In vivo antitumor effects of murine interferon- γ -inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites

Interferon-γ-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 μg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon γ. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4⁺ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4⁺ cells. Received: 17 September 1996 / Accepted: 8 November 1996     

Targeting HER2

The potential of the HER2-targeting antibody trastuzumab as a radioimmunoconjugate useful for both imaging and therapy was investigated. Conjugation of trastuzumab with the acyclic bifunctional chelator CHX-A”-DTPA yielded a chelate:protein ratio of 3.4±0.3; the immunoreactivity of the antibody unaffected. Radiolabeling was efficient, routinely yielding a product with high specific activity. Tumor targeting was evaluated in mice bearing subcutaneous (s.c.) xenografts of colorectal, pancreatic, ovarian, and prostate carcinomas. High uptake of the radioimmunoconjugate, injected intravenously (i.v.), was observed in each of the models, and the highest tumor %ID/g (51.18±13.58) was obtained with the ovarian (SKOV-3) tumor xenograft. Specificity was demonstrated by the absence of uptake of ¹¹¹In-trastuzumab by melanoma (A375) s.c. xenografts and ¹¹¹In-HuIgG by s.c. LS-174T xenografts. Minimal uptake of i.v. injected ¹¹¹In-trastuzumab in normal organs was confirmed in non-tumor-bearing mice. The in vivo behavior of ¹¹¹In-trastuzumab in mice bearing intraperitoneal (i.p.) LS-174T tumors resulted in a tumor %ID/g of 130.85±273.34 at 24 h. Visualization of tumor, s.c. and i.p. xenografts, was achieved by γ-scintigraphy and PET imaging. Blood pool was evident as expected, but cleared over time. The blood pharmacokinetics of i.v. and i.p. injected ¹¹¹In-trastuzumab was determined in mice with and without tumors. The data from these in vitro and in vivo studies supported advancement of radiolabeled trastuzumab into two clinical studies, a Phase 0 imaging study in the Molecular Imaging Program of the National Cancer Institute and a Phase 1 radioimmunotherapy study at the University of Alabama.     

Targeting HER2: A report on the in vitro and in vivo pre-clinical data supporting trastuzumab as a radioimmunoconjugate for clinical trials

The potential of the HER2-targeting antibody trastuzumab as a radioimmunoconjugate useful for both imaging and therapy was investigated. Conjugation of trastuzumab with the acyclic bifunctional chelator CHX-A″-DTPA yielded a chelate:protein ratio of 3.4 ± 0.3; the immunoreactivity of the antibody unaffected. Radiolabeling was efficient, routinely yielding a product with high specific activity. Tumor targeting was evaluated in mice bearing subcutaneous (s.c.) xenografts of colorectal, pancreatic, ovarian and prostate carcinomas. High uptake of the radioimmunoconjugate, injected intravenously (i.v.), was observed in each of the models and the highest tumor %ID/g (51.18 ± 13.58) was obtained with the ovarian (SKOV-3) tumor xenograft. Specificity was demonstrated by the absence of uptake of ¹¹¹In-trastuzumab by melanoma (A375) s.c. xenografts and ¹¹¹In-HuIgG by s.c. LS-174T xenografts. Minimal uptake of i.v. injected ¹¹¹In-trastuzumab in normal organs was confirmed in non-tumor-bearing mice. The in vivo behavior of ¹¹¹In-trastuzumab in mice bearing intraperitoneal (i.p.) LS-174T tumors resulted in a tumor %ID/g of 130.85 ± 273.34 at 24 h. Visualization of tumor, s.c. and i.p. xenografts was achieved by γ-scintigraphy and PET imaging. Blood pool was evident as expected but cleared over time. The blood pharmacokinetics of i.v. and i.p. injected ¹¹¹In-trastuzumab was determined in mice with and without tumors. The data from these in vitro and in vivo studies supported advancement of radiolabeled trastuzumab into two clinical studies, a Phase 0 imaging study in the Molecular Imaging Program of the National Cancer Institute and a Phase 1 radioimmunotherapy study at the University of Alabama.Key words: monoclonal antibody, HER2, trastuzumab, radioimmunodiagnosis, radioimmunotherapy     

Superparamagnetic Iron Oxide Nanoparticles Function as a Long-Term,Multi-Modal Imaging Label for Non-Invasive Tracking of Implanted Progenitor Cells

The purpose of this study was to determine the ability of superparamagnetic iron oxide (SPIO) nanoparticles to function as a long-term tracking label for multi-modal imaging of implanted engineered tissues containing muscle-derived progenitor cells using magnetic resonance imaging (MRI) and X-ray micro-computed tomography (μCT). SPIO-labeled primary myoblasts were embedded in fibrin sealant and imaged to obtain intensity data by MRI or radio-opacity information by μCT. Each imaging modality displayed a detection gradient that matched increasing SPIO concentrations. Labeled cells were then incorporated in fibrin sealant, injected into the atrioventricular groove of rat hearts, and imaged in vivo and ex vivo for up to 1 year. Transplanted cells were identified in intact animals and isolated hearts using both imaging modalities. MRI was better able to detect minuscule amounts of SPIO nanoparticles, while μCT more precisely identified the location of heavily-labeled cells. Histological analyses confirmed that iron oxide particles were confined to viable, skeletal muscle-derived cells in the implant at the expected location based on MRI and μCT. These analyses showed no evidence of phagocytosis of labeled cells by macrophages or release of nanoparticles from transplanted cells. In conclusion, we established that SPIO nanoparticles function as a sensitive and specific long-term label for MRI and μCT, respectively. Our findings will enable investigators interested in regenerative therapies to non-invasively and serially acquire complementary, high-resolution images of transplanted cells for one year using a single label.     

Pharmacokinetics and in vitro and in vivo anti-tumor response of an interleukin-2-human serum albumin fusion protein in mice

Purpose: Albuleukin fusion protein is a recombinant human interleukin-2 (rIL-2) genetically fused to recombinant human serum albumin (rHSA). The pharmacokinetics and pharmacologic activity of Albuleukin were examined in mice to determine whether the fusion protein had the immunomodulatory and anti-tumor properties of rIL-2 as well as a prolonged serum half-life due to the rHSA. Methods: The effect of Albuleukin on lymphocyte proliferation, IL-2 receptor binding, and release of IFN- from human NK cells were examined in vitro. For the pharmacokinetic analysis, Albuleukin and rIL-2 were administered intravenously (i.v.) and subcutaneously (s.c.) to BALB/c mice, both at a single dose of 500 g/kg. The anti-tumor properties of Albuleukin were evaluated in a Renca tumor model in BALB/c mice and in a metastatic liver model of B16F10 melanoma in C57B1/6 mice. In the Renca tumor model, BALB/c mice were dosed intraperitoneally (i.p.) and s.c. with Albuleukin on days 12, 14, 16, 19, 21, and 23 and i.p. with rIL-2 daily for two periods of 5 days (days 10–14 and 17–21). In the B16 melanoma model, C57B1/6 mice were dosed s.c. with rIL-2 twice daily or Albuleukin every 48 h for 14 days. Results: In vitro, Albuleukin induced the proliferation of primary human and mouse T cells and B cells and primary human NK cells, competed with rIL-2 for binding to the IL-2 receptors, and induced the production of IFN- from primary human NK cells. The s.c. bioavailability of Albuleukin was about 45% relative to the i.v. dose. Plasma half-life was prolonged and ranged from 6 to 8 h with Albuleukin, compared to 19–57 min with rIL-2. Total clearance of Albuleukin was about 50-fold slower than that of rIL-2 after i.v. dosing. In vivo, Albuleukin suppressed the growth of Renca tumors and induced a dense infiltration of CD4⁺ and CD8⁺ T cells. Both Albuleukin and rIL-2 significantly reduced the tumor burden in mice with hepatic B16F10 metastases. Albuleukin significantly reduced the incidence of residual macroscopic hepatic tumors, resulting in improved survival relative to controls and rIL-2. Conclusion: Results from these studies suggest that the therapeutic efficacy of rIL-2 is improved in mice by prolonging its in vivo half-life through genetic fusion to albumin. Albuleukin, the fusion protein, had pronounced anti-tumor effects in Renca and hepatic melanoma tumor models without an increase in mortality. On the basis of its preclinical effects, Albuleukin was brought to the clinic to assess its therapeutic benefit in a variety of cancers.     

MRI Detection of Nonproliferative Tumor Cells in Lymph Node Metastases Using Iron Oxide Particles in a Mouse Model of Breast Cancer

Cell tracking with magnetic resonance imaging (MRI) and iron nanoparticles is commonly used to monitor the fate of implanted cells in preclinical disease models. Few studies have employed these methods to study cancer cells because proliferative iron-labeled cancer cells will lose the label as they divide. In this study, we evaluate the potential for retention of the iron nanoparticle label, and resulting MRI signal, to serve as a marker for slowly dividing cancer cells. Green fluorescent protein-transfected MDA-MB-231 breast cancer cells were labeled with red fluorescent micron-sized superparamagnetic iron oxide (MPIO) nanoparticles. Cells were examined in vitro at multiple time points after labeling by staining for iron-labeled cells and by flow cytometric detection of the fluorescent MPIO. Severe combined immune deficiency (SCID) mice were implanted with 5 x 10⁵ MPIO-labeled or unlabeled cells in the mammary fat pad and MRI was performed weekly until 28 days after injection. Microscopy was performed to validate MRI. In vitro assays revealed a very small percentage of cells that retained MPIO at 14 days after labeling. Regions of signal loss were observed in MRI of primary tumors that developed from iron-labeled cancer cells. Small focal regions of signal loss were detected in images of the axillary and brachial nodes in six of eight mice, at day 14 or later, with microscopy confirming the presence of iron-labeled cancer cells. Our data suggest an interesting role for cell tracking with iron particles since label retention leads to persistent signal void, allowing proliferative status to be determined.     

Cell Surface Expression of I-A Products Is Required for Contact Sensitivity Induction by Trinitrophenyl-Coupled Epidermal Cells

Trinitrophenyl (TNP)-coupled epidermal cells (EC) injected subcutaneously (s.c.) were more capable of inducing contact sensitivity (CS) to 2, 4, 6-trinitro-1-chlorobenzene (TNCB) than similarly substituted spleen cells (TNP-SC). Furthermore, the intravenous (i.v.) or intraperitoneal (i.p.) injection of TNP-EC also induced CS responses, whereas the i.v. or i.p. injection of TNP-SC failed to induce them. Treatment of mice with cyclophosphamide (Cy; 50 mg/kg) or anti I-J serum allowed animals injected with TNP-SC i.v. to develop significant CS responses, suggesting that Cy-sensitive and I-J positive regulatory cells were involved in the induction of unresponsiveness by the i.v. injection of TNP-SC. Mapping studies of the major histocompatibility gene complex (MHC) region demonstrated that identity at the I-A subregion alone between EC donor and recipient mice was sufficient for the induction of CS by TNP-EC given i.v. Blocking experiments using antisera in the absence of complement indicated that I-A subregion-encoded antigens on the surface of TNP-EC apparently are involved in the induction of CS, and are not simply phenotypic markers on the surface of accessory cells.     

A calcium channel blocker flunarizine attenuates the neurotoxic effects of iron

Iron is a metal highly concentrated in liver and brain tissue, and known to induce neuronal hyperactivity and oxidative stress. It has been established that iron levels rise in the brain in some neurodegenerative diseases such as Parkinson's and Alzheimer's diseases (AD). A body of evidence indicates a link between neuronal death and intracellular excessive calcium accumulation. The aim of the present study was to investigate the effects of a calcium antagonist, flunarizine, on neurotoxicity induced by intracerebroventricular (i.c.v.) iron injection. For this reason rats were divided into three groups as control, iron and iron+flunarizine groups. Animals in iron and iron+flunarizine groups received i.c.v. FeCl₃ injection (200 mM, 2.5 μl), while control rats received the same amount of saline into the cerebral ventricles. Rats in iron+flunarizine group also received i.c.v. flunarizine (1 μM, 2 μl) following FeCl₃ injection. All animals were kept alive for ten days following the operation and animals in iron+flunarizine group received intraperitoneal (i.p.) flunarizine injections once a day (10 mg/kg/day) during this period. After ten days, rats were sacrificed. The total numbers of neurons in hippocampus of all rats were estimated with the latest, unbiased stereological techniques. Findings of the present study suggest that flunarizine may attenuate the neurotoxic effects of iron injection by inhibiting the cellular influx of excessive calcium ions.     

铁过负荷对大鼠眼、泪腺中铁血黄素的沉积及转铁蛋白表达的影响

通过饲喂高铁饲料、腹腔注射或静脉注射多糖铁复合物的方法复制SD(Sprague Dawley)大鼠(Rattus norregicus)的铁过负荷模型。取动物的眼和泪腺组织染色显微观察发现,静脉注射多糖铁复合物大鼠眼巩膜、虹膜、睫状体、脉络膜间质细胞及泪腺小叶间结缔组织中巨噬细胞铁血黄素沉积明显;而腹腔注射多糖铁复合物或饲喂高铁饲料大鼠的眼和泪腺中铁血黄素沉积较少或未见沉积。不同方法造成的铁过负荷大鼠眼和泪腺组织中均未见转铁蛋白的表达。结果表明,铁血黄素在眼及泪腺的沉着很可能是眼晶状体混浊和眼底血管样条纹产生的主要原因。     

SP/NK1R system regulates carcinogenesis in prostate cancer: Shedding light on the antitumoral function of aprepitant

AimsProstate cancer continues to be one of the main global health issues in men. Neuropeptide substance P (SP) acting via neurokinin-1receptor (NK1R) promotes tumorigenicity in many human malignant tumors. However, its pro-tumorigenic functions and the therapeutic effects of its inhibition in prostate cancer remain unclear.MethodsMTT assay was employed for measuring cellular proliferation and cytotoxicity. mRNAs and proteins expression levels were evaluated by qRT-PCR and western blot assay, respectively. Gelatinase activity was assessed by zymography. The migration ability was defined using wound-healing assay. Flow cytometry was employed to evaluate the cell cycle distribution. We also performed an in vivo experiment in a mouse model of prostate cancer to confirm the in vitro therapeutic effect of targeting the SP/NK1R system.ResultsWe found a noticeable increase in the expression of the truncated isoform of NK1R as an oncogenic NK1R splice variant in tumor cells. We also demonstrated that SP promotes both proliferative and migrative phenotypes of prostate cancer through modifying cell cycle-related proteins (c-Myc, cyclin D1, cyclin B1, p21), and apoptosis-related genes (Bcl-2 and Bax), promoting cell migration and increasing MMP-2 and MMP-9 expression and activity, while aprepitant administration could remarkably reverse these effects. SP also stimulated tumor growth in vivo, which was correlated with shorter survival times, while aprepitant reversed this effect and led to significantly longer survival time.SignificanceOur findings suggest that SP/NK1R system may serve as a novel therapeutic target in prostate cancer and support the possible candidacy of aprepitant in future prostate cancer therapy.     

Enterostatin (VPDPR) Has Anti-analgesic and Anti-amnesic Activites

Enterostatin (VPDPR), an anorexigenic peptide derived from the amino terminus of procolipase, significantly inhibited analgesia induced by the μ-opioidagonist morphine (5 mg/kg, s.c.) after i.c.v. administration to mice at a dose of 100 nmol. On the other hand, VPDPR (～200 nmol, i.c.v.) did not attenuate analgesia induced by the κ-opioid agonist D-Phe-D-Phe-D-Nle-D-Arg-NH₂ (100 μg/mouse, i.c.v.) or δ-opioid agonist DTLET (4 nmol/mouse, i.c.v.). VPDPR (100 nmol, i.c.v.) significantly improved amnesia induced by scopolamine (0.2 mg/kg, i.p.) in mice. However, VPDPR did not enhance memory in normal mice at the same dose.     

脂连素对小鼠急性肝损伤的保护作用

目的观察脂连素对刀豆蛋白A（ConA）所致小鼠急性肝损伤的干预作用,并对其机制进行初步探讨。方法雄性BALB／C小鼠17只,随机分为3组：ConA组6只：尾静脉注射ConA。脂连素组5只：在静脉注射ConA前腹腔内注射脂连素。对照组6只：尾静脉注射生理盐水。注射ConA后8小时留取血清检测各组ALT和TNF-α水平,肝组织进行HE染色,并检测NF-κB活性及肝细胞凋亡率。结果HE染色可见ConA组肝细胞水肿、变性,肝组织内淋巴细胞浸润、淤血等,脂连素组上述病理变化减轻（P〈0．01）。脂连素组ALT和TNF-α水平、NF-κB活性、肝细胞凋亡率均低于ConA组（P〈0．01或P〈0．05）。结论脂连素具有抗炎作用,其机制可能与减少TNF-α的产生、抑制肝组织NF-κB活性及抗肝细胞凋亡有关。     

Cancer-Specific Inhibitory Effects of Genetically Engineered Stem Cells Expressing Cytosine Deaminase and Interferon-β Against Choriocarcinoma in Xenografted Metastatic Mouse Models

Cancer treatments using stem cells expressing therapeutic genes have been identified for various types of cancers. In this study, we investigated inhibitory effects of HB1.F3.CD and HB1.F3.CD.IFN-β cells expressing Escherichia coli cytosine deaminase (CD) and human interferon-β (IFN-β) genes in intravenously (i.v.) injected mice with a metastasis model. In this treatment, pro-drug 5-fluorocytosine (5-FC) is converted to cytotoxic drug 5-fluorouracil by hNSCs expressing the CD gene, which inhibits DNA synthesis in cancer cells. Moreover, IFN-β induces apoptosis and reduces the growth of cancer cells. Upon MTT assay, proliferation of choriocarcinoma (JEG-3) cells decreased when co-cultured with hNSCs expressing CD and IFN-β genes. To confirm the cancer-tropic effect of these stem cells, chemoattractant factors (VEGF, CXCR4, and C-kit) secreted from JEG-3 cells were identified by polymerase chain reaction. hNSCs migrate toward JEG-3 cells due to ligand-receptor interactions of these factors. Accordingly, the migration capability of hNSCs toward JEG-3 cells was confirmed using an in vitro Trans-well assay, in vivo subcutaneously (s.c.) injected mice groups (xenograft model), and metastasis model. Intravenously injected hNSCs migrated freely to other organs when compared to s.c. injected hNSCs. Thus, we confirmed the inhibition of lung and ovarian metastasis of choriocarcinoma by i.v. injected HB1.F3.CD or HB1.F3.CD.IFN-β cells in the presence of 5-FC. Treatment of these stem cells also increased the survival rates of mice. In conclusion, this study showed that metastatic cancer was diminished by genetically engineered hNSCs and noncytotoxic drug 5-FC. This is the first report of the therapeutic potential of i.v. injected hNSCs in a metastasis model; therefore, the results indicate that this stem cell therapy can be used as an alternative novel tool to treat metastatic choriocarcinoma.     

Attenuation of systemic morphine-induced analgesia by central administration of ghrelin and related peptides in mice

Ghrelin, an acylated 28-amino peptide secreted in the gastric endocrine cells, has been demonstrated to stimulate the release of growth hormone, increase food intake, and inhibit pro-inflammatory cascade, etc. Ghrelin mainly combines with its receptor (GHS-R1α) to play the role in physiological and pathological functions. It has been reported that ghrelin plays important roles in the control of pain through interaction with the opioid system in inflammatory pain and acute pain. However, very few studies show the effect of supraspinal ghrelin system on antinociception induced by intraperitoneal (i.p.) administration of morphine. In the present study, intracerebroventricular (i.c.v.) injection of ghrelin (0.1, 1, 10 and 100 nmol/L) produced inhibition of systemic morphine (6 mg/kg, i.p.) analgesia in the tail withdrawal test. Similarly, i.c.v. injection GHRP-6 and GHRP-2 which are the agonists of GHS-R1α, also decreased analgesia effect induced by morphine injected intraperitoneally in mice. Furthermore, these anti-opioid activities of ghrelin and related peptides were not blocked by pretreatment with the GHS-R1α selective antagonist [d-Lys³]-GHRP-6 (100 nmol/L, i.c.v.). These results demonstrated that central ghrelin and related peptides could inhibit the analgesia effect induced by intraperitoneal (i.p.) administration of morphine. The anti-opioid effects of ghrelin and related peptides do not interact with GHS-R1a. These findings may pave the way for a new strategy on investigating the interaction between ghrelin system and opioids on pain modulation.     

Involvement of potassium channels in the antidepressant-like effect of venlafaxine in mice

AimsStudies have shown that the acute administration of venlafaxine elicits an antidepressant-like effect in the mouse forced swim test (FST) by a mechanism dependent on the l-arginine–nitric oxide (NO)–cyclic guanosine monophosphate (cGMP) pathway. Because it has been reported that NO activates different types of potassium (K⁺) channels in the brain, this study investigated the involvement of K⁺ channels in the antidepressant-like effect of venlafaxine in the mouse FST.Main methodsMale adult Swiss mice were pretreated with different K⁺ channel inhibitors or openers 15 min before venlafaxine administration. After 30 min, the open-field test (OFT) and FST were carried out.Key findingsIntracerebroventricular (i.c.v.) pretreatment of mice with subeffective doses of tetraethylammonium (TEA, a non-specific inhibitor of K⁺ channels, 25 pg/site), glibenclamide (an ATP-sensitive K⁺ channel inhibitor, 0.5 pg/site), charybdotoxin (a large- and intermediate-conductance calcium-activated K⁺ channel inhibitor, 25 pg/site) or apamin (a small-conductance calcium-activated K⁺ channel inhibitor, 10 pg/site) was able to potentiate the action of a subeffective dose of venlafaxine (2 mg/kg, i.p.). Moreover, the reduction in the immobility time elicited by an effective dose of venlafaxine (8 mg/kg, i.p.) in the FST was prevented by the pretreatment of mice with the K⁺ channel openers cromakalim (10 µg/site, i.c.v.) and minoxidil (10 µg/site, i.c.v.). The drugs used in this study did not produce any change in locomotor activity.SignificanceThe results demonstrate that the neuromodulatory effects of venlafaxine, via the inhibition of K⁺ channels, are possibly involved in its anti-immobility activity in the mouse FST.     

Non-invasive monitoring of the kinetic infiltration and therapeutic efficacy of nanoparticle-labeled chimeric antigen receptor T cells in glioblastoma via 7.0-Tesla magnetic resonance imaging

Background aimsChimeric antigen receptor (CAR) T-cell therapy is a promising treatment strategy in solid tumors. In vivo cell tracking techniques can help us better understand the infiltration, persistence and therapeutic efficacy of CAR T cells. In this field, magnetic resonance imaging (MRI) can achieve high-resolution images of cells by using cellular imaging probes. MRI can also provide various biological information on solid tumors.MethodsThe authors adopted the amino alcohol derivatives of glucose-coated nanoparticles, ultra-small superparamagnetic particles of iron oxide (USPIOs), to label CAR T cells for non-invasive monitoring of kinetic infiltration and persistence in glioblastoma (GBM). The specific targeting CARs included anti-human epidermal growth factor receptor variant III and IL13 receptor subunit alpha 2 CARs.ResultsWhen using an appropriate concentration, USPIO labeling exerted no negative effects on the biological characteristics and killing efficiency of CAR T cells. Increasing hypointensity signals could be detected in GBM models by susceptibility-weighted imaging MRI ranging from 3 days to 14 days following the injection of USPIO-labeled CAR T cells. In addition, nanoparticles and CAR T cells were found on consecutive histopathological sections. Moreover, diffusion and perfusion MRI revealed significantly increased water diffusion and decreased vascular permeability on day 3 after treatment, which was simultaneously accompanied by a significant decrease in tumor cell proliferation and increase in intercellular tight junction on immunostaining sections.ConclusionThese results establish an effective imaging technique that can track CAR T cells in GBM models and validate their early therapeutic effects, which may guide the evaluation of CAR T-cell therapies in solid tumors.     

IgE production after four routes of injections of Japanese cedar pollen allergen without adjuvant: crucial role of resident cells at intraperitoneal or intranasal injection site in the production of specific IgE toward the allergen

The production of specific IgE antibodies directed toward cedar pollen correlates well with the onset of allergic rhinitis; but the mechanisms of allergen recognition as nonself and Ig class switch to IgE by the immune system are still not fully understood. In the present study, we injected cedar pollen into mice through 4 different routes (intranasal (i.n.), intraperitoneal (i.p.), intravenous (i.v.), and subcutaneous (s.c.)) without adjuvant 1 to 3 times, and determined time-dependent changes in the total and specific serum IgE levels compared with those in the serum levels of other isotype Igs. After an i.p. or i.n. injection of allergen into the mice, they produced a 1.5-to 1.7-fold increase in total IgE, but none in IgG, IgM, or IgA antibodies in their serum, whereas an i.v. or s.c. injection of allergen was inactive as an inducer of total IgE antibodies. Upon a 2nd (s.c.) injection of the allergen into the i.p. or i.n. sensitized mice, a large amount of allergen-specific IgE antibodies was found in the serum. In the case of i.v. or s.c. sensitized mice, however, they produced total, but not specific, IgE antibodies; and a 3rd (s.c.) injection of the allergen resulted in a large amount of specific IgE antibodies in the serum. These results imply that resident cells at the i.p. or i.n. injection site may play a crucial role in the efficient production of total and specific IgE antibodies toward the allergen.     

Evidence of reduced uptake of convulsant in brain following prostaglandin E2

The toxic and convulsant effects of the acetylcholinesterase (AChE) inhibitor Soman, were examined in mice pretreated with various doses of prostglandin E₂ (PGE₂), administered by either intraperitoneal injection (i.p.) or by intracerebroventricular (i.c.v.) infusion. PGE₂ (i.p.) reduced the lethal effects of Soman slightly. PGE₂ (i.p. and i.c.v.) delayed the onset and reduced the severity of cholinergically-induced convulsions, resulting from Soman. Whole brain AChE was measured at various times after Soman or Soman preceded by PGE₂. PGE₂ (i.p. or i.c.v.) reduced the rate at which Soman inhibited brain AChE, which appeared to be related to the increased time to onset of convulsive activity. Repeated injections of PGE₂ did not delay convulsions indefinitely nor were convulsions terminated once they had started. The results suggest that the anticonvulsant properties of PGE₂ may have been due, in part, to decreased cerebral circulation with subsequent reduction in the access of the convulsant to the brain and in part to direct neuronal effects.     

Fe3O4-PEG-CD56/Avastin@Ce6纳米探针的合成及细胞MR成像初步研究

目的:探讨Fe_3O_4-PEG-CD56/Avastin@Ce6靶向探针与NK92细胞的结合能力并进行细胞体外MRI成像。方法:制备Fe_3O_4-PEG-CD56/Avastin@Ce6纳米探针,对合成的材料进行表征。应用凋亡试剂盒测定不同浓度的材料对NK92的细胞毒性,通过流式细胞术分析纳米材料与NK92细胞的结合能力和应用MRI对细胞进行体外成像并分析其T2信号强度的改变。结果:合成的纳米探针具有较好的生物相容性,且对NK92细胞的影响较小,不同浓度下细胞凋亡水平基本一致,与NK92细胞结合的材料随浓度的增加而逐渐增加。MRI检查提示不同浓度探针孵育的NK92细胞T2加权像(T2WI)的信号均降低。结论:Fe_3O_4-PEG-CD56/Avastin@Ce6探针对NK92细胞具有靶向性,3.0 T MR扫描仪可对其进行体外监测。     

Generation of tumor-specific cytotoxic T lymphocytesin vivo by combined treatment with inactivated tumor cells and recombinant interleukin-2

In order to search for a new therapy that would maximize the effect of interleukin-2 (IL-2) in evoking antitumor immunity in vivo, the therapeutic effect of a combination of mitomycin-C(MMC)-treated tumor cells and recombinant IL-2 was examined for its induction of antitumor activity against established melanoma metastasis. In C57BL/6 mice intravenously (i. v.) injected with B16 melanoma cells on day 0, the combined treatment with an intraperitoneal (i. p.) injection of MMC-treated melanoma cells on day 6 and 2500 U rIL-2 (twice daily) on days 7 and 8 markedly reduced the number of pulmonary metastases. This antitumor activity was more effective than that in untreated controls and mice that were injected with MMC-treated melanoma cells alone or rIL-2 alone. When the i. p. injection of MMC-treated tumor cells was replaced by other syngeneic tumor cells, antitumor activity against metastatic melanoma was not induced. The antitumor activity induced by this treatment increased in parallel with an increase in the dose of rIL-2 injected. In contrast, an i. p. injection of soluble tumor-specific antigens alone could induce only a marginal level of antitumor activity, and this activity was not augmented by subsequent i. p. injections of rIL-2. In vivo treatment with anti-CD8 monoclonal antibody (mAb), but not with anti-CD4 mAb or anti-asialo-GM1 antibody, abrogated the antitumor activity induced by this combined therapy. This suggests that the antitumor effect was dependent on CD8⁺ T cells. Lung-infiltrating lymphocytes from mice that had been i. v. injected with melanoma cells 11 days before and were treated with this combined therapy, showed melanoma-specific cytolytic activity. This combined therapy also showed significant antitumor activity against subcutaneously inoculated melanoma cells. These results demonstrate that the combined therapy of an i. p. injection of MMC-treated tumor cells and subsequent and consecutive i. p. administration of rIL-2 increases antitumor activity against established metastatic melanoma by generating tumor-specific CD8⁺ CTL in vivo.