Functional modulation of CD8+ T cell by approved novel immune enhancer: Nocardia rubra Cell-Wall Skeletons (Nr-CWS)

Nocardia rubra cell wall skeleton (Nr-CWS) has been reported to have innate immunostimulating and anti-tumor activities. However, the immunomodulatory effects of Nr-CWS on CD8+ T cells and their related mechanisms are still unknown. In this work, our team purified CD8⁺T cells from spleen cells and explored the phenotype and function of NR-CWS in vitro on CD8⁺T cells. We observed that Nr-CWS can significantly up-regulate the expression of CD69 and CD25 on CD8⁺T cells, with no significant effect on apoptosis or cell death of CD8⁺T cells that occurs in vitro during culture. In addition, the effect of perforin and granzyme B was increased after Nr-CWS treatment, but did not substantially alter the expression of TRAIL and FasL. A variety of cytokine analyses have shown that of the cytokines examined (IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6 and IL-10), only IFN-γ and TNF The increase in -α was more pronounced, and the effect of Nr-CWS in CD8⁺T cell culture medium on CD8+ T cells was independent of Th cells. Our results demonstrated that Nr-CWS could up-regulate CD69 and CD25 expression on CD8⁺T cells, promoting IFN-γ and TNF-α secretion, and enhancing perforin and granzyme B production. Thus Nr-CWS may have Immunoaugmenting therapeutic activity via an increase in CD8⁺T cells response.     

CXCR5+ CD8+ T cells present elevated capacity in mediating cytotoxicity toward autologous tumor cells through interleukin 10 in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a common and aggressive subtype of non-Hodgkin's lymphomas, with limited treatment options in refractory and relapsed patients. Growing evidence supports the notion that CD8⁺ T cell immunity could be utilized to eliminate B cell lymphomas. CXCR5⁺ CD8⁺ T cell is a novel cell subtype and share CXCR5 expression with CD19⁺ tumor cells. In this study, we investigated the frequency and function of existing CXCR5⁺ CD8⁺ T cells in DLBCL patients. We found that DLBCL patients as a group demonstrated significantly higher level of CXCR5⁺ CD8⁺ T cells than healthy individuals, with huge variability in each patient. Using anti-CD3/CD28-stimulated CD8⁺ T cells as effector (E) cells and autologous CD19⁺ tumor cells as target (T) cells, at high E:T ratio, no difference between the intensities of CXCR5⁺ CD8⁺ T cell- and CXCR5⁻ CD8⁺ T cell-mediated cytotoxicity were observed. However, at intermediate and low E:T ratios, the CXCR5⁺ CD8⁺ T cells presented stronger cytotoxicity than CXCR5⁻ CD8⁺ T cells. The expressions of granzyme A, granzyme B, and perforin were significantly higher in CXCR5⁺ CD8⁺ T cells than in CXCR5⁻ CD8⁺ T cells, with no significant difference in the level of degranulation. Tumor cells in DLBCL were known to secrete high level of interleukin 10 (IL-10). We therefore blocked the IL-10/IL-10R pathway, and found that the expressions of granzyme A, granzyme B, and perforin by CXCR5⁺ CD8⁺ T cells were significantly elevated. Together, these results suggest that CXCR5⁺ CD8⁺ T cells are potential candidates of CD8⁺ T cell-based immunotherapies, could mediate elimination of autologous tumor cells in DLBCL patients, but are also susceptible to IL-10-mediated suppression.     

Astilbin promotes the induction of regulatory NK1.1− CD4+ NKG2D+ T cells through the PI3K,STAT3, and MAPK signaling pathways

Astilbin is a potential agent for autoimmune and inflammatory diseases and has a protective effect in mice with DSS-induced colitis. NK1.1⁻ CD4⁺ NKG2D⁺ T cells are a subpopulation of regulatory T cells that produce TGF-β1 and IL-10. Whether astilbin directly promotes the induction of NK1.1⁻ CD4⁺ NKG2D⁺ T cells and whether these astilbin-stimulated T cells exert an immune-regulatory role remain unclear. Here, we show that astilbin efficiently induces the production of NK1.1⁻ CD4⁺ NKG2D⁺ T cells with high expressions of TGF-β1, IL-10, CCR6, and CCR9 in a dose-dependent manner ex vivo. These regulatory T cells also substantially inhibit the activities of CD8⁺ T cells and macrophages. Intraperitoneal injection of astilbin ameliorates the severity of colitis with an increase in the frequency of NK1.1⁻ CD4⁺ NKG2D⁺ T cells in the colon tissue of DSS-treated mice. Moreover, adoptive transfer of NK1.1⁻ CD4⁺ NKG2D⁺ T cells induced by astilbin remarkably protects against the onset of DSS-induced colitis. Finally, the PI3K, STAT3, and MAPK signaling pathways are involved in the induction of NK1.1⁻ CD4⁺ NKG2D⁺ T cells by astilbin. Taken together, our study elucidates a new immune-regulatory mechanism of astilbin by inducing the regulatory NK1.1⁻ CD4⁺ NKG2D⁺ T cells and indicates a potential clinical use of astilbin for patients with inflammatory bowel diseases.     

Downregulation and altered function of natural killer cells in hepatitis B virus patients treated with entecavir

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Effects of Vitamin D3, Calcipotriol and FTY720 on the Expression of Surface Molecules and Cytolytic Activities of Human Natural Killer Cells and Dendritic Cells

We describe here the effects of three drugs that are either approved or have the potential for treating multiple sclerosis (MS) patients through the in vitro activities of human natural killer (NK) cells and dendritic cells (DCs). Our results indicate that 1,25(OH)₂D₃, the biologically active metabolite vitamin D₃, calcipotriol and FTY720 augment IL-2-activated NK cell lysis of K562 and RAJI tumor cell lines as well as immature (i) and mature (m) DCs, with variable efficacies. These results are corroborated with the ability of the drugs to up-regulate the expression of NK cytotoxicity receptors NKp30 and NKp44, as well as NKG2D on the surfaces of NK cells. Also, they down-regulate the expression of the killer inhibitory receptor CD158. The three drugs down-regulate the expression of CCR6 on the surface of iDCs, whereas vitamin D₃ and calcipotriol tend to up-regulate the expression of CCR7 on mDCs, suggesting that they may influence the migration of DCs into the lymph nodes. Finally, vitamin D₃, calcipotriol and FTY720 enhance NK17/NK1 cell lysis of K562 cells, suggesting that a possible mechanism of action for these drugs is via activating these newly described cells. In conclusion, our results show novel mechanisms of action for vitamin D₃, calcipotriol and FTY720 on cells of the innate immune system.     

Exposure to particulate matter 2.5 (PM2.5) induced macrophage-dependent inflammation,characterized by increased Th1/Th17 cytokine secretion and cytotoxicity

Particulate matter PM2.5 is a class of airborne particles and droplets with sustained high levels in many developing countries. Epidemiological studies have shown the association between sustained high level of PM2.5 and the risk of many diseases in the respiratory system, including lung cancer. However, the precise mechanisms through which PM2.5 induces respiratory diseases are still unclear. In this study, we demonstrated that CD4⁺ and CD8⁺ T cells following PM2.5 treatment demonstrated significantly elevated mRNA and protein levels of interferon (IFN)-γ, interleukin (IL)-10, IL-17, and IL-21 production. This increase in cytokines required the presence of macrophages, such that CD4⁺ and CD8⁺ T cells treated with PM2.5 in the absence of macrophages did not present higher IFN-γ, IL-10, or IL-21 expression. In contrast, PM2.5-treated macrophages could significantly upregulate T cell cytokine secretion, even when excess PM2.5 was removed from cell culture. We also observed a macrophage-dependent upregulation of granzyme A and granzyme B expression by CD4⁺ and CD8⁺ T cells following PM2.5 treatment. These PM2.5-stimulated CD4⁺ and CD8⁺ T cells potently induced the death of human bronchial epithelial (HBE) cells. Interestingly, the CD4⁺ and CD8⁺ T cells presented synergistic effects at inducing HBE cytotoxicity, such that CD4⁺ T cells and CD8⁺ T cells combined resulted in higher HBE cell death than the sum of the separate effects of CD4⁺ T cells and CD8⁺ T cells. While blocking cytotoxic molecule release significantly compromised the T cell-mediated cytotoxicity against HBE cells, blocking IFN-γ, but not IL-10, could also slightly but significantly reduce T cell-mediated cytotoxicity. Together, these data demonstrated that PM2.5 could promote the inflammation of cytotoxicity of T cells in a macrophage-dependent manner. In addition, PM2.5-treated macrophages presented long-lasting proinflammatory effects on T cells.     

Inhibition of CD8+ T cells and elimination of myeloid cells by CD4+ Foxp3− T regulatory type 1 cells in acute respiratory distress syndrome

Acute lung injury and acute respiratory distress syndrome (ARDS) are caused by rapid‐onset bilateral pulmonary inflammation. We therefore investigated the potential role of interleukin (IL)‐10⁺CD4⁺ Tr1 cells, a regulatory T cell subset with previously identified immunosuppressive functions, in ARDS patients. We first showed that circulating Tr1 cells were upregulated in active and resolved ARDS patients compared to healthy controls and pneumonia patient controls. A significant fraction of these Tr1 cells expressed granzyme B and perforin, while most Tr1 cells did not express interferon gamma (IFN‐γ), IL‐4, IL‐17 or FOXP3, suggesting that the effector functions of these Tr1 cells were primarily mediated by IL‐10, granzyme B, and perforin. Indeed, Tr1 cells effectively suppressed CD8⁺ T cell IFN‐γ production and induced lysis of monocytes and dendritic cells in vitro. The elimination of myeloid antigen‐presenting cells depended on granzyme B production. We also discovered that Tr1 cells could be identified in the bronchoalveolar lavage fluid collected from ARDS patients. All these results suggested that Tr1 cells possessed the capacity to downregulate inflammation in ARDS. In support of this, we found that ARDS patients who resolved the inflammation and survived the syndrome contained significantly higher levels of Tr1 cells than ARDS patients who succumbed to the syndrome. Overall, this report added a novel piece of evidence that ARDS could be intervened by regulatory T cell‐mediated suppressive mechanisms.     

Interleukin-7 promotes lung-resident CD14+ monocytes activity in patients with lung squamous carcinoma

Interleukin (IL)-7 enhances cytokines secretion by CD14⁺ monocytes, and induces recruitment of monocytes to endothelium. As an important regulator to different types of immune cells, the role of IL-7 in modulation of CD14⁺ monocytes is still not fully elucidated. Thus, the aim of current study was to investigate the immunoregulatory activity of IL-7 to peripheral and lung-resident CD14⁺ monocytes in lung squamous carcinoma patients. Thirty-seven lung squamous carcinoma patients and eighteen healthy individuals were enrolled. CD14⁺ monocytes and CD4⁺ T cells were purified from both peripheral bloods and bronchoalveolar lavage fluids (BALF). IL-7 expression in plasma and BALF was measured by ELISA, and CD127 expression in peripheral and lung-resident CD14⁺ monocytes was investigated by real-time PCR and flow cytometry, respectively. Cellular proliferation, cytokine production, and molecules in IL-7 signaling pathway was assessed in CD14⁺ monocytes in response to IL-7 stimulation. IL-7-induced CD14⁺ monocytes activity to CD4⁺ T cells was also assessed in direct and indirect contact co-culture system. There were no remarkable differences of plasma IL-7 concentration or CD127 level between healthy individuals and lung squamous carcinoma patients. However, IL-7 expression was significantly reduced in BALF from tumor site in squamous carcinoma patients, especially in stage III and IV. IL-7 stimulation not only promoted proliferation, cytokines secretion, and STAT-5 phosphorylation in lung-resident CD14⁺ monocytes, but also enhanced CD14⁺ monocytes-induced Th1 and T follicular helper cells activation, which presented as elevated interferon-γ and IL-21 secretion by CD4⁺ T cells. This process required direct cell-to-cell contact, and was dependent on IL-6 secretion. The current data revealed a potential immunopromotive property of IL-7 to lung-resident CD14⁺ monocytes in lung squamous carcinoma.     

Alcohol consumption decreases IL-2-induced NF-kappaB activity in enriched NK cells from C57BL/6 mice.

Previously we showed that ethanol (EtOH) consumption suppressed IL-2-induced cytolytic activity of murine splenic natural killer (NK) cells. Although IL-2 receptor signaling is involved in activation of NK cells, neither the mechanism for this activation nor the role of EtOH consumption in modulating activation is completely understood. In this study we show by electrophoretic mobility-shift assay (EMSA) that enriched splenic NK cells from EtOH-consuming C57BL/6 mice exhibit reduced NF-kappaB and AP-1 binding activity in response to IL-2 stimulation as compared to the water-drinking mice. Semiquantitative RT-PCR and real-time PCR analyses indicated that EtOH consumption inhibits the induction of perforin, granzyme A, and granzyme B in response to IL-2. Pyrrolidine dithiocarbamate (PDTC) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) blocked NFkappaB and AP-1 binding activity in nuclear extracts of IL-2-stimulated NK cells in an EMSA and also inhibited the IL-2-induced expression of perforin, granzyme A, and granzyme B gene expression in enriched NK cells. These inhibitors dramatically suppressed IL-2-stimulated NK cytolytic activity against YAC-1 lymphoma target cells. Taken together, these results suggest that NFkappaB and AP-1 are important regulators of NK cell cytolytic function through regulation of perforin, granzyme A, and granzyme B gene expression. The findings further suggest that the decreased cytolytic activity of IL-2-stimulated NK cytolytic activity in EtOH-consuming mice is due at least in part to impaired transactivation of these and possibly other genes involved in control of NK-cell target lysis.     

Role of CD8+ T lymphocyte cells: Interplay with stromal cells in tumor microenvironment

CD8⁺ T lymphocytes are pivotal cells in the host response to antitumor immunity. Tumor-driven microenvironments provide the conditions necessary for regulating infiltrating CD8⁺ T cells in favor of tumor survival, including weakening CD8⁺ T cell activation, driving tumor cells to impair immune attack, and recruiting other cells to reprogram the immune milieu. Also in tumor microenvironment, stromal cells exert immunosuppressive skills to avoid CD8⁺ T cell cytotoxicity. In this review, we explore the universal function and fate decision of infiltrated CD8⁺ T cells and highlight their antitumor response within various stromal architectures in the process of confronting neoantigen-specific tumor cells. Thus, this review provides a foundation for the development of antitumor therapy based on CD8⁺ T lymphocyte manipulation.KEY WORDS: CD8⁺ T lymphocyte, Stromal cell, Tumor microenvironment, Immunosuppression, Immunotherapy, Antitumor     

PGE2 inhibits natural killer and γδ T cell cytotoxicity triggered by NKR and TCR through a cAMP-mediated PKA type I-dependent signaling

Natural killer (NK) and unconventional γδ T cells, by their ability to sense ligands induced by oncogenic stress on cell surface and to kill tumor cells without a need for clonal expansion, show a great therapeutic interest. They use numerous activating and inhibitory receptors which can function with some independence to trigger or inhibit destruction of target cells. Previous reports demonstrated that PGE₂ is able to suppress the destruction of some tumor cell lines by NK and γδ T cells but it remained uncertain if PGE₂ interferes with the different activating receptors governing the cytolytic responses of NK and γδ T cells. In this report, using the model of specific redirected lysis of the mouse FcγR⁺ cell line P815, we clearly demonstrate that the major NK receptors (NKR): NKG2D, CD16 and natural cytotoxicity receptors (NCR: NKp30, NKp44, NKp46) and γδ T cell receptors TCR Vγ9Vδ2, NKG2D and CD16 are all inhibited by PGE₂. As is the case with γδ T cells, we show that PGE₂ binds on E-prostanoid 2 (EP2) and EP4 receptors on NK cells. Finally, we delineate that the signaling of the blockade by PGE₂ is mediated through a cAMP-dependent activation of PKA type I which inhibits early signaling protein of cytotoxic cells. In the discussion, we focused on how these data should impact particular approaches in the treatment of cancer.     

乳腺癌NKG2D表达及对NK细胞杀伤活性的影响

目的分析乳腺癌NKG2D表达及对NK细胞细胞毒的影响。方法应用免疫荧光技术和流式细胞术（FCM）分选25例乳腺癌、25例健康对照的外周血NK细胞,定量分析NKG2D蛋白表达。用MTT比色法检测抗NKG2DpAb加入前后NK细胞的细胞毒效应。结果乳腺癌患者NK细胞的NKG2D蛋白表达量均低于正常对照,差异有显著性（P〈0.05）。抗NKG2DpAb能显著抑制NK细胞对K562细胞的细胞毒效应。结论NK细胞NKG2D表达异常与乳腺癌的发生发展可能有关。     

Immunosenescence in chronic HIV infected patients impairs essential functions of their natural killer cells

The HIV/AIDS pandemic still represents an important global health issue. There is no sterilizing cure, therefore a continuous treatment is necessary, which caused the emerged idea of HIV as a chronic inflammatory disease that may also affect healthy aging. Considering that the activation profile of some innate cells such as natural killer cells has previously been associated to HIV progression, it remains to be better defined this activation status of NK cells considering the time of HIV infection. In this study, we characterized NK cell phenotype and function during acute and chronic HIV infection and also investigated markers of immunosenescence in these cells. Our results showed that chronic infected patients remained with elevated levels of some plasma inflammatory molecules (IP-10, sCD14) and a concurrent expansion of the non-functional NK cell subset (CD3^-CD56^-CD16⁺). NK cells from the chronic infected group displayed an activated profile with higher levels of cytokines and chemokines production (TNF-α, IL-12, IFN-α2, IFN-γ, IL-6, RANTES, MCP-1, IL-10, IL-4 and IL-5). The production of these molecules was positively correlated to the time of infection. Moreover, we noted a possible association of higher global DNA methylation frequency of NK cells in two HIV patients in the advanced stage of disease. Chronic infected patients also showed a trend towards higher production of reactive oxygen species by their NK cells which altogether suggest the evolution of these cells to a senescent state that might be further evaluated.     

Invariant NKT cells increase drug-induced osteosarcoma cell death

BACKGROUND AND PURPOSE

In osteosarcoma (OS) patients, only a limited number of drugs are active and the regimens currently in use include a combination of at least two of these drugs: doxorubicin, cisplatin, methotrexate and ifosfamide. Today, 30–40% of patients still die of OS highlighting the urgent need for new treatments. Invariant NKT (iNKT) cells are a lymphocyte lineage with features of both T and NK cells, playing important roles in tumour suppression. Our aim was to test whether the cytoxicity induced by cisplatin, doxorubicin and methotrexate against OS cells can be enhanced by iNKT cell treatment.

EXPERIMENTAL APPROACH

iNKT cells were purified from human peripheral blood mononuclear cells by cell sorting (Vα24Vβ11⁺ cells) and used as effector cells against OS cells (U2-OS, HOS, MG-63). Cell death (calcein-AM method), perforin/granzyme B and Fas/FasL expressions were determined by flow cytometry. CD1d expression was analysed at both the gene and protein level.

KEY RESULTS

iNKT cells were cytotoxic against OS cells through a CD1d-dependent mechanism. This activity was specific for tumour cells, because human CD1d⁺ mesenchymal stem cells and CD1d^- osteoblasts were not affected. iNKT cell treatment enhanced drug-induced OS cell death in a concentration-dependent manner and this effect was reduced in CD1d-silenced OS cells.

CONCLUSION AND IMPLICATIONS

iNKT cells kill malignant, but not non-malignant, cells. iNKT cell treatment enhances the cytotoxicity of anti-neoplastic drugs against OS cells in a CD1d-dependent manner. The present data encourage further studies on the use of iNKT cells in OS therapy.     

IL-18诱导外周血单个核细胞对Raji细胞的杀伤效应及其机制的实验研究

目的 探讨IL-18诱导PBMC对Raji细胞的杀伤效应及其作用机制。方法 IL-18在体外诱导PBMC后,以MTT法测定其对Raji细胞的杀伤效应。在杀伤活性最大时,以BAS-ELISA法检测培养上清液sFas-L的含量,以流式细胞仪检测PBMC的CD_(16)/CD_(56)、Fas-L、穿孔素、颗粒酶B表达。结果 PBMC在体外经IL-18诱导后,能明显增强其对Raji细胞的杀伤活性,在PBMC经IL-18诱导后的d 4,这种杀伤活性最高。在杀伤活性最高时,PBMC表达CD16/CD_(56)、Fas-L、穿孔素、颗粒酶B的能力明显增加,但培养上清液中sFas-L的含量无明显增加。结论 本实验的结果显示,IL-18能诱导PBMC杀伤细胞的增殖和活化,并使其对Raji细胞杀伤活性增强,这种杀伤机制可能与穿孔素、颗粒酶B和Fas-L的高表达有关。     

The anti-lung cancer activity of SEP is mediated by the activation and cytotoxicity of NK cells via TLR2/4 in vivo

Strongylocentrotus nudus egg polysaccharide (SEP) has been reported to display antitumor activity. However, the effects of SEP and its underlying mechanism in the treatment of lung cancer remain unclear, particularly with an immunodeficient mouse model of human non-small cell lung cancer (NSCLC). In the present study, we investigated the anti-lung cancer effects of SEP and its underlying mechanism of action in both Lewis lung cancer (LLC)-bearing C57/BL6J mice and human NSCLC H460-bearing nude mice. Although SEP showed no inhibitory effects on tumor cells in vitro, it markedly stimulated the percentage of CD3−NK1.1⁺ cells and natural killer (NK) cell cytotoxicity in the spleens of nude mice and C57/BL6J mice. In LLC-bearing mice, SEP not only inhibited tumor growth but also promoted NK-mediated cytotoxicity, the NK1.1⁺ cell population, and IL-2 and IFN-γ secretion. SEP significantly suppressed H460 growth in nude mice, which was abrogated by the selective depletion of NK cells via the intraperitoneal injection of anti-asialo GM-1 antibodies. Furthermore, anti-TLR2/4 antibodies blocked both SEP and NK cell binding and SEP-induced perforin secretion. SEP-induced proliferation and IFN-γ secretion by NK cells in wild type mice were partially impaired in TLR2 or TLR4 knockout mice. These results suggest that SEP-promoted NK cytotoxicity, which was partially mediated via TLR2 and TLR4, was the main contributing factor to lung cancer inhibition in vivo and that SEP may be a potential immunotherapy candidate for the treatment of lung cancer.     

Δ9-Tetrahydrocannabinol (THC) Impairs CD8+ T Cell-Mediated Activation of Astrocytes

CD8⁺ T cells can contribute to neuroinflammation by secretion of inflammatory cytokines like interferon γ (IFNγ) and tumor necrosis factor α (TNFα). Astrocytes, a glial cell in the brain, can be stimulated by IFNγ and TNFα to secrete the inflammatory cytokines, monocyte chemotactic protein 1 (MCP-1), interleukin 6 (IL-6), and interferon-γ inducible protein 10 (IP-10). Δ⁹-Tetrahydrocannabinol (THC), the primary psychoactive cannabinoid in Cannabis sativa, possesses potent anti-inflammatory activity. The objective of this investigation was to assess the effects of THC treatment on CD8⁺ T cell-mediated activation of astrocytes. CD3/CD28/IFNα- stimulated CD8⁺ T cells were treated with vehicle (0.03% EtOH) or THC and cocultured with U251 astrocytes. IP-10⁺, MCP-1⁺, and IL-6⁺ astrocytes were quantified by flow cytometry. LegendPlex™ was used to measure cytokine secretion by CD8⁺ T cells and flow cytometry was employed to quantify IFNγ, TNFα, and lysosomal-associated membrane protein 1 (LAMP-1) expression. Recombinant TNFα and IFNγ were used to stimulate MCP-1, IP-10, IL-6 responses in U251 astrocytes, which were measured by flow cytometry. Treatment with THC reduced CD8⁺ T cell-mediated induction of IP-10 and IL-6 responses in U251 astrocytes but had no effect on MCP-1. THC treatment differentially affected T cell effector functions such that IFNγ and degranulation responses were sensitive to THC-mediated ablation while TNFα was not. Lastly, THC treatment reduced the IFNγ-induced IP-10 response but had no effect on TNFα-induced MCP-1 response in U251 astrocytes. The results suggest that cannabinoid treatment can selectively reduce certain CD8⁺ T cell responses that contribute to stimulation of astrocytes.

Treatment with THC can abate CD8⁺ T cell-dependent neuroinflammatory processes by inhibiting CD8⁺ cell differentiation into effector cells, suppressing CD8⁺ effector cell function, and reducing activation of astrocytes by CD8⁺ T cell-derived inflammatory cytokines.