.01), IFN-γ (P < 0.01), COX-2 (P < 0.01) and MIP-2 (P < 0.01) in peripheral blood of VD rats. CONCLUSION: This study indicated that Huangdisan grain could decrease the activation of microglia/macrophages, regulate the proportions of lymphocyte subsets and the level of cytokines, which could adjust the immunologic abnormalities of VD rats, and ultimately improve cognitive function.

Impact of Antarctic krill oil supplementation on skeletal muscle injury recovery after resistance exercise.

BACKGROUND: Antarctic krill oil (KO) is a natural source of n-3 polyunsaturated fatty acids (n-3 PUFAs), and is rich in phospholipids, Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA), astaxanthin, flavonoids, vitamins, trace elements, and other bioactive substances. KO has been confirmed to have anti-inflammatory and immunomodulatory effects. n-3 PUFAs also have been purported to improve the recovery of muscular performance. Moreover, the phospholipids present in KO can enhance n-3 PUFA bioavailability because of its higher absorption rate in plasma compared to fish oil. Astaxanthin, found in Antarctic KO, is a red carotenoid and powerful antioxidant that inhibits oxidative stress after intense exercise. Hence, we examined the effect of KO supplementation on the recovery of exercise by measuring muscular performance, oxidant/antioxidant and anti-inflammatory activity, and the markers of muscle damage following a rigorous bout of resistance exercise. METHODS: 30 college-aged resistance-trained males (20.4 ± 0.92 years, 74.09 ± 7.23 kg, 180.13 ± 4.72 cm) were randomly supplemented with 3 g/d KO or placebo (PL) for 3 days and continued to consume after resistance exercise for 3 days until the experiment finished. Before supplementation, pre-exercise performance assessments of knee isokinetic strength, 20 m sprint, hexagon test, and blood serum creatine kinase (CK), lactate dehydrogenase (LDH), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), reactive oxygen species (ROS), malondialdehyde (MDA), interleukin-2 (IL-2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were completed. Then after 3 days of supplementation, participants completed a bout of muscle-damaging exercise, and subsequently, they performed and repeated the exercise performance assessments and blood-related indicators tests immediately (0 h), as well as at 6, 24, 48, and 72 h post-muscle-damaging exercise. RESULTS: Compared to the PL group, the serum CK of KO group was significantly lower at 24 h and 48 h post-exercise; the hexagon test time of the KO group was significantly lower than that of the PL group at 6 h and 24 h post-exercise; the KO group's isokinetic muscle strength showed different degrees of recovery than that of the PL group at 24 h and 48 h, and even over-recovery at 72 h post-exercise; the SOD level of the KO group was significantly higher than that of the PL group at 0, 6, and 24 h after exercise; the T-AOC level of the KO group was significantly higher than that of the PL group at 0, 6, and 72 h after exercise; the MDA level of the KO group was significantly lower than that of the PL group at 6 h; and there was no significant difference in serum IL-2, IL-6, and TNF-α between the two groups. CONCLUSION: Our results demonstrated that 3 g/d KO supplementation and continued supplementation after exercise can alleviate exercise-induced muscle damage (EIMD) and promote post-exercise recovery.

Oral administration of Helianthus annuus leaf extract ameliorates atopic dermatitis by modulation of T cell activity in vivo.

BACKGROUND: Atopic dermatitis (AD) is multifactorial disease that is highly involved in the activity of T cells from the skin lesion. Seeds of Helianthus annuus extract have been traditionally used as anti-inflammatory reagent but few studies have been reported on leaf of H. annuus that are discarded uselessly as an immunomodulator. PURPOSE: Therefore, here, the regulatory effect of Helianthus annuus extract (HAE) on AD via suppression of T cell activity was investigated. METHODS: The efficacy of HAE was evaluated in T cells stimulated with CD3/CD28 antibody and PMA/A23187. And demonstration of the alleviating effect of HAE on AD in the ears of Balb/c female mice stimulated with mite extract and DNCB. RESULTS: Pre-treatment with HAE abrogates IL-2 production from activated T cells. It was also found that HAE suppresses the expression of surface molecules in activated T cells. Cell viability results demonstrated that HAE is not associated with cytotoxicity in resting and activated T cells. Besides, we exhibited that regulated phosphorylation of MAPK through TAK1-IKKα-NFκB by pre-treatment with HAE leads to the suppressive effect of HAE on T cell activation. Oral administration of HAE attenuates manifestations of AD including reduced thickness of dermis and epidermis, decreased IgE level in serum, and declined mRNA levels of atopic cytokines on ear tissues. The ameliorative effect of HAE on AD was found to be associated with suppressed activity of T cells from draining lymph nodes. CONCLUSION: Therefore, our results provide that HAE alleviates AD symptoms via modulation of T cell activity. In addition, these results suggest the immunomodulatory effect of HAE on T-cell mediated diseases.

The Lipid-Soluble Forms of Choline Enhance Ex Vivo Responses from the Gut-Associated Immune System in Young Female Rat Offspring.

BACKGROUND: In humans, the development of gut-associated lymphoid tissue (GALT) occurs in the first years of life and can be influenced by diet. OBJECTIVES: The objective of this study was to determine the effect of dietary choline on the development of gut-associated lymphoid tissue (GALT). METHODS: Three feeding trials were conducted in female Sprague-Dawley rats. Beginning 3 d before parturition (studies 1 and 3) or at day 10 of gestation (study 2), control dams consumed a 100% free choline (FC) diet until the end of the lactation period. In studies 1 and 3, test dams consumed a high-glycerophosphocholine (HGPC) diet [75% glycerophosphocholine (GPC), 12.5% phosphatidylcholine (PC), 12.5% FC] and a 100% PC diet, respectively (both 1 g of choline/kg diet). In study 2, test dams consumed a high-sphingomyelin (SM) and PC (SMPC) diet (34% SM, 37% PC, 17% GPC, 7% FC, 5% phosphocholine) or a 50% PC diet (50% PC, 25% FC, 25% GPC), both 1.7 g of choline/kg diet. Immune cell phenotypes and ex vivo cytokine production by mitogen-stimulated immune cells were measured. RESULTS: Feeding of the HGPC diet lowered T-cell IL-2 (44%), IFN-γ (34%), and TNF-α (55%) production in mesenteric lymph nodes (MLNs) compared with control. Feeding both SMPC and 50% PC diets during the lactation and weaning periods increased IL-2 (54%) and TNF-α (46%) production after T-cell stimulation compared with control. There was a lower production of IL-2 (46%), IL-6 (66%), and TNF-α (45%), and a higher production of IL-10 (44%) in both SMPC and 50% PC groups following ovalbumin stimulation compared with control in MLNs. Feeding a diet containing 100% PC increased the production of IFN-γ by 52% after T-cell stimulation compared with control. CONCLUSION: Feeding a diet containing a mixture of choline forms with a high content of lipid-soluble forms during both the lactation and weaning periods enhances ex vivo immune responses from the GALT in female Sprague-Dawley offspring.

Natural Killer Cell Viability After Hyperthermia Alone or Combined with Radiotherapy with or without Cytokines.

BACKGROUND: The effects of hyperthermia and irradiation, alone and in combination, on natural killer (NK) cell viability were investigated in vitro. The roles of interleukin-2 (IL-2) and interferon (IFN) α, ß and γ in rescuing NK cells from hyperthermia and irradiation were studied. MATERIALS AND METHODS: Non-selected NK cells were used as effector cells and K-562 cells as target cells. NK and K-562 cells were treated at 37 to 45°C for 0 to 180 min. The cells were irradiated at room temperature using single doses from 0 to 60 Gy. Recombinant IL-2 at 100 to 450 U/ml and recombinant IFNα, ß and γ at 1,000 U/ml were used for different periods of time. NK cell viability was measured by intracellular adenosine tri-, and diphosphate (ATP, ADP) levels via luminometer, trypan blue exclusion and propidium iodide (PI) staining. Binding capacity of NK effector cells to target K-562 cells was also microscopically assessed. RESULTS: Thermal treatments between 37 and 41°C did not significantly affect the ATP levels of NK cells. Between 41°C and 42°C, ATP levels significantly decreased, whilst there was an insignificant reduction up to 45°C. At 42°C or higher, no recovery was detectable. At 42°C, the ATP level of NK cells rescued by IL-2 were significantly higher than those of controls at 37°C. IFNα, ß and γ had no significant effects. A combination of heating at 42°C and irradiation at 20 Gy significantly reduced the ATP levels (p<0.001) more than heating and irradiation alone. At 42°C, IL-2 abolished the reduction of ATP levels by heating and irradiation. This effect was dependent on heating time and irradiation dose. The ATP/ADP ratio did not significantly change when NK cells were heated for different times at 42°C. Thermal treatment of target K-562 cells at temperatures from 37 to 45°C reduced the number of NK cells binding K-652 cells. CONCLUSION: In vitro, NK cell viability was strongly reduced between 41°C and 42°C. At 42°C, the combination of irradiation and thermal treatment reduced the ATP levels in NK cells. However, IL-2 restored cell viability depending on thermal and radiation doses.

Comparative Analysis of Protocols to Induce Human CD4+Foxp3+ Regulatory T Cells by Combinations of IL-2, TGF-beta, Retinoic Acid, Rapamycin and Butyrate.

Regulatory T cells (Tregs) suppress other immune cells and are critical mediators of peripheral tolerance. Therapeutic manipulation of Tregs is subject to numerous clinical investigations including trials for adoptive Treg transfer. Since the number of naturally occurring Tregs (nTregs) is minute, it is highly desirable to develop a complementary approach of inducing Tregs (iTregs) from naïve T cells. Mouse studies exemplify the importance of peripherally induced Tregs as well as the applicability of iTreg transfer in different disease models. Yet, procedures to generate iTregs are currently controversial, particularly for human cells. Here we therefore comprehensively compare different established and define novel protocols of human iTreg generation using TGF-ß in combination with other compounds. We found that human iTregs expressed several Treg signature molecules, such as Foxp3, CTLA-4 and EOS, while exhibiting low expression of the cytokines Interferon-γ, IL-10 and IL-17. Importantly, we identified a novel combination of TGF-ß, retinoic acid and rapamycin as a robust protocol to induce human iTregs with superior suppressive activity in vitro compared to currently established induction protocols. However, iTregs generated by these protocols did not stably retain Foxp3 expression and did not suppress in vivo in a humanized graft-versus-host-disease mouse model, highlighting the need for further research to attain stable, suppressive iTregs. These results advance our understanding of the conditions enabling human iTreg generation and may have important implications for the development of adoptive transfer strategies targeting autoimmune and inflammatory diseases.

Cognitive and Affective Symptoms Experienced by Cancer Patients Receiving High-Dose Intravenous Interleukin 2 Therapy: An Integrative Literature Review.

BACKGROUND: Alterations in cognitive/affective functioning are among the most challenging adverse effects experienced by 80% of patients with metastatic melanoma and metastatic renal cell carcinoma undergoing high-dose interleukin 2 (IL-2) therapy. OBJECTIVE: The purpose of this literature review is to describe what is known about IL-2-induced cognitive/affective symptoms, their prevalence, and level of severity and synthesize findings to determine areas for future research to address symptom management challenges. This review describes the IL-2 patient experience and the pathophysiology leading to these changes. METHODS: An online electronic search using PubMed was performed to identify relevant literature published between 1992 and 2015. Of the original 113 articles, information was extracted from 9 articles regarding cognitive symptoms, affective symptoms, sample size, research design, reliability, and validity. RESULTS: Our review suggests that the trajectories, breadth, and depth of cognitive/affective symptoms have yet to be described. Despite intervention studies designed to address the psychosocial complications of IL-2, an understanding of the level of altered cognitive/affective symptoms experienced by IL-2 patients remains unclear. CONCLUSION: Our literature review reveals a lack of standardization when assessing, reporting, and managing cognitive/affective symptoms. Patients/family members have reported cognitive/affective symptoms to be the most alarming and difficult symptoms, yet these symptoms are not adequately screened for, and patients were not informed about potential changes. IMPLICATIONS FOR PRACTICE: Assessing patients for cognitive/affective alterations is important to reduce anxiety while improving outcomes. Education about the illness trajectory (what to expect during/after treatment) can help care partners/patients set realistic shared expectations and increase coping.

The T cell-selective IL-2 mutant AIC284 mediates protection in a rat model of Multiple Sclerosis.

Targeting regulatory T cells (Treg cells) with interleukin-2 (IL-2) constitutes a novel therapeutic approach for autoimmunity. As anti-cancer therapy with IL-2 has revealed substantial toxicities a mutated human IL-2 molecule, termed AIC284 (formerly BAY 50-4798), has been developed to reduce these side effects. To assess whether AIC284 is efficacious in autoimmunity, we studied its therapeutic potential in an animal model for Multiple Sclerosis. Treatment of Lewis rats with AIC284 increased Treg cell numbers and protected the rats from Experimental Autoimmune Encephalomyelitis (EAE). AIC284 might, thus, also efficiently prevent progression of autoimmune diseases in humans.

[Amplification efficency and optimization of culture conditions of γδ T cells in peripheral blood by different phosphate compounds].

OBJECTIVE: To compare the efficiency of pamidronate (PAM) and isopentenyl pyrophosphate (IPP) to stimulate γδ T cell expansion from human peripheral blood and explore the optimized expansion conditions. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque gradient centrifugation, and then cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, IPP (1.0, 5.0, 10.0, 15.0 µg/mL) or PAM (2.0, 5.0, 8.0, 12.0 µg/mL), and IL-2 (100.0, 200.0, 500.0 IU/mL). The cells were observed and collected. The number and proportion of CD3⁺TCRδ2⁺ γδ T cells stimulated by PAM or IPP in total lymphocytes were evaluated by flow cytometry and the expansion efficiency was calculated. RESULTS: After 14 days, the ratios of γδ T cells in total lymphocytes in IPP group and PAM group increased to 81.3% and 78.5%, respectively. This indicated that both IPP and PAM could effectively stimulate γδ T cell expansion and there was no significant difference in the efficiency of expansion between the two groups (P>0.05). CONCLUSION: PAM has the similar ability with IPP to stimulate γδ T cell expansion in vitro. PAM could become more economical and practical choice for stimulating γδ T cell expansion.

Bioactive compounds or metabolites from black raspberries modulate T lymphocyte proliferation, myeloid cell differentiation and Jak/STAT signaling.

Bioactive phytochemicals from natural products, such as black raspberries (BRB; Rubus occidentalis), have direct anticancer properties on malignant cells in culture and in xenograft models. BRB components inhibit cancer progression in more complex rodent carcinogenesis models. Although mechanistic targets for BRB phytochemicals in cancer cells are beginning to emerge, the potential role in modulating host immune processes impacting cancer have not been systematically examined. We hypothesized that BRB contain compounds capable of eliciting potent immunomodulatory properties that impact cellular mediators relevant to chronic inflammation and tumor progression. We studied both an ethanol extract from black raspberries (BRB-E) containing a diverse mixture of phytochemicals and two abundant phytochemical metabolites of BRB produced upon ingestion (Cyanidin-3-Rutinoside, C3R; Quercitin-3-Rutinoside, Q3R). BRB-E inhibited proliferation, and viability of CD3/CD28 activated human CD4(+) and CD8(+) T lymphocytes. BRB-E also limited in vitro expansion of myeloid-derived suppressor cells (MDSC) and their suppressive capacity. Pre-treatment of immune cells with BRB-E attenuated IL-6-mediated phosphorylation of signal transducer and activator of transcription-3 (STAT3) and IL-2-induced STAT5 phosphorylation. In contrast, pre-treatment of immune cells with the C3R and Q3R metabolites inhibited MDSC expansion, IL-6-mediated STAT3 signaling, but not IL-2-induced STAT5 phosphorylation and were less potent inhibitors of T cell viability. Together these data indicate that BRB extracts and their physiologically relevant metabolites contain phytochemicals that affect immune processes relevant to carcinogenesis and immunotherapy. Furthermore, specific BRB components and their metabolites may be a source of lead compounds for drug development that exhibits targeted immunological outcomes or inhibition of specific STAT-regulated signaling pathways.

PTEN-inhibition by zinc ions augments interleukin-2-mediated Akt phosphorylation.

Free zinc ions (Zn(2+)) participate in several signaling pathways. The aim of the present study was to investigate a potential involvement of Zn(2+) in the PI3K/Akt pathway of interleukin (IL)-2 signaling in T-cells. The IL-2 receptor triggers three major pathways, ERK1/2, JAK/STAT5, and PI3K/Akt. We have previously shown that an IL-2-mediated release of lysosomal Zn(2+) into the cytoplasm activates ERK1/2, but not STAT5. In the present study, Akt phosphorylation in response to IL-2 was abrogated by the Zn(2+) chelator N,N,N',N'-tetrakis-2(pyridyl-methyl)ethylenediamine, and was induced by treatment with Zn(2+) and the ionophore pyrithione. The latter were ineffective in cells that were treated with siRNA against the phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a phosphatase that degrades the lipid second messenger PI(3,4,5)P3, which is produced by PI3K and leads to activation of Akt. Inhibition of recombinant PTEN by Zn(2+)in vitro yielded an IC50 of 0.59 nM. Considering a resting free cytoplasmic Zn(2+) level of 0.2 nM in the T-cell line CTLL-2, this seems ideally suited for dynamic regulation by cellular Zn(2+). Oxidation with H2O2 and supplementation with Zn(2+) led to similar changes in the CD spectrum of PTEN. Moreover, Zn(2+) partially prevented the oxidation of cysteines 71 and 124. Hence, we hypothesize that zinc signals affect the IL-2-dependent PI3K/Akt pathway by inhibiting the negative regulator PTEN through binding with a sub-nanomolar affinity to cysteine residues that are essential for its catalytic activity.

[Medical treatment of renal cell carcinoma]. / Les médicaments du cancer du rein.

AIM: To describe drugs used in renal cell carcinoma. METHOD: Pubmed search for efficacy, mode of action and side effects for each molecule. Additional data were searched from the French regulatory agencies websites (HAS and ANSM). RESULTS: Since 2007, a total of three different therapeutic classes in the management of metastatic renal cell carcinoma are available. These three classes are tyrosine kinase inhibitors with sunitinib and sorafenib, the anti-VEGF antibodies (bevacizumab which is associated with alpha interferon in the treatment of advanced kidney cancer) and mTOR inhibitors with temsirolimus and everolimus. These targeted therapies are a major progress in the treatment of patients with metastatic kidney cancer. The side effects encountered with these molecules are numerous but serious side effects are less than 5% of all reported side effects. CONCLUSIONS: A better understanding of molecular mechanisms has enabled the development of new therapies for the treatment of metastatic renal cell carcinoma. In the future, a personalized approach taking into account the biology of each tumor could be created to provide a more targeted treatment.

Pathomorphosis of experimental infection in mice, infected by Streptococcus pneumoniae, under the effect of immunotropic drugs.

Pathomorphological changes in the organs of animals intranasally infected with Streptococcus pneumoniae were studied under conditions of immunotropic therapy added to antibiotic therapy. The pathomorphosis in the lungs, spleen, and thymus in animals treated with likopid, tinrostim, and roncoleukin was described. A positive time course of the pathological process in experimental animals in comparison with intact animals and animals receiving no immunotropic drugs was demonstrated.

TLR2 agonist PSK activates human NK cells and enhances the antitumor effect of HER2-targeted monoclonal antibody therapy.

PURPOSE: The therapeutic effect of trastuzumab monoclonal antibody (mAb) therapy has been shown to be partially dependent on functional natural killer (NK) cells. Novel agents that enhance NK cell function could potentially improve the antitumor effect of trastuzumab. We recently identified polysaccharide krestin (PSK), a natural product extracted from medicinal mushroom Trametes versicolor, as a potent toll-like receptor 2 (TLR2) agonist. This study was undertaken to evaluate the effect of PSK on human NK cells and the potential of using PSK to enhance HER2-targeted mAb therapy. EXPERIMENTAL DESIGN: Human peripheral blood mononuclear cells were stimulated with PSK to evaluate the effect of PSK on NK cell activation, IFN-γ production, cytotoxicity, and trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). Whether the effect of PSK on NK cells is direct or indirect was also investigated. Then, in vivo experiment in neu transgenic (neu-T) mice was carried out to determine the potential of using PSK to augment the antitumor effect of HER2-targeted mAb therapy. RESULTS: PSK activated human NK cells to produce IFN-γ and to lyse K562 target cells. PSK also enhanced trastuzumab-mediated ADCC against SKBR3 and MDA-MB-231 breast cancer cells. Both direct and interleukin-12-dependent indirect effects seem to be involved in the effect of PSK on NK cells. Oral administration of PSK significantly potentiated the antitumor effect of anti-HER2/neu mAb therapy in neu-T mice. CONCLUSION: These results showed that PSK activates human NK cells and potentiates trastuzumab-mediated ADCC. Concurrent treatment with PSK and trastuzumab may be a novel way to augment the antitumor effect of trastuzumab.

Deferiprone modulates in vitro responses by peripheral blood T cells from control and relapsing-remitting multiple sclerosis subjects.

T cells are important mediators of autoimmune inflammation in relapsing-remitting multiple sclerosis (RRMS). Previous studies found that deferiprone, an iron chelator, suppressed disease activity in a mouse model of multiple sclerosis, and inhibition of T cell proliferation was implicated as a putative mechanism. The objective of the present study was to examine the effects of deferiprone on suppressing in vitro responses of T cells from control and RRMS subjects. Peripheral blood T cells were co-stimulated with anti-CD3+anti-CD28 and cultured with or without interleukin 2 (IL-2). Proliferating CD4+ T cells from control and RRMS subjects, cultured with or without IL-2, decreased in response to 75 µM deferiprone, although the extent of decreased proliferation of CD4+ T cells from RRMS subjects was less than for control subjects. Proliferating CD8+ T cells from control subjects, cultured with or without IL-2, also decreased in response to 75 µM deferiprone, and this decrease was seen in proliferating CD8+ T cells from RRMS cultured with IL-2. CD4+CD25+ and CD8+CD25+ cells from control subjects, cultured with or without IL-2, declined in 75 µM deferiprone, but the decrease was smaller than for the CD4+ and CD8+ proliferative responses. CD4+CD25+ and CD8+CD25+ cells from RRMS subjects showed more variability than for control subjects, but CD4+CD25+ cultured with IL-2 and CD8+CD25+ cells cultured without IL-2 significantly declined in 75 µM deferiprone. CD4+FoxP3+ and CD4+CD25+FoxP3+ cells tended to remain constant or increase. In summary, deferiprone induced declines in proliferative responses at a dosage that is within peak serum pharmacological concentrations.

Additive melanoma suppression with intralesional phospholipid-conjugated TLR7 agonists and systemic IL-2.

There remains a compelling need for the development of treatments for unresectable melanoma. Agents that stimulate the innate immune response could provide advantages for cell-based therapies. However, there are conflicting reports concerning whether toll-like receptor (TLR) signaling controls tumor growth. The objective of this study was to evaluate the effect of intralesional administration of a TLR7 agonist in melanoma therapy. B16cOVA melanoma was implanted to TLR7 mice to evaluate the roles of stromal TLR7 on melanoma growth. To capitalize on the potential deleterious effects of TLR7 stimulation on the tumor growth, we injected melanoma tumor nodules with a newly developed and potent TLR7 agonist. B16 melanoma nodules expanded more rapidly in TLR7-deficient and MyD88 mice compared with TLR9 and wild type mice. Repeated injections with low doses of unconjugated TLR7 agonist were more effective at attenuating nodule size than a single high dose injection. To improve the efficacy we conjugated the agonist to phospholipid or phospholipids-polyethylene glycol, which retained TLR7 specificity. The phospholipid conjugate was indeed more effective in reducing lesion size. Furthermore, intralesional administration of the phospholipid TLR7 agonist conjugate enhanced the antimelanoma effects of systemic treatment with interleukin (IL)-2 and prolonged the survival of mice compared with IL-2 alone. Our study showed that: (1) TLR7/MyD88 signaling in the stroma is involved in melanoma growth; and (2) intralesional administration of a TLR7 agonist reduces the growth of melanoma nodules and enhances the antimelanoma effects of IL-2.

In vivo manipulation of Vgamma9Vdelta2 T cells with zoledronate and low-dose interleukin-2 for immunotherapy of advanced breast cancer patients.

The potent anti-tumour activities of gammadelta T cells have prompted the development of protocols in which gammadelta-agonists are administered to cancer patients. Encouraging results from small Phase I trials have fuelled efforts to characterize more clearly the application of this approach to unmet clinical needs such as metastatic carcinoma. To examine this approach in breast cancer, a Phase I trial was conducted in which zoledronate, a Vgamma9Vdelta2 T cell agonist, plus low-dose interleukin (IL)-2 were administered to 10 therapeutically terminal, advanced metastatic breast cancer patients. Treatment was well tolerated and promoted the effector maturation of Vgamma9Vdelta2 T cells in all patients. However, a statistically significant correlation of clinical outcome with peripheral Vgamma9Vdelta2 T cell numbers emerged, as seven patients who failed to sustain Vgamma9Vdelta2 T cells showed progressive clinical deterioration, while three patients who sustained robust peripheral Vgamma9Vdelta2 cell populations showed declining CA15-3 levels and displayed one instance of partial remission and two of stable disease, respectively. In the context of an earlier trial in prostate cancer, these data emphasize the strong linkage of Vgamma9Vdelta2 T cell status to reduced carcinoma progression, and suggest that zoledronate plus low-dose IL-2 offers a novel, safe and feasible approach to enhance this in a subset of treatment-refractory patients with advanced breast cancer.

MS-1020 is a novel small molecule that selectively inhibits JAK3 activity.

In order to identify Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling inhibitors, a cell-based high throughput screening was performed using a plant extract library that identified Nb-(alpha-hydroxynaphthoyl)serotonin called MS-1020 as a novel JAK3 inhibitor. MS-1020 potently inhibited persistently-active STAT3 in a cell type-specific manner. Further examination showed that MS-1020 selectively blocked constitutively-active JAK3 and consistently suppressed interleukin-2-induced JAK3/STAT5 signalling but not prolactin-induced JAK2/STAT5 signalling. Furthermore, MS-1020 affected cell viability only in cancer cells harbouring persistently-active JAK3/STATs, and in vitro kinase assays showed MS-1020 binds directly with JAK3, blocking its catalytic activity. Therefore, the present study suggested that this reagent selectively inhibits JAK3 and subsequently leads to a block in STAT signalling. Finally, MS-1020 decreased cell survival by inducing apoptosis via down-regulation of anti-apoptotic gene expression. These results suggest that MS-1020 may have therapeutic potential in the treatment of cancers harbouring aberrant JAK3 signalling.