Core-Shell type lipid/rPAA-Chol polymer hybrid nanoparticles for in vivo siRNA delivery

Our previous study had reported that cholesterol-grafted poly(amidoamine) (rPAA-Chol polymer) was able to self-assemble into cationic nanoparticles and act as a potential carrier for siRNA transfection. In this study, the core–shell type lipid/rPAA-Chol hybrid nanoparticles (PEG-LP/siRNA NPs and T7-LP/siRNA NPs) were developed for improving in vivo siRNA delivery by modifying the surface of rPAA-Chol/siRNA nanoplex core with a lipid shell, followed by post-insertion of polyethylene glycol phospholipid (DSPE-PEG) and/or peptide (HAIYPRH, named as T7) modified DSPE-PEG-T7. The integrative hybrid nanostructures of LP/siRNA NPs were evidenced by dynamic light scattering (DLS), confocal laser scanning microscope (CLSM), cryo-transmission electron microscope (Cryo-TEM) and surface plasmon resonance (SPR) assay. It was demonstrated that the T7 peptide modified LP/siRNA NPs (T7-LP/siRNA NPs) exhibited uniform and spherical structures with particle size of 99.39 ± 0.65 nm and surface potential of 42.53 ± 1.03 mV, and showed high cellular uptake efficiency and rapid endosomal/lysosomal escape ability in MCF-7 cells. Importantly, in vitro gene silencing experiment demonstrated that both of pegylated and targeted LP/siEGFR NPs exhibited significantly stronger downregulation of EGFR protein expression level in MCF-7 cells, compared to that of the physical mixture of siRNA lipoplexes and rPAA-Chol/siRNA nanoplexes. In vivo tumor therapy on nude mice bearing MCF-7 tumors further confirmed that the targeted T7-LP/siEGFR NPs exhibited the greatest inhibition on tumor growth via transferrin receptor-mediated targeting delivery, without any activation of immune responses and significant body weight loss following systemic administration. These findings indicated that the core-shell type T7-LP/siRNA nanoparticles would be promising siRNA delivery systems for in vivo tumor-targeted therapy.     

Inhibition of Interferon Regulatory Factor 4 Attenuates Acute Liver Allograft Rejection in Mice

Acute liver allograft rejection is a serious complication after liver transplantation. Interferon regulatory factor 4 (IRF4) is expressed predominantly in the immune system and plays an important role in its development and function. However, the role of IRF4 in liver transplantation has never been investigated. In our current study, to evaluate the effect of IRF4 inhibition on recipient survival, IRF4 siRNA, or control siRNA, or PBS was injected into the liver allograft recipients through caudal vein. The survival time of mice treated with IRF4 siRNA (MST = 31.5 days) was prolonged significantly compared with that of mice treated with PBS (MST = 6 days) or control siRNA (MST = 6.5 days) (P < 0.001). IRF4 siRNA treatment displays lower induction of pro‐inflammatory levels, including TNF‐α, IL‐6 and IFN‐γ, and higher induction of anti‐inflammatory IL‐10 levels. Administration of anti‐IL‐10 into IRF4 siRNA‐treated mice resulted in shortened allograft survival and increased rejection scores. Furthermore, IRF4 inhibition promotes M2 macrophage differentiation in vivo and in vitro. And inhibition of macrophages with GdCl₃ reverses the prolonged liver allograft survival and decreased liver rejection scores induced by IRF4 siRNA. In conclusion, inhibition of IRF4 attenuates acute liver allograft rejection in mice, and this is associated with promoted M2 macrophage differentiation.     

PEGylated carboxymethyl chitosan/calcium phosphate hybrid anionic nanoparticles mediated hTERT siRNA delivery for anticancer therapy

Lack of safe and effective delivery vehicle is the main obstacle for siRNA mediated cancer therapy. In this study, we synthesized a pH-sensitive polymer of PEG grafted carboxymethyl chitosan (PEG-CMCS) and developed anionic-charged hybrid nanoparticles of PEG-CMCS and calcium phosphate (CaP) for siRNA delivery through a single-step self-assembly method in aqueous condition. The formed nanoparticles with charge of around −8.25 mv and average diameter of 102.1 nm exhibited efficient siRNA encapsulation and enhanced colloidal and serum stability. The test in vitro indicated that the nanoparticles entered into HepG2 cells by endocytosis, and achieved endosomal escape of siRNA effectively due to the pH-responsive disassembly of nanoparticles and dissolution of CaP in the endosome. Reporter gene silencing assay showed that luciferase siRNA delivered by the anionic nanoparticles could achieve gene silencing efficacy comparable to that of conventional Lipofectamine 2000. Additionally, dramatic hTERT knockdown mediated by the anionic nanoparticles transfection induced significant apoptosis of HepG2 cells in vitro. After intravenous injection in tumor-bearing BALB/c nude mice, the nanoparticles specifically accumulated into tumor regions by EPR effect, leading to efficient and specific gene silencing sequentially. Most importantly, the nanoparticles carrying hTERT siRNA inhibited tumor growth significantly via silencing hTERT expression and inducing cells apoptosis in HepG2 tumor xenograft. Moreover, comprehensive safety studies of the nanoparticles confirmed their superior safety both in vitro and in vivo. We concluded that the PEG-CMCS/CaP hybrid anionic nanoparticles possessed potential as a safe and effective siRNA delivery system for anticancer therapy.     

Inhibition of blood vessel formation in tumors by IL‐18‐polarized M1 macrophages

We previously showed that interleukin (IL)‐18 produced by NFSA cells induced the M1 type of macrophages in NFSA tumors, caused the destruction of endothelial cells in vitro and may have resulted in the necrosis of NFSA tumors by enhancing macrophage phagocytosis and cytotoxicity. However, the effect of IL‐18 on blood vessel formation in vivo has not been elucidated. MS‐K cells do not express il‐18, and they form tumors with well‐developed blood vessels. Here, we established IL‐18‐over‐expressing MS‐K cell clones (MS‐K‐IL‐18) to address the roles of IL‐18 in angiogenesis. The over‐expression of IL‐18 inhibited the proliferation rate of the MS‐K‐IL‐18 cells in vitro and blood vessel formation in the MS‐K‐IL‐18 tumors. Interestingly, CD14‐positive cells from the MS‐K‐IL‐18 tumor had up‐regulated expression of the M1‐type macrophage marker il‐6 and down‐regulated expression of interferon (ifn)‐γ. Furthermore, FACS analysis showed more accumulation of CD11b+/CD80+ M1 macrophages in the MS‐K‐IL‐18 tumors than in the parental MS‐K tumor. Moreover, an in vitro coculture assay showed that MS‐K‐IL‐18‐conditioned medium (CM) stimulated macrophages to induce the apoptosis of endothelial cells. Cumulatively, our data showed that IL‐18 inhibited tumor blood vessel formation in vivo.     

In vitro Monocyte IL‐6 Secretion Levels Following Stimulation with Autologous Spheroids Derived from Tumour or Benign Mucosa Predict Long‐term Survival in Head and Neck Squamous Cell Carcinoma Patients

MCP‐1/IL‐6 in vitro monocyte secretion upon coculture with autologous fragment spheroids was studied in relation to patient 5‐ and 10‐year overall survival rates in head and neck squamous cell carcinoma (HNSCC) patients (n = 65) diagnosed between 1998 and 2005, nine of whom had an human papilloma virus (HPV) tumour infection. The spheroids were harvested from malignant or benign tissue during primary surgery. Two weeks following surgery, freshly isolated autologous monocytes and benign or malignant spheroids were cocultured 24 h in vitro. The IL‐6 secretion was expressed as a fraction of the lipopolysaccharide (LPS) response from the same batch of monocytes. HPV status was obtained by employing PCR analyses of primary diagnostic blocks. IL‐6/MCP‐1 response levels were not found to be dependent on HPV infection status. MCP‐1 secretion did not predict prognosis, nor did in vitro IL‐6 monocyte background or LPS‐stimulated IL‐6 secretion. At 5‐year observation, dichotomized IL‐6 levels following monocyte coculture, with both malignant and benign spheroids, showed a strong trend towards predicting survival, that is a low monocyte malignant coculture response showed a survival of 31 ± 17 versus 58 ± 17% with a high such response (P = 0.057). When studying monocyte IL‐6 coculture responses evaluating benign and malignant spheroid results statistically together, a prediction of survival up to 10 years was found (hazard ratio = 0.48; confidence interval = 0.24–0.96; P < 0.05) with double low IL‐6 responses. This survival prediction was also present after an adjustment for HPV tumour infection status. In conclusion, monocyte IL‐6 in vitro secretion in cocultures with autologous spheroids/serum from HNSCCs predicted 5‐ and 10‐year survivals, both with and without tumour HPV tumour adjustment.     

Multi-parametric hydrogels support 3D in vitro bioengineered microenvironment models of tumour angiogenesis

Tumour microenvironment greatly influences the development and metastasis of cancer progression. The development of three dimensional (3D) culture models which mimic that displayed in vivo can improve cancer biology studies and accelerate novel anticancer drug screening. Inspired by a systems biology approach, we have formed 3D in vitro bioengineered tumour angiogenesis microenvironments within a glycosaminoglycan-based hydrogel culture system. This microenvironment model can routinely recreate breast and prostate tumour vascularisation. The multiple cell types cultured within this model were less sensitive to chemotherapy when compared with two dimensional (2D) cultures, and displayed comparative tumour regression to that displayed in vivo. These features highlight the use of our in vitro culture model as a complementary testing platform in conjunction with animal models, addressing key reduction and replacement goals of the future. We anticipate that this biomimetic model will provide a platform for the in-depth analysis of cancer development and the discovery of novel therapeutic targets.     

Inhibition of Interferon Regulatory Factor 4 Suppresses Th1 and Th17 Cell Differentiation and Ameliorates Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease that is characterized by recurrent episodes of T‐cell‐mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability. Interferon regulatory factor 4 (IRF4) is expressed predominantly in the immune system and plays an important role in its development and function. Recent study demonstrated that IRF4 was critical for the generation of IL‐17‐producing Th17 cells. However, the effect of IRF4 on experimental autoimmune encephalomyelitis (EAE), an animal model of MS, needs to be further investigated. In our current study, inhibition of IRF4 with IRF4 siRNA (SiIRF4) decreases EAE scores and infiltration of Th1 and Th17 cells, but increases Treg infiltration. SiIRF4 inhibits Th1 and Th17 cell differentiation in vivo and in vitro. In our DC‐T‐cell coculture system, SiIRF4‐treated DCs resulted in significantly less IFN‐γ and IL‐17 production from T cells. Next, we adoptively transfer CD11c⁺ DCs from SiIRF4‐treated mice into recipient mice and found that these CD11c⁺ DCs ameliorated EAE. Furthermore, CD11c⁺ DCs from SiIRF4‐treated naive mice exhibited significantly reduced expression of pro‐inflammatory cytokines TNF‐α, IL‐1β, IL‐6 and IL‐12/IL‐23 (p40), and a corresponding increase in anti‐inflammatory IL‐10 expression. In conclusion, inhibition of IRF4 suppresses Th1 and Th17 cell differentiation and ameliorates EAE, via a direct regulation of DCs.     

Circulating Dendritic Cells,Farm Exposure and Asthma at Early Age

Farm environment has been shown to protect from childhood asthma. Underlying immunological mechanisms are not clear yet, including the role of dendritic cells (DCs). The aim was to explore whether asthma and farm exposures are associated with the proportions and functional properties of DCs from 4.5‐year‐old children in a subgroup of the Finnish PASTURE birth cohort study. Myeloid DCs (mDCs), plasmacytoid DCs (pDCs) and CD86 expression on mDCs ex vivo (n = 100) identified from peripheral blood mononuclear cells (PBMCs) were analysed using flow cytometry. MDCs and production of interleukin (IL)‐6 and tumour necrosis factor alpha (TNF‐α) by mDCs were analysed after 5 h in vitro stimulation with lipopolysaccharide (LPS) (n = 88). Prenatal and current farm exposures (farming, stables, hay barn and farm milk) were assessed from questionnaires. Asthma at age 6 years was defined as a doctor′s diagnosis and symptoms; atopic sensitization was defined by antigen‐specific IgE measurements. Asthma was positively associated with CD86 expression on mDCs ex vivo [adjusted odds ratio (aOR) 4.83, 95% confidence interval (CI) 1.51–15.4] and inversely with IL‐6 production in mDCs after in vitro stimulation with LPS (aOR 0.19, 95% CI 0.04–0.82). In vitro stimulation with LPS resulted in lower percentage of mDCs in the farm PBMC cultures as compared to non‐farm PBMC cultures. Our results suggest an association between childhood asthma and functional properties of DCs. Farm exposure may have immunomodulatory effects by decreasing mDC proportions.     

miR‐1, regulated by LMP1, suppresses tumour growth and metastasis by targeting K‐ras in nasopharyngeal carcinoma

There is evidence to show that downregulation of miR‐1 expression is closely related to cancer progression, including in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms underlying miR‐1 downregulation in NPC remain largely unknown, especially its association with Epstein–Barr virus (EBV). In this study we found that restoration of miR‐1 dramatically inhibited cell invasion in vitro, together with tumour growth and metastasis in vivo. Importantly, we found that LMP1, an Epstein–Barr virus (EBV)‐associated protein, suppressed miR‐1 expression. Furthermore, we identified K‐ras as a novel direct target of miR‐1. Our results demonstrated for the first time that miR‐1 was suppressed by LMP1 and its tumour‐suppressive effects were mediated chiefly by repressing K‐ras expression. We propose that miR‐1 could serve as an independent biomarker to identify patients with different clinical characteristics.     

Cytokine gene polymorphisms and breast cancer susceptibility and prognosis*

Single nucleotide polymorphisms (SNPs) in the promoter regions of cytokine genes are associated with differential levels of cytokine expression. We hypothesized that these SNPs might influence breast tumour development and progression by affecting the efficiency of the antitumour immune response and/or pathways of angiogenesis. A total of 144 female breast cancer patients and 263 cancer‐free population controls were genotyped for the interleukin (IL)‐1β?511 (T/C), IL‐6 ?174 (G/C), tumour necrosis factor (TNF)‐α?308 (A/G), IL‐10 ?1082 (A/G), IL‐8 ?251 (A/T) and vascular endothelial growth factor (VEGF) ?1154 (A/G) SNPs, using amplification refractory mutation system polymerase chain reaction (ARMS‐PCR) and TaqMan® (Applied Biosystems, Foster City, CA, USA) 5′ nuclease assays for allelic discrimination. No significant associations were seen. Patient–control comparisons revealed a non‐significant trend for association between the TNF‐α?308 GG genotype and breast cancer compared to controls (79.7 vs. 68.2%, P = 0.03, P_c = 0.54). Stratification of the patient group according to the Nottingham Prognostic Index and individual prognostic factors revealed trends for association between IL‐6 ?174 GC and IL‐8 ?251 AA genotypes and markers of poor prognosis (P = 0.04, P_c = 0.72 and P = 0.02, P_c = 0.36, respectively). There were also trends for associations between VEGF ?1154 AG and IL‐1β?511 TC genotypes and markers of good prognosis (P = 0.02, P_c = 0.36 and P = 0.05, P_c = 0.90, respectively). These results suggest that the role of cytokine promoter SNPs in both susceptibility to and prognosis in breast cancer requires further investigation in a larger study.     

Transient activation of mucosal effector immune responses by resident intestinal bacteria in normal hosts is regulated by interleukin‐10 signalling

Interleukin‐10 (IL‐10) is a key regulator of mucosal homeostasis. In the current study we investigated the early events after monoassociating germ‐free (GF) wild‐type (WT) mice with an Escherichia coli strain that we isolated previously from the caecal contents of a normal mouse housed under specific pathogen‐free conditions. Our results show that interferon‐γ (IFN‐γ) secreted by mesenteric lymph node (MLN) cells from both IL‐10 deficient mice and WT mice, stimulated ex vivo with E. coli lysate, was dramatically higher at day 4 after monoassociation compared with IFN‐γ secreted by cells from GF mice without E. coli colonization. Production of IFN‐γ rapidly and progressively declined after colonization of WT but not IL‐10‐deficient mice. The E. coli lysate‐stimulated WT MLN cells also produced IL‐10 that peaked at day 4 and subsequently declined, but not as precipitously as IFN‐γ. WT cells that express CD4, CD8 and NKp46 produced IFN‐γ; WT CD4‐positive cells and B cells produced IL‐10. Recombinant IL‐10 added to E. coli‐stimulated MLN cell cultures inhibited IFN‐γ secretion in a dose‐dependent fashion. MLN cells from WT mice treated in vivo with neutralizing anti‐IL‐10 receptor antibody produced more IFN‐γ compared with MLN cells from isotype control antibody‐treated mice. These findings show that a resident E. coli that induces chronic colitis in monoassociated IL‐10‐deficient mice rapidly but transiently activates the effector immune system in normal hosts, in parallel with induction of protective IL‐10 produced by B cells and CD4⁺ cells that subsequently suppresses this response to mediate mucosal homeostasis.     

Syndecan‐1 (CD138) contributes to prostate cancer progression by stabilizing tumour‐initiating cells

Increasing evidence suggests that tumour‐initiating cells (TICs) contribute to the development of prostate cancer. Here, we identified syndecan‐1 as a key molecule maintaining the stability of prostate cancer TICs. Holoclones harbouring the biological properties of stemness were derived from single‐cell cultures of the PC3 human prostate cancer cell line. These holoclones over‐expressed syndecan‐1, but showed reduced expression of NADPH oxidase (NOX) and synthesis of hydrogen peroxide and oxygen radicals. Stable RNA‐mediated silencing of syndecan‐1 gene expression up‐regulated NOX‐dependent generation of reactive oxygen species and reduced the survival of holoclones in vitro. Syndecan‐1 down‐regulation also strongly reduced the number of CD133⁺/CD44⁺ primitive cancer cells and tumour growth in vivo. Interestingly, syndecan‐1 gene knockdown significantly enhanced the tumour‐suppressive effects of docetaxel by inhibiting the docetaxel‐induced increase in CD133⁺/CD44⁺ cells in vivo. In the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model of prostate cancer, early intervention with a syndecan‐1 inhibitor (OGT2115) or syndecan‐1 RNAi reduced the incidence of adenocarcinoma and the number of c‐kit⁺/CD44⁺ cells in cancer foci. Finally, we found that syndecan‐1 immunopositivity in prostate cancer cells was significantly associated with biochemical recurrence after radical prostatectomy. Taken together, our results show that syndecan‐1 contributes to prostatic carcinogenesis by maintaining TICs and may be a target molecule for therapy. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The Inflammation Induced by Lipopolysaccharide can be Mitigated by Short‐chain Fatty Acid,Butyrate, through Upregulation of IL‐10 in Septic Shock

Short‐chain fatty acids (SCFAs) with the anti‐inflammatory capacity are produced by intestinal bacteria; however, their effect on the acute systematical inflammation remains unclear. This study aimed to investigate the effects of SCFAs, acetate, propionate and butyrate, on septic shock and the underlying mechanism. The LPS‐induced septic model was used to evaluate the function of SCFAs by survival rate observation. Only butyrate, but not acetate or propionate, significantly decrease the mortality of septic mice. At 2 h and 6 h of LPS administration, the levels of TNF‐α, IL‐6 and IL‐1β in plasma were measured by ELISA to estimate the effects of butyrate pretreatment on excessive inflammation. And the anti‐inflammatory mediators including TGF‐β, IL‐10 and LXT4 in plasma were detected for further mechanism study in septic mice. Moreover, the murine macrophage‐like RAW 264.7 cells were stimulated by LPS to further confirm the finding in vivo. Pretreatment with butyrate led to significant attenuation of the LPS‐induced elevation of TNF‐α, IL‐6 and IL‐1β levels. However, when detecting the anti‐inflammatory factors, a significant increase in IL‐10, but not TGF‐β or LXT4, was shown in butyrate‐pretreated group. Pretreatment of RAW 264.7 cells with butyrate led to downregulation of LPS‐induced pro‐inflammatory mediators, IL‐6 and IL‐1β, but did not affect the level of TNF‐α, and increased IL‐10 (P < 0.01). In conclusion, SCFA butyrate significantly attenuated the inflammation against sepsis through upregulation of anti‐inflammatory IL‐10.     

Nanovaccine loaded with poly I:C and STAT3 siRNA robustly elicits anti-tumor immune responses through modulating tumor-associated dendritic cells in vivo

Although cancer vaccine-based immunotherapy holds great potential for cancer treatment, tumor-induced dendritic cell (DC) dysfunction remains to be the major obstacle for developing effective vaccines. Compared with normal DCs, tumor-associated DCs (TADCs) are less matured with poor responsiveness to Toll-like receptor (TLR) stimulation, which has been related with STAT3 hyperactivity. In the present study, Poly I:C (PIC, a TLR3 agonist), STAT3 siRNA and OVA antigen were co-encapsulated by poly (ethylene glycol)-b-poly (l-lysine)-b-poly (l-leucine) (PEG-PLL-PLLeu) polypeptide micelles to generate PMP/OVA/siRNA nanovaccine, which was aimed to effectively overcome DC dysfunction in vivo by deleting STAT3 gene in situ. The results showed that PMP/OVA/siRNA simultaneously facilitated the cellular uptake of OVA antigen and siRNA about 3–200 folds, and decreased STAT3 expression in TADCs over 50% both in vitro and in vivo. PMP/OVA/siRNA also elevated CD86 and CD40 expression as well as IL-12 production by TADCs more effectively than PMP/OVA did, indicating its strong potency of inducing TADC maturation and activation. Moreover, the immunization of PMP/OVA/siRNA rather than PMP/OVA effectively abrogated immunosuppression in the tumor microenvironment by increasing mature DCs and decreasing immunosuppressive cells in tumor-draining lymph nodes, which thereby led to potent anti-tumor immune responses and dramatic tumor regression with prolonged survival. Hence, in vivo co-delivery of immunopotentiator (PIC) and immunosuppressive gene silencer (STAT3 siRNA) by nanovaccines are expected to be a promising strategy to improve the therapeutic efficacy of cancer vaccines by modulating TADCs and overcoming tumor immunosupression.     

Genetically modified human CD4+ T cells can be evaluated in vivo without lethal graft‐versus‐host disease

Adoptive cell immunotherapy for human diseases, including the use of T cells modified to express an anti‐tumour T‐cell receptor (TCR) or chimeric antigen receptor, is showing promise as an effective treatment modality. Further advances would be accelerated by the availability of a mouse model that would permit human T‐cell engineering protocols and proposed genetic modifications to be evaluated in vivo. NOD‐scid IL2rγ^null (NSG) mice accept the engraftment of mature human T cells; however, long‐term evaluation of transferred cells has been hampered by the xenogeneic graft‐versus‐host disease (GVHD) that occurs soon after cell transfer. We modified human primary CD4⁺ T cells by lentiviral transduction to express a human TCR that recognizes a pancreatic beta cell‐derived peptide in the context of HLA‐DR4. The TCR‐transduced cells were transferred to NSG mice engineered to express HLA‐DR4 and to be deficient for murine class II MHC molecules. CD4⁺ T‐cell‐depleted peripheral blood mononuclear cells were also transferred to facilitate engraftment. The transduced cells exhibited long‐term survival (up to 3 months post‐transfer) and lethal GVHD was not observed. This favourable outcome was dependent upon the pre‐transfer T‐cell transduction and culture conditions, which influenced both the kinetics of engraftment and the development of GVHD. This approach should now permit human T‐cell transduction protocols and genetic modifications to be evaluated in vivo, and it should also facilitate the development of human disease models that incorporate human T cells.     

Immunomodulatory glc/man‐directed Dolichos lablab lectin (DLL) evokes anti‐tumour response in vivo by counteracting angiogenic gene expressions

Neovascularization and jeopardized immunity has been critically emphasized for the establishment of malignant progression. Lectins are the diverse class of carbohydrate interacting proteins, having great potential as immunopotentiating and anti‐cancer agents. The present investigation sought to demonstrate the anti‐proliferative activity of Dolichos lablab lectin (DLL) encompassing immunomodulatory attributes. DLL specific to glucose and mannose carbohydrate moieties has been purified to homogeneity from the common dietary legume D. lablab. Results elucidated that DLL agglutinated blood cells non‐specifically and displayed striking mitogenicity to human and murine lymphocytes in vitro with interleukin (IL)‐2 production. The DLL‐conditioned medium exerted cytotoxicity towards malignant cells and neoangiogenesis in vitro. Similarly, in‐vivo anti‐tumour investigation of DLL elucidated the regressed proliferation of ascitic and solid tumour cells, which was paralleled with blockade of tumour neovasculature. DLL‐treated mice showed an up‐regulated immunoregulatory cytokine IL‐2 in contrast to severely declined levels in control mice. Mechanistic validation revealed that DLL has abrogated the microvessel formation by weakening the proangiogenic signals, specifically nuclear factor kappa B (NF‐κB), hypoxia inducible factor 1α (HIF‐1 α), matrix metalloproteinase (MMP)‐2 and 9 and vascular endothelial growth factor (VEGF) in malignant cells leading to tumour regression. In summary, it is evident that the dietary lectin DLL potentially dampens the malignant establishment by mitigating neoangiogenesis and immune shutdown. For the first time, to our knowledge, this study illustrates the critical role of DLL as an immunostimulatory and anti‐angiogenic molecule in cancer therapeutics.     

Increased Levels of Cytokines in Cerebrospinal Fluid of Children with Aseptic Meningitis Caused by Mumps Virus and Echovirus 30

We measured levels of pro‐inflammatory cytokines in the cerebrospinal fluid (CSF) of patients with mumps meningitis, enteroviral echovirus 30 meningitis and children without central nervous system infection to investigate whether these molecules were involved in the pathogenesis of viral meningitis. The CSF was obtained from 62 children suspected with meningitis. These patients were classified to the mumps meningitis (n = 19), echovirus 30 meningitis (n = 22) and non‐meningitis (n = 21) groups. The concentrations of interleukin‐1 (IL‐1), interleukin‐1 soluble receptor type 2 (IL‐1R2), interleukin‐8 (IL‐8), human interferon gamma (IFN‐γ) and human tumour necrosis factor alpha (TNF‐α) were determined by immunoassay. A significant increase was noted in the levels of IL‐8, TNF‐α and IL‐1R2 in the CSF of both meningitis groups as compared to controls. The concentrations of IFN‐γ and IL‐1 differed significantly only between the mumps group and control. The levels of IL‐1, IFN‐γ and TNF‐α were significantly higher in mumps meningitis when compared to the echovirus 30 group. Of all cytokines examined, only IFN‐γ correlated with pleocytosis (r = 0.58) in the mumps meningitis group. The increased CSF cytokine levels are markers of meningeal inflammation, and each virus may cause a specific profile of the cytokine pattern.     

IL‐7 is superior to IL‐2 for ex vivo expansion of tumour‐specific CD4+ T cells

It is well established that tumours hinder both natural and vaccine‐induced tumour‐specific CD4⁺ T‐cell responses. Adoptive T‐cell therapy has the potential to circumvent functional tolerance and enhance anti‐tumour protective responses. While protocols suitable for the expansion of cytotoxic CD8⁺ T cells are currently available, data on tumour‐specific CD4⁺ T cells remain scarce. We report here that CD4⁺ T cells sensitized to tumour‐associated Ag in vivo, proliferate in vitro in response to IL‐7 without the need for exogenous Ag stimulation and accumulate several folds while preserving a memory‐like phenotype. Both cell proliferation and survival accounts for the outgrowth of tumour‐sensitized T cells among other memory and naive lymphocytes following exposure to IL‐7. Also IL‐2, previously used to expand anti‐tumour CTL, promotes tumour‐specific CD4⁺ T‐cell accumulation. However, IL‐7 is superior to IL‐2 at preserving lymphocyte viability, in vitro and in vivo, maintaining those properties, that are required by helper CD4⁺ T cells to confer therapeutic efficacy upon transplantation in tumour‐bearing hosts. Together our data support a unique role for IL‐7 in retrieving memory‐like CD4⁺ T cells suitable for adoptive T‐cell therapy.