Gold nanoparticles-conjugation of irisin enhances therapeutic effect by improving cardiac function and attenuating inflammation in sepsis.

Irisin is considered to be a promising therapeutic approach for cardiac depression and inflammatory disorders. The short half-life of irisin impeded its use and drug efficacy in the treatment. This study aimed to examine if pegylated gold nanoparticles-conjugated to irisin would improve therapeutic effects in cecal ligation and puncture (CLP)-induced sepsis in mice. Recombinant irisin were conjugated to a pegylated gold nanoparticle, which was given to mice exposed to CLP. The cecal ligation procedure and sham on mice were operated and assigned to one of following five groups: (I) CLP group: The mouse models underwent the CLP surgical procedure and received only vehicle saline treatment (n = 5); (II) CLP + soluble Irisin: The mouse underwent the CLP and received an intramuscular injection (i.m) (TA) injection of 1 ug of soluble irisin into each tibialis anterior (TA) leg (n = 5); (III) CLP + Gold nanoparticle-conjugated to Irisin: The mouse models underwent the CLP and received an i.m (TA) injection of 1 µg of Gold nanoparticle-irisin via intramuscular injection (TA) into each leg (n = 5); (IV) CLP + Gold nanoparticles- conjugated to IgG: The mouse underwent the CLP and received an i.m (TA) injection of gold nanoparticles conjugated to IgG (n = 5). (V) Sham: The mouse underwent the surgical operation without conducting the CLP (n = 10). The post-operated animals were observed for one week, and survival rates were estimated. Echocardiography was performed to measure cardiac function at 12 h following CLP. TUNEL was employed to detect apoptosis in both cardiac and skeletal muscles; histology was conducted to assess tissue injury in muscles. Enzyme linked immunosorbent assay (ELISA) was conducted to examine release of interleukin 6 (IL6) and the tumor necrosis factor (TNF) alpha. Compared to the CLP control, soluble irisin treatment improved cardiac function recovery, as indicated by the fractional shortening (FS) and ejection fraction (EF). Irisin treatment exhibited reduced IL6 and TNF-alpha release in association with less apoptosis, lower muscle injury index and improved survival post-CLP. However, compared to soluble irisin treatment, gold nanoparticles-conjugated to irisin showed a significant improvement in cardiac function, suppression of apoptosis, reduced IL6 and TNF-alpha releases, decreased muscle injury and an improved survival rate of post-CLP. This study reveals that gold nanoparticles-conjugated irisin can serve to improve irisin's therapeutic effects over a longer course of treatment.

Inhibition of integrin alpha v/beta 5 mitigates the protective effect induced by irisin in hemorrhage.

INTRODUCTION: Irisin plays an important role in regulating tissue stress, cardiac function, and inflammation. Integrin αvß5 was recently identified as a receptor for irisin to elicit its physiologic function. It remains unknown whether integrin αvß5 is required for irisin's function in modulating the physiologic response to hemorrhage. The objective of this study is to examine if integrin αvß5 contributes to the effects of irisin during the hemorrhagic response. METHODS: Hemorrhage was induced in mice by achieving a mean arterial blood pressure of 35-45 mmHg for one hour, followed by two hours of resuscitation. Irisin (0.5  µg/kg) was administrated to assess its pharmacologic effects in hemorrhage. Cilengitide, a cyclic Arg-Gly-Asp peptide (cRGDyK) which is an inhibitor of integrin αvß5, or control RGDS (1 mg/kg) was administered with irisin. In another cohort of mice, the irisin-induced protective effect was examined after knocking down integrin ß5 with nanoparticle delivery of integrin ß5 sgRNA using CRSIPR/Cas-9 gene editing. Cardiac function and hemodynamics were measured using echocardiography and femoral artery catheterization, respectively. Systemic cytokine releases were measured using Enzyme-linked immunosorbent assay (ELISA). Histological analyses were used to determine tissue damage in myocardium, skeletal muscles, and lung tissues. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out to assess apoptosis in tissues. RESULTS: Hemorrhage induced reduction of integrin αvß5 in skeletal muscles and repressed recovery of cardiac performance and hemodynamics. Irisin treatment led to significantly improved cardiac function, which was abrogated by treatment with Cilengitide or knockdown of integrin ß5. Furthermore, irisin resulted in a marked suppression of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1), muscle edema, and inflammatory cells infiltration in myocardium and skeletal muscles, which was attenuated by Cilengitide or knockdown of integrin ß5. Irisin-induced reduction of apoptosis in the myocardium, skeletal muscles, and lung, which were attenuated by either the inhibition of integrin αvß5, or knockdown of integrin ß5. CONCLUSION: Integrin αvß5 plays an important role for irisin in modulating the protective effect during hemorrhage.

Irisin Preserves Cardiac Performance and Insulin Sensitivity in Response to Hemorrhage.

Irisin, a cleaved product of the fibronectin type III domain containing protein-5, is produced in the muscle tissue, which plays an important role in modulating insulin resistance. However, it remains unknown if irisin provides a protective effect against the detrimental outcomes of hemorrhage. Hemorrhages were simulated in male CD-1 mice to achieve a mean arterial blood pressure of 35-45 mmHg, followed by resuscitation. Irisin (50 ng/kg) and the vehicle (saline) were administrated at the start of resuscitation. Cardiac function was assessed by echocardiography, and hemodynamics were measured through femoral artery catheterization. A glucose tolerance test was used to evaluate insulin sensitivity. An enzyme-linked immunosorbent assay was performed to detect inflammatory factors in the muscles and blood serum. Western blot was carried out to assess the irisin production in skeletal muscles. Histological analyses were used to determine tissue damage and active-caspase 3 apoptotic signals. The hemorrhage suppressed cardiac performance, as indicated by a reduced ejection fraction and fractional shortening, which was accompanied by enhanced insulin resistance and hyperinsulinemia. Furthermore, the hemorrhage resulted in a marked decrease in irisin and an increase in the production of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1). Additionally, the hemorrhage caused marked edema, inflammatory cell infiltration and active-caspase 3 positive signals in skeletal muscles and cardiac muscles. Irisin treatment led to a significant improvement in the cardiac function of animals exposed to a hemorrhage. In addition, irisin treatment improved insulin sensitivity, which is consistent with the suppressed inflammatory cytokine secretion elicited by hemorrhages. Furthermore, hemorrhage-induced tissue edema, inflammatory cell infiltration, and active-caspase 3 positive signaling were attenuated by irisin treatment. The results suggest that irisin protects against damage from a hemorrhage through the modulation of insulin sensitivity.

Cancer-associated fibroblasts secreted miR-103a-3p suppresses apoptosis and promotes cisplatin resistance in non-small cell lung cancer.

BACKGROUND: The cisplatin resistance of non-small cell lung cancer (NSCLC) patients results in low response rate and overall survival rate. Exosomes contribute to pathological processes of multiple cancers. OBJECTIVE: In this study, we explored the function and mechanisms of exosomal miR-103a-3p derived from cancer-associated fibroblast (CAF) in cisplatin resistance in NSCLC. RESULTS: MiR-103a-3p was highly expressed in CAFs and CAF exosomes, and exosomal miR-103a-3p derived from CAFs in NSCLC. CAFs exosomes co-cultured with NSCLC cells promoted miR-103a-3p expression both in NSCLC cells and its exosomes. Functional experiments showed that exo-miR-103a-3p derived from CAFs promoted cisplatin resistance and inhibited apoptosis in NSCLC cells. Pumilio2 (Pum2) bound with miR-103a-3p in cytoplasm and nucleus, and facilitated packaging into CAF-derived exosomes in NSCLC cells. Further analysis showed Bak1 was a direct target of miR-103a-3p, and miR-103a-3p accelerated cisplatin resistance in NSCLC cells via Bak1 downregulation. In vivo tumorigenesis assay showed CAF-derived exosomal miR-103a-3p enhanced cisplatin resistance and inhibited cell apoptosis in NSCLC. CONCLUSION: Our study revealed that CAFs-derived exosomal miR-103a-3p promoted cisplatin resistance by suppressing apoptosis via targeting Bak1, which provided a potential therapeutic target for cisplatin resistance in NSCLC.

Qishen Yiqi Dropping Pills Ameliorates Doxorubicin-induced Cardiotoxicity in Mice via Enhancement of Cardiac Angiogenesis.

BACKGROUND Qishen Yiqi Dropping Pills (QYDP) is a Chinese traditional medicine that has been applied to treat coronary heart disease and ischemic heart failure in China. However, few studies have explored whether QYDP exerted an effect on doxorubicin (Doxo)-induced cardiotoxicity. Hence, in this study we investigated the effect of QYDP on cardiotoxicity induced by doxorubicin (Doxo) and its potential mechanism. MATERIAL AND METHODS Male C57BL/6 mice (20-25 g, 8-10 weeks old) were randomly assigned to 4 groups: Control group, QYDP group, Doxo group, and QYDP+Doxo group. The mice were intraperitoneal injected with Doxo weekly for 4 weeks to mimic the chronic toxicity. Four weeks after Doxo injection, echocardiography was applied to evaluate the left ventricular (LV) function, and the structure of the cardiac muscle fibers was analyzed with anti-actinin-2 antibody staining by immunofluorescence. Moreover, TUNEL staining and western blot analysis of Bax protein, Bcl-2 protein, and cleaved caspase-3 protein expression levels were conducted to explore whether QYDP exerted effect on cardiac apoptosis. In addition, Masson trichrome staining and western blot analysis of alpha-SMA protein expression levels were used to evaluate whether QYDP exerted an effect on cardiac fibrosis. Western blots and quantitative real-time polymerase chain reaction were applied to detect the vascular endothelial growth factor (VEGF) protein and mRNA levels in the myocardial tissue, and anti-CD31 antibody staining by immunohistochemistry was employed to explore whether QYDP exerted an effect on cardiac angiogenesis. RESULTS QYDP effectively attenuated cardiac dysfunction and cardiac muscle fibers disruption in Doxo treated mice. Moreover, QYDP reduced myocardial apoptosis and myocardial fibrosis in Doxo treated mice, accompanied with elevated protein levels of VEGF and enhancement of myocardial microvessel density. CONCLUSIONS QYDP could protect against Doxo-induced cardiotoxicity, which may be closely associated with enhanced cardiac angiogenesis. Hence, QYDP could be a promising alternative for the treatment of Doxo-induced cardiotoxicity.

Application of novel targeted molecular imaging probes in the early diagnosis of upper urinary tract epithelial carcinoma.

Imaging techniques of upper tract urothelial carcinoma (UTUC) are presently limited. Upconversion particles (UCPs) could be used to target tumors for imaging. The present study aimed to assess the value of a nano-UCP as a diagnostic probe for deep tumor tissue, including UTUC. Polymer-coated water-soluble UCPs were synthesized. The pH Low Insertion Peptide (pHLIP) polypeptide was synthesized using the solid phase method. The silane shell surface was modified to present amino or carboxyl groups. Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate was used for the coupling of the polypeptide to the UCPs. An animal model of subcutaneous tumor was established in 4-week old nude mice using UTUC cells. Urinary tract epithelial cancer T24 cells were injected into the diaphragm below the heart. PHLIP-UCP solution (1 ml) was injected into the abdominal cavity of each animal. Optical detection was performed using a small animal living body multispectral imaging system. UCPs dispersed in chloroform emitted no light under natural light, while they emitted a green light when excited with a 980-nm laser. The maximum emission wavelength of Ho3+-doped UCPs was ~550 nm and the red emission region was ~650 nm. As the coated UCPs possessed a tendency to agglomerate and precipitate, the yield of the UCPs in the aqueous phase was reduced. Tumors could be successfully imaged in tumor-bearing mice. NaYF4: Yb, Ho3+ UPCs could be used for the detection of UTUC, thus further studies are required to determine if it could be used in larger animals with deeper tumors.

Doublecortin-Like Kinase 1 (DCLK1) Regulates B Cell-Specific Moloney Murine Leukemia Virus Insertion Site 1 (Bmi-1) and is Associated with Metastasis and Prognosis in Pancreatic Cancer.

BACKGROUND/AIMS: Cancer stem cells (CSCs) are largely responsible for tumor relapse and metastatic behavior. Doublecortin-like kinase 1 (DCLK1) was recently reported to be a biomarker for gastrointestinal CSCs and involved in the epithelial-mesenchymal transition (EMT) and tumor progression. B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1) is a crucial regulator of CSC self-renewal, malignant transformation and EMT, and a previous study from our group showed that Bmi-1 is upregulated in pancreatic cancer progression and participates in EMT. However, it remains unclear whether DCLK1 is involved in pancreatic cancer or whether DCLK1 is associated with the altered level of Bmi-1 expression. METHODS: The correlation of DCLK1 expression and clinical features of pancreatic cancer was analyzed in 210 paraffin-embedded archived pancreatic cancer specimens by immunohistochemical analysis. The biological effects of DCLK1 siRNA on cells were investigated by examining cell proliferation using a cell counting kit and cell colony assays, cell migration by wound healing assay and cell invasion by Transwell invasion assay. We further investigated the effect of therapeutic siRNA targeting DCLK1 on pancreatic cancer cell growth in vivo. Moreover, the molecular mechanism by which DCLK1 upregulates Bmi-1 expression was explored using real-time PCR, western blotting and Co-immunoprecipitation assay. RESULTS: DCLK1 is overexpressed in pancreatic cancer and is related to metastasis and prognosis. Knockdown of DCLK1 markedly suppressed cell growth in vitro and in vivo and also inhibited the migration and invasion of pancreatic cancer cells. Furthermore, we found that DCLK1 silencing could inhibit EMT in cancer cells via downregulation of Bmi-1 and the mesenchymal markers Snail and Vimentin and upregulation of the epithelial marker E-cadherin. Moreover, high DCLK1 expression in human pancreatic cancer samples was associated with a mesenchymal phenotype and increased cell proliferation. Further co-immunoprecipitation indicated that DCLK1 did not interact with Bmi-1 directly. CONCLUSION: Our data suggest that upregulation of DCLK1 may contribute to pancreatic cancer metastasis and poor prognosis by increasing Bmi-1 expression indirectly. The findings indicate that inhibiting DCLK1 expression might be a novel strategy for pancreatic cancer therapy.

Silencing circular RNA hsa_circ_0000977 suppresses pancreatic ductal adenocarcinoma progression by stimulating miR-874-3p and inhibiting PLK1 expression.

Circular RNAs (CircRNAs) are a novel type of endogenous noncoding RNAs that regulate target gene expression by interacting with microRNA (miRNA). Emerging evidence shows that dysregulation of circRNAs plays important roles in biological and pathological processes, including cancer development and progression. The functional role of circRNA in PDAC (pancreatic ductal adenocarcinoma) remains to be investigated. In this study, high throughput microarray assay revealed that hsa_circ_0000977 was aberrantly up-regulated in pancreatic cancer tissues; this was also validated by qRT-PCR. Silencing hsa_circ_0000977 suppressed pancreatic cancer cell proliferation and induced cell cycle arrest, which was simulated by hsa-miR-874-3p mimics and blocked by hsa-miR-874-3p inhibitor. Bioinformatics analysis predicted that there is an hsa_circ_0000977/hsa-miR-874-3p/PLK1 (Polo like kinase 1) axis in pancreatic cancer progression. Dual-luciferase reporter system and FISH assay validated the direct interaction of hsa_circ_0000977, hsa-miR-874-3p, and PLK1. Western blot verified that inhibition of hsa_circ_0000977 decreased PLK1 expression. Furthermore, silencing hsa_circ_0000977 suppressed pancreatic cancer growth in vivo. Altogether, silencing hsa_circ_0000977 suppresses progression of pancreatic cancer by interacting with hsa-miR-874-3p and decreasing inhibiting PLK1 expression. Our results may provide a promising strategy for future diagnosis and treatment of pancreatic cancer.

Delivery of paclitaxel using nanoparticles composed of poly(ethylene oxide)-b-poly(butylene oxide) (PEO-PBO).

An amphiphilic block copolymer poly(ethylene oxide)-b-poly(butylene oxide) (PEO-PBO) was evaluated as a carrier for therapeutic delivery of paclitaxel (PTX). PEO-PBO and PTX form nanoparticles (NPs) by self-assembly upon hydration. The size of these NPs was about 92.71nm and the zeta potential was -5.06mV, which met the requirements for passive tumor targeting through the enhanced permeability and retention effect. Compared with a commonly used block copolymer poly(ethylene glycol)-b-poly-D,L-(lactic acid) (PEG-PDLLA), PEO-PBO forms nanoparticles with superior pharmacokinetic, biodistribution, and tumor inhibitory properties. Meanwhile, results of hemolysis study and CMC determination showed that PEO-PBO had better biocompatibility and stability than PEG-PDLLA. These data suggest that PEO-PBO has potential for application in drug delivery and warrant further evaluation.

New Generation Nanomedicines Constructed from Self-Assembling Small-Molecule Prodrugs Alleviate Cancer Drug Toxicity.

The therapeutic index for chemotherapeutic drugs is determined in part by systemic toxicity, so strategies for dose intensification to improve efficacy must also address tolerability. In addressing this issue, we have investigated a novel combinatorial strategy of reconstructing a drug molecule and using sequential drug-induced nanoassembly to fabricate supramolecular nanomedicines (SNM). Using cabazitaxel as a target agent, we established that individual synthetic prodrugs tethered with polyunsaturated fatty acids were capable of recapitulating self-assembly behavior independent of exogenous excipients. The resulting SNM could be further refined by PEGylation with amphiphilic copolymers suitable for preclinical studies. Among these cabazitaxel derivatives, docosahexaenoic acid-derived compound 1 retained high antiproliferative activity. SNM assembled with compound 1 displayed an unexpected enhancement of tolerability in animals along with effective therapeutic efficacy in a mouse xenograft model of human cancer, compared with free drug administered in its clinical formulation. Overall, our studies showed how attaching flexible lipid chains to a hydrophobic and highly toxic anticancer drug can convert it to a systemic self-deliverable nanotherapy, preserving its pharmacologic efficacy while improving its safety profile. Cancer Res; 77(24); 6963-74. ©2017 AACR.

Comparative effect of a stannous fluoride toothpaste and a sodium fluoride toothpaste on a multispecies biofilm.

OBJECTIVES: This paper aimed to compare the mode of action of a stannous fluoride-containing toothpaste with a conventional sodium fluoride-containing toothpaste on anti-biofilm properties. METHODS: A three-species biofilm model that consists of Streptococcus mutans, Streptococcus sanguinis and Porphyromonas gingivalis was established to compare the anti-biofilm properties of a stannous fluoride-containing toothpaste (CPH), a conventional sodium fluoride-containing toothpaste (CCP) and a negative control (PBS). The 48h biofilms were subjected to two-minute episodes of treatment with test agents twice a day for 5 consecutive days. Crystal violet staining and XTT assays were used to evaluate the biomass and viability of the treated biofilm. Live/dead staining and bacteria/extracellular polysaccharides (EPS) double-staining were used to visualize the biofilm structure and to quantify microbial/extracellular components of the treated biofilms. Species-specific fluorescent in situ hybridization and quantitative polymerase chain reaction (qPCR) were used to analyze microbial composition of the biofilms after treatment. RESULTS: The biomass and viability of the biofilms were significantly reduced after CPH toothpaste treatment. The inhibitory effect was further confirmed by the live/dead staining. The EPS amounts of the three-species biofilm were significantly reduced by CCP and CPH treatments, and CPH toothpaste demonstrated significant inhibition on EPS production. More importantly, CPH toothpaste significantly suppressed S. mutans and P. gingvalis, and enriched S. sanguinis in the three-species biofilm. In all experiments CPH had a significantly greater effect than CCP (p<0.05) and CCP had a greater effect than PBS (p<0.05). CONCLUSIONS: Stannous fluoride-containing toothpaste not only showed better inhibitory effect against oral microbial biofilm, but was also able to modulate microbial composition within multi-species biofilm compared with conventional sodium fluoride-containing toothpaste.

Rhizotomy targeting the intermediate nerve, the glossopharyngeal nerve and the upper 1st to 2nd rootlets of the vagus nerve for the treatment of laryngeal neuralgia combined with intermediate nerve neuralgia--a case report.

BACKGROUND: In neurosurgery, the most common type of facial and pharyngeal pain is trigeminal neuralgia. In contrast, glossopharyngeal neuralgia is relatively rare, and laryngeal neuralgia is the most rarely observed. CASE PRESENTATION: A case of laryngeal neuralgia combined with intermediate nerve neuralgia that was admitted to our hospital in May 2012 was reported here. The patient was a 58-year-old middle-aged female, who experienced 2 years of paroxysmal burning and stabbing pain near the thyroid perichodrium, in the skin covering the right front side of the neck, and deep in inner ear. CONCLUSION: The surgical treatment plan similar to that for glossopharyngeal neuralgia could be applied if laryngeal neuralgia is associated with glossopharyngeal neuralgia and intermediate neuralgia or if no obvious improvement is achieved with the above mentioned treatment approaches.

[Comparation of inflammation between wild mice and severe combined immunodeficiency mice with endotoxemia induced by lipopolysaccharide].

AIM: To compare the inflammation between wild BALB/c mice and mice with severe combined immunodeficiency (SCID) with endotoxemia induced by lipopolysaccharide (LPS). METHODS: Endotoxemia models of wild and SCID mice were established by injecting LPS intraperitoneally. Serum was taken before and 3 h, 6 h, 12 h after injecting LPS, liver and lung were taken 12 h after injecting LPS. Alanine transarninase (ALT), aspartate aminotransferase (AST) and blood urea nitrogen (BUN) levels were measured by automatic biochemical analyzer. Liver and lung inflammation injury were observed by H.E staining. TNF-α, IFN-γ, IL-6 and MCP-1 levels in serum were detected by Cytometric Bead Array (CBA). RESULTS: (1) All of SCID mice (8/8) were dead at 12-24 h after injecting LPS, and only one BALB/c mouse (1/8) was dead. (2) ALT and AST levels of SCID mice 12 h after injecting LPS were higer than those of BALB/c mice(P<0.05), but there was no difference of BUN levels between them. (3) The blind liver and lung pathology scores of SCID mice were higher than those of BALB/c mice (P<0.05). (4) TNF-α, IFN-γ, IL-6 and MCP-1 levels in serum at 3 h, 6 h, 12 h after injecting LPS increased significantly, and the cytokine levels of SCID mice were higher than those of BALB/c mice(P<0.05). CONCLUSION: SCID mice don't only excessively secrete inflammatory cytokines after injecting LPS, but also lead to more severe endotoxemia and inflammation injury of organs, which are the important cause of mouse death. The above results also show that the adjustment deficiency of adaptive immunoresponse, abnormal augmentation of innate immunoresponse may be an important cause of severe SIRS of endangering life.

[Benign fibrous histiocytoma involving the skull: a case report and literature review].

OBJECTIVE: Benign fibrous histiocytomas (BFH) represent a rare group of tumors with a common origin from the tissue histiocytes, often causing pain and space-occupying effect. BFH of bone causes diagnostic difficulties due to its atypical clinical symptoms, radiographic features and cytological characteristics, which can be easily confused with other benign lesions such as non-ossifying fibroma (NOF), giant cell tumor (GCT), and fibrous dysplasia. The lesions are prone to relapse, and the patients often show poor response to radiotherapy and chemotherapy, therefore radical lesion resection should be the therapeutic target of this disease. This paper reported a case of BFH involving the skull and reviewed the associated literatures.

TYchi, a novel chitinase with RNA N-glycosidase and anti-tumor activities.

Chitinases which catalyze hydrolysis of chitin are believed to be antifungal proteins in plant. Nevertheless, a variety of functions and some new enzymatic activities of chitinases have been found in recent years. We cloned a novel protein from Trichosanthes kirilowii Maximowicz (Family Cucurbitaceae) named TYchi. Expression of TYchi gene in T. kirilowii plants was induced by F. oxysporum, an important cucurbitaceous fungal pathogen, which indicated that TYchi involved in the pathogen-induced plant defense reaction. In addition to its chitin-hydrolytic activity, the recombinant TYchi protein also had RNA N-glycosidase property. In cell-free rabbit reticulocyte lysate system, TYchi inhibited protein synthesis with an IC50 of approximate 5 nM. TYchi also exhibited efficient cytotoxicities to leukemia U937 and choriocarcinoma JAR cells with IC50 about 54 microg ml(-1) and 73 microg ml(-1), respectively. Structure analyses indicated that the putative domain of TYchi is highly similar to the well known active domain of the N-glycosidase trichosanthin (TCS). This bifuntional protein should be useful in diverse applications like RIP-based immunotoxin agent and genetic engineering of plant resistance.

Construction of hu-PBL/SCID chimeras and development of EBV-related lymphomas.

OBJECTIVE: To construct hu-PBL/SCID chimeras and to investigate the development of lymphoma and oncogenicity of the Epstein-Barr virus (EBV). METHODS: Human peripheral blood lymphocytes (PBLs) were isolated from healthy adult donors and transplanted intraperitoneally into severe combined immunodeficient (SCID) mice. Mice with hu-PBL engraftment from healthy EBV seronegative donors were injected intraperitoneally with EBV-containing supernatant from suspension culture of B95-8 cell line (active infection), whereas mice receiving lymphocytes from healthy EBV seropositive donors were not re-infected with B95-8 derived EBV (latent infection). Pathological examination and molecular analysis were performed on experimental animals and induced neoplasms. RESULTS: In the early stage of this experiment, 12 mice died of acute graft-versus-host disease, mortality was 34.3% (12/35 mice) with an average life span of 17.5 days. In 19 survival hu-PBL/SCID chimeric recipients from 12 healthy donors, tumor incidence was 84.2% (16/19 mice). The average survival time of tumor-bearing mice was 65.5 days. EBV-related neoplasms in SCID mice were nodular tumors with aggressive and fatal features. Histological morphology of tumors exhibited diffuse large cell lymphomas. Immunohistochemistry revealed that LCA (CD45) and L26 (CD20) were positive, but both PS1 (CD3) and UCHL-1 (CD45RO) were negative, and EBV products ZEBRA, LMP1, and EBNA2 were expressed in a small number of tumor cells. EB virus particles were seen in the nuclei of some tumor cells by electron microscopy, and EBV DNA could be amplified in the tumor tissues by PCR. In situ hybridization indicated that the nuclei of tumor cells contained human-specific Alu sequence. CONCLUSIONS: EBV-induced tumors were human B-cell malignant lymphomas. We obtained direct causative evidence dealing with EBV-associated tumor deriving from normal human cells.

Cyclosporine A effectively inhibits graft-versus-host disease during development of Epstein-Barr virus-infected human B cell lymphoma in SCID mouse.

We previously constructed human peripheral blood lymphocyte (hu-PBL)/severe combined immunodeficiency mouse (SCID) chimeras and induced human B-cell lymphomas associated with Epstein-Barr virus (EBV) in SCID mice. However, a number of SCID mice died of graft-versus-host disease (GVHD) during the early experimental course. The aim of this study was to test the efficacy of cyclosporine A (CSA) for prevention of GVHD and to define how CSA inhibits the occurrence of GVHD and the production of soluble interleukin (IL) 2 receptor (sIL-2R) in hu-PBL/SCID mice. No mouse died in the active EBV infection group with CSA administration, while 17 mice in three groups without CSA administration died of GVHD. Mortalities in these three groups were 55.56% (5/9), 30.43% (7/23), and 27.78% (5/18), and the medium life span was 17 days. Over the first 33 days after hu-PBL transplantation, serum level of human sIL-2R in hu-PBL/SCID chimeras was stable in the active EBV infection plus CSA group, while sIL-2R concentration gradually increased in the sera of mice with active EBV infection without CSA administration and peaked at 22 days. Thirty-two mice developed tumors among the 43 surviving SCID mice. There was no significant difference of tumor incidence between the active EBV infection groups with CSA and without CSA administration (P > 0.05). From their morphological and immunohistochemical features, as well as detection of human Alu-sequence and EBV in tumor cells, these EBV-induced tumors were identified as human B-cell lymphomas. Thus, CSA can strikingly inhibit GVHD in hu-PBL/SCID chimeras, and should therefore be effective to establish a stable SCID mouse model of human lymphoma associated with EBV. Treatment with CSA had no effect on the tumor incidence in hu-PBL/SCID chimeras after active EBV infection. Accordingly, serum level of sIL-2R is a valuable indicator of GVHD occurrence in hu-PBL/SCID chimeras.