AFG1-induced TNF-α-mediated inflammation enhances gastric epithelial cell injury via CYP2E1.

Aflatoxin G1 (AFG1), a member of the aflatoxin family with cytotoxic and carcinogenic properties, is one of the most common mycotoxins occurring in various agricultural products, animal feed, and human foods and drinks worldwide. Epithelial cells in the gastrointestinal tract are the first line of defense against ingested mycotoxins. However, the toxicity of AFG1 to gastric epithelial cells (GECs) remains unclear. In this study, we explored whether and how AFG1-induced gastric inflammation regulates cytochrome P450 to contribute to DNA damage in GECs. Oral administration of AFG1 induced gastric inflammation and DNA damage in mouse GECs associated with P450 2E1 (CYP2E1) upregulation. Treatment with the soluble TNF-α receptor sTNFR:Fc inhibited AFG1-induced gastric inflammation, and reversed CYP2E1 upregulation and DNA damage in mouse GECs. TNF-α-mediated inflammation plays an important role in AFG1-induced gastric cell damage. Using the human gastric cell line GES-1, AFG1 upregulated CYP2E1 through NF-κB, causing oxidative DNA damage in vitro. The cells were also treated with TNF-α and AFG1 to mimic AFG1-induced TNF-α-mediated inflammation. TNF-α activated the NF-κB/CYP2E1 pathway to promote AFG1 activation, which enhanced DNA cellular damage in vitro. In conclusion, AFG1 ingestion induces TNF-α-mediated gastric inflammation, which upregulates CYP2E1 to promote AFG1-induced DNA damage in GECs.

Intramyocardial injection of hypoxia-preconditioned adipose-derived stromal cells treats acute myocardial infarction: an in vivo study in swine.

Hypoxic preconditioning is a promising method for improving the anti-apoptotic and paracrine signaling capabilities of adipose-derived stromal cells (ADSCs). The purpose of this study was to analyze the influence of different hypoxic conditions on ADSCs and the therapeutic effects of hypoxia-preconditioned ADSCs (HPADSCs) on an animal model of myocardial infarction (MI). For the in vitro studies, ADSCs were divided into five groups and cultured in different oxygen concentrations (1, 3, 5, 10, and 21 %). After 24 h, RT-PCR and western blots showed that 3 % oxygen preconditioning could improve the viability and cytokine secretion of the ADSCs. A Matrigel assay indicated that the HPADSC-conditioned medium could stimulate endothelial cells to form capillary-like tubes. For the in vivo studies, MI was induced by coronary occlusion in 24 mature Chinese minipigs. The animals were divided into three groups and treated by intramyocardial injection with vehicle alone (saline group), with 1 × 10(8) ADSCs cultured in normoxic conditions (ADSCs group) or with 1 × 10(8) ADSCs precultured in 3 % oxygen (HPADSCs group). SPECT and echocardiography demonstrated that cardiac function was improved significantly in the HPADSC transplant group compared with the vehicle control group (P < 0.05). Immunofluorescence showed fewer apoptotic cells and more small- to medium-sized vessels in the HPADSC transplantation group (P < 0.05). Three percent oxygen is the optimum preconditioning treatment for ADSCs. HPADSC transplantation can prevent ventricular remodeling and reduce the infarct size.

Umbilical cord-derived mesenchymal stem cells regulate thymic epithelial cell development and function in Foxn1(-/-) mice.

Thymic microenvironments are essential for the maturation of thymocytes, which can be anatomically compartmentalized into cortical and medullar regions. The absence of the gene encoding the transcription factor forkhead box n1 (Foxn1) causes epithelial differentiation to stall in the precursor stage, resulting in the formation of an abnormal thymus. In this study, we used human umbilical cord-derived mesenchymal stem cells (UC-MSCs) to treat Foxn1(-/-) mice, and then analyzed the maturation and distribution of thymic epithelial cells in the Foxn1(-/-) thymic rudiment and the thymopoiesis of this newly developed rudiment. Our data showed a well-organized cortex-medulla architecture and an obvious improvement in the maturation of thymic epithelial cells along with the appearance of UEA-1(+)MHCII(hi) thymic epithelial cells in the rudiment. We further demonstrated improved thymopoiesis and the enhanced export of mature T cells with increased numbers of regulatory T cells into the peripheral blood. Furthermore, we observed that MSCs can engraft into thymic tissue and express many cytokines or proteins, particularly keratinocyte growth factor (KGF) and CD248, which are essential for thymic development. Collectively, our data identified a new mechanism for MSCs, which may provide a proper microenvironment for the reconstitution and functional maturation of the thymus in Foxn1(-/-) mice. Additionally, we elicited additional insights into the therapeutic efficacy of MSCs in several autoimmune diseases.

How important is differentiation in the therapeutic effect of mesenchymal stromal cells in liver disease?

BACKGROUND AIMS: The protocols for differentiation of hepatocyte-like cells (HLCs) from mesenchymal stromal cells (MSCs) have been well established. Previous data have shown that MSCs and their derived HLCs were able to engraft injured liver and alleviate injuries induced by carbon tetrachloride. The goal of the current study was to determine the differences of MSCs and their derived HLCs in terms of therapeutic functions in liver diseases. METHODS: After hepatic differentiation of umbilical cord-derived MSCs in vitro, we detected both MSC and HLC expressions of adhesion molecules and chemokine receptor CXCR4 by flow cytometry; immunosuppressive potential and hepatocyte growth factor expression were determined by means of enzyme-linked immunosorbent assay. We compared the therapeutic effect for fulminant hepatic failure in a mouse model. RESULTS: MSC-derived-HLCs expressed lower levels of hepatocyte growth factor, accompanied by impaired immunosuppression in comparison with MSCs. Furthermore, undifferentiated MSCs showed rescuing potentials superior to those in HLCs for the treatment of fulminant hepatic failure. CONCLUSIONS: After differentiation, HLCs lost several major properties in comparison with undifferentiated MSCs, which are beneficial for their application in liver diseases. Undifferentiated MSCs may be more appropriate than are HLCs for the treatment of liver diseases.

Intrathecal Administration of Autologous CD34 Positive Cells in Patients with Past Cerebral Infarction: A Safety Study.

Regenerative strategies in treatment of stroke have great potential. The goal of the current study was to investigate safety of intrathecal administration of autologous CD34 positive cells in treatment of patients with poststroke. A total of eight male patients with a history of stroke were enrolled. The patients were treated subcutaneously with 5 µ g/kg body weight rhG-CSF for 5 consecutive days, and then leukapheresis was performed to concentrate cells for CD34 positive immunoselection. All patients underwent intrathecal administration of CD34 positive cells via lumbar puncture. The primary outcome was safety evaluation for 12-month followup. In addition, behavioral function was evaluated with NIH stroke scale and Barthel index 1, 6, and 12 months after the last treatment, respectively. There were no major adverse events, and abnormal changes of blood tests during the whole treatment process included intrathecal administration and 12-month followup. The main message from the current study was that administration of G-CSF-mobilized autologous CD34 positive cells in patients with poststroke was safe. Future studies with larger population and control group are needed to confirm the safety and investigate the efficacy.

Human umbilical cord mesenchymal stem cell therapy for patients with active rheumatoid arthritis: safety and efficacy.

This study was designed to assess the safety and efficacy of human umbilical cord mesenchymal stem cells (UC-MSCs) in the treatment of rheumatoid arthritis (RA). In this ongoing cohort, 172 patients with active RA who had inadequate responses to traditional medication were enrolled. Patients were divided into two groups for different treatment: disease-modifying anti-rheumatic drugs (DMARDs) plus medium without UC-MSCs, or DMARDs plus UC-MSCs group (4×10(7) cells per time) via intravenous injection. Adverse events and the clinical information were recorded. Tests for serological markers to assess safety and disease activity were conducted. Serum levels of inflammatory chemokines/cytokines were measured, and lymphocyte subsets in peripheral blood were analyzed. No serious adverse effects were observed during or after infusion. The serum levels of tumor necrosis factor-alpha and interleukin-6 decreased after the first UC-MSCs treatment (P<0.05). The percentage of CD4(+)CD25(+)Foxp3(+) regulatory T cells of peripheral blood was increased (P<0.05). The treatment induced a significant remission of disease according to the American College of Rheumatology improvement criteria, the 28-joint disease activity score, and the Health Assessment Questionnaire. The therapeutic effects maintained for 3-6 months without continuous administration, correlating with the increased percentage of regulatory T cells of peripheral blood. Repeated infusion after this period can enhance the therapeutic efficacy. In comparison, there were no such benefits observed in control group of DMARDS plus medium without UC-MSCs. Thus, our data indicate that treatment with DMARDs plus UC-MSCs may provide safe, significant, and persistent clinical benefits for patients with active RA.

Wharton's jelly-derived mesenchymal stem cells promote myocardial regeneration and cardiac repair after miniswine acute myocardial infarction.

OBJECTIVE: To investigate the therapeutic effects of Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) on myocardial regeneration and cardiac repair after acute myocardial infarction (AMI). MATERIALS AND METHODS: WJ-MSCs were isolated from human umbilical cord under sterile conditions and were cultured and expanded until passage 5. WJ-MSCs were labeled with CM-Dil before transplantation. The mid-third of the left anterior descending artery was ligated surgically to establish a mini-swine AMI model. The miniswines were divided randomly into three groups (n=6 in each): control group, PBS group, and transplantation group. Six weeks later, evaluation of the changes in myocardial perfusion and function was performed by single-photon emission computed tomography and echocardiography in each group. Then, the animals were euthanized and the tissues in the infarcted area were harvested for histopathological examination. RESULTS: The changes in myocardial perfusion and function were significantly improved in the transplantation group compared with the control and PBS groups (P<0.001). Immunofluorescence results confirmed that the transplanted WJ-MSCs were still alive and part of them appeared to have differentiated into cardiomyocytes and vascular endothelia 6 weeks after transplantation. In the meantime, it was also observed that resident cardiac stem cells also recruited and differentiated into neonatal cardiomyocytes and vascular endothelia. Masson's trichrome showed more viable myocardium and less fibrous tissue in the transplantation group compared with the other two groups (P<0.001). Vessel density was augmented and cell apoptosis was reduced in the transplantation group compared with the control and PBS groups (P<0.001). CONCLUSION: WJ-MSCs transplanted by a direct injection into the infarcted area could survive and differentiate into cardiomyocytes and endothelial cells. They also promoted recruitment and differentiation of cardiac stem cells in a porcine model with AMI. In addition, WJ-MSC transplantation reduced apoptosis and fibrosis, enhanced viable myocardium, and thus improved ventricular remodeling and function.

Secreted galectin-3 as a possible biomarker for the immunomodulatory potential of human umbilical cord mesenchymal stromal cells.

BACKGROUND AIMS: Human umbilical cord-derived mesenchymal stromal cells (UC-MSCs) possess broad and potent immunomodulatory activities and have shown great potential in anti-inflammatory therapies. However, a biomarker that can be used to assess quickly and efficiently the immunomodulatory function of UC-MSCs has not been identified. Several studies have revealed that galectin-3 (Gal-3), a member of the human galectin family, is involved in the immunosuppressive function of MSCs. METHODS: Gal-3 gene expression in UC-MSCs was analyzed using quantitative reverse transcriptase polymerase chain reaction and Western blotting. Blocking of Gal-3 expression in UC-MSCs with small interfering RNA was employed to analyze whether the immunosuppressive function of UC-MSCs was affected. RESULTS: We found that UC-MSCs expressed Gal-3 both on the cell surface and in secreted form, and the expression levels of Gal-3 did not show significant variation after cell passaging. We further showed that Gal-3 expression correlated with the immunosuppressive function of UC-MSCs because knock-down of Gal-3 expression with small interfering RNA significantly abrogated the inhibitory effects of UC-MSCs on mitogen-stimulated and alloantigen-stimulated proliferation of human peripheral blood mononuclear cells; meanwhile, the inhibitory effect of UC-MSCs was reversed by adding back recombinant Gal-3 to the co-culture systems. The inhibitory activities of human UC-MSCs were not reduced even when they were separated from human peripheral blood mononuclear cells in a transwell co-culture system, indicating that the soluble form of Gal-3 was the major effector. CONCLUSIONS: The Gal-3 protein secreted by UC-MSCs shows good correlation with immunosuppressive potential and may serve as a possible biomarker for the potency test of UC-MSCs.

Therapeutic potential of umbilical cord mesenchymal stromal cells transplantation for cerebral palsy: a case report.

Cerebral palsy is the most common motor disability in childhood. In current paper, we first report our clinical data regarding administration of umbilical cord mesenchymal stem cells (MSCs) transplantation in treatment of cerebral palsy. A 5-year-old girl with cerebral palsy was treated with multiple times of intravenous and intrathecal administration of MSCs derived from her young sister and was followed up for 28 months. The gross motor dysfunction was improved. Other benefits included enhanced immunity, increased physical strength, and adjusted speech and comprehension. Temporary low-grade fever was the only side effect during the treatment. MSCs may be a safe and effective therapy to improve symptoms in children with cerebral palsy.

Flame speed and self-similar propagation of expanding turbulent premixed flames.

In this Letter we present turbulent flame speeds and their scaling from experimental measurements on constant-pressure, unity Lewis number expanding turbulent flames, propagating in nearly homogeneous isotropic turbulence in a dual-chamber, fan-stirred vessel. It is found that the normalized turbulent flame speed as a function of the average radius scales as a turbulent Reynolds number to the one-half power, where the average radius is the length scale and the thermal diffusivity is the transport property, thus showing self-similar propagation. Utilizing this dependence it is found that the turbulent flame speeds from the present expanding flames and those from the Bunsen geometry in the literature can be unified by a turbulent Reynolds number based on flame length scales using recent theoretical results obtained by spectral closure of the transformed G equation.

Dexamethasone has variable effects on mesenchymal stromal cells.

BACKGROUND AIMS: Dexamethasone (Dex) is a potent synthetic member of the glucocorticoid class of steroid drugs. Frequently, Dex has been used to enhance osteogenic, chondrogenic and adipogenic differentiation of mesenchymal stromal cells (MSC). Recently, Dex was applied to promote MSC proliferation, because of the rare frequency of MSC in bone marrow, and could protect the cells from apoptosis. The effects of Dex on MSC cytobiology behavior needs to be investigated. METHODS: MSC were obtained from human umbilical cord. The surface phenotype and functional characterization of MSC cultured with different concentrations of Dex were investigated, in comparison with a control group, including MSC proliferation, apoptosis, cytokine expression and immunosuppression. RESULTS: Different concentrations of Dex exerted diverse effects on MSC proliferation and apoptosis. Dex was also able to affect the pattern of cytokine expression of MSC. Furthermore, Dex impaired immunosuppression of MSC on peripheral blood mononuclear cells. CONCLUSIONS: A low dose of Dex favors MSC expansion in vitro, and protects against apoptosis. It is not suitable for MSC to be pre-treated with Dex when they are to be used to treat immunologic disease. However, when MSC are applied to promote angiogenesis, it is beneficial for them to be pre-treated with 10(-9) mol/L Dex.

Human umbilical cord mesenchymal stem cells ameliorate mice trinitrobenzene sulfonic acid (TNBS)-induced colitis.

Mesenchymal stem cells (MSCs), which are poorly immunogenic and have potent immunosuppressive activities, have emerged as a promising candidate for cellular therapeutics for the treatment of disorders caused by abnormal immune responses. In this study we investigated whether human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) could ameliorate colitis in a trinitrobenzene sulfonic acid (TNBS)-induced colitis model. TNBS-treated colitic mice were infused with hUC-MSCs or vehicle control. The mice were sacrificed on day 1, 3, and 5 after infusion, and their clinical and pathological conditions were evaluated by body weight, colon length, and histological analysis. The expression levels of proinflammatory cytokine proteins in colon were examined by ELISA. The homing of hUC-MSCs was studied by live in vivo imaging and immunofluorescent microscopy. hUC-MSCs were found to migrate to the inflamed colon and effectively treated the colitic mice with improved clinical and pathological signs. The levels of IL-17 and IL-23 as well as IFN-γ and IL-6 were significantly lower in the colon tissues of the hUC-MSC-treated mice in comparison with the vehicle-treated mice. Coculture experiments showed that hUC-MSCs not only could inhibit IFN-γ expression but also significantly inhibit IL-17 production by lamina propria mononuclear cells (LPMCs) or splenocytes of the colitic mice or by those isolated from normal animals and stimulated with IL-23. Systemically infused hUC-MSCs could home to the inflamed colon and effectively ameliorate colitis. In addition to the known suppressive effects on Th1-type immune responses, hUC-MSC-mediated modulation of IL-23/IL-17 regulated inflammatory reactions also plays an important role in the amelioration of colitis.

CD14+ monocytes promote the immunosuppressive effect of human umbilical cord matrix stem cells.

Here, the effect of CD14(+) monocytes on human umbilical cord matrix stem cell (hUC-MSC)-mediated immunosuppression was studied in vitro. hUC-MSCs exerted a potent inhibitory effect on the proliferation and interferon-gamma (IFN-gamma) secretion capacities of CD4(+) and CD8(+) T cells in response to anti-CD3/CD28 stimulation. Transwell co-culture system revealed that the suppressive effect was primarily mediated by soluble factors. Addition of prostaglandin synthesis inhibitors (indomethacin or NS-398) almost completely abrogated the immunosuppression activity of hUC-MSCs, identifying prostaglandin E(2) (PGE(2)) as an important soluble mediator. CD14(+) monocytes were found to be able to enhance significantly the immunosuppressive effect of hUC-MSCs in a dose-dependent fashion. Moreover, the inflammatory cytokine IL-1beta, either exogenously added or produced by CD14(+) monocytes in culture, could trigger expression of high levels of PGE(2) by hUC-MSCs, whereas inclusion of the IL-1 receptor antagonist (IL-1RA) in the culture down-regulated not only PGE(2) expression, but also reversed the promotional effect of CD14(+) monocytes and partially restored CD4(+) and CD8(+) T cell proliferation and IFN-gamma secretion. Our data demonstrate an important role of monocytes in the hUC-MSC-induced immunomodulation, which may have important implications in future efforts to explore the clinical potentials of hUC-MSCs.

Human umbilical cord mesenchymal stem cells hUC-MSCs exert immunosuppressive activities through a PGE2-dependent mechanism.

Human umbilical-cord-derived mesenchymal stem cells (hUC-MSCs) constitute an attractive alternative to bone-marrow-derived MSCs for potential clinical applications because of easy preparation and lower risk of viral contamination. In this study, both proliferation of human peripheral blood mononuclear cells (hPBMCs) and their IFN-gamma production in response to mitogenic or allogeneic stimulus were effectively inhibited by hUC-MSCs. Co-culture experiments in transwell systems indicated that the suppression was largely mediated by soluble factor(s). Blocking experiments identified prostaglandin E(2) (PGE(2)) as the major factor, because inhibition of PGE(2) synthesis almost completely mitigated the immunosuppressive effects, whereas neutralization of TGF-beta, IDO, and NO activities had little effects. Moreover, the inflammatory cytokines, IFN-gamma and IL-1beta, produced by hPBMCs upon activation notably upregulated the expression of cyclooxygenase-2 (COX-2) and the production of PGE(2) by hUC-MSCs. In conclusion, our data have demonstrated for the first time the PGE(2)-mediated mechanism by which hUC-MSCs exert their immunomodulatory effects.

Stem/progenitor cells in liver injury repair and regeneration.

Morbidity and mortality from cirrhosis is increasing rapidly in the world. Currently, orthotopic liver transplantation is the only definitive therapeutic option. However, its clinical use is limited, because of poor long-term graft survival, donor organ shortage and high costs associated with the procedure. Stem cell replacement strategies are therefore being investigated as an attractive alternative approach to liver repair and regeneration. In this review we discuss recent preclinical and clinical investigations that explore the therapeutic potential of stem cells in repair of liver injuries. Several types of stem cells. including embryonic stem cells, haematopoietic stem cells and mesenchymal stem cells, can be induced to differentiate into hepatocyte-like cells by defined culture conditions in vitro. Stem cell transplantation has been shown to significantly improve liver function and increase animal survival in experimentally-induced liver-injury models. Moreover, several pilot clinical studies have reported encouraging therapeutic effects in patients treated with stem cells. Although there remain many unresolved issues, the available data support the notion that stem cell technology may lead to the development of effective clinical modalities for human liver diseases.

Amplification of immune responses against a DNA-delivered idiotypic lymphoma antigen by fusion to the B subunit of E. coli heat labile toxin.

The pentameric B-subunit of Escherichia coli heat-labile enterotoxin (EtxB) is not only highly immunogenic itself, but can also act as a potent adjuvant or carrier to increase immune responses to other antigens. In this study, we investigated the ability of EtxB to promote anti-tumor immune responses using a fusion DNA vaccine design. EtxB was genetically linked to a single chain Fv sequence derived from the idiotypic immunoglobulin antigen (Id) of the mouse BCL1 B-cell lymphoma. We found that the EtxB-BCL1scFv fusion protein with a specifically selected linker retained the ability to pentamerize and to bind the GM1 ganglioside. Immunization of mice with the pEtxB-BCL1scFv DNA construct generated high levels of Id-specific antibody and protected against lethal tumor challenge. The immuno-enhancing activities of EtxB were highly dependent on GM1 binding, since fusion constructs unable to pentamerize or to bind to GM1 were less effective. Thus, the EtxB fusion vaccine approach may be an attractive strategy to increase the potency of tumor vaccines.

A DNA fusion vaccine induces bactericidal antibodies to a peptide epitope from the PorA porin of Neisseria meningitidis.

An experimental DNA plasmid vaccine was developed based on a well-characterized and protective peptide epitope derived from a bacterial porin protein. For this study, we used the P1.16b serosubtype epitope, located in variable region (VR)2 in loop 4 of the PorA outer membrane (OM) porin from Neisseria meningitidis serogroup B strain MC58. A plasmid that encoded the entire loop (pPorAloop4) was prepared, as well as a fusion plasmid that encoded the loop in tandem with the fragment C (FrC) immunostimulatory sequence from tetanus toxin (pPorAloop4-FrC). The constructs were used for intramuscular immunization without exogenous adjuvant. Murine antisera raised to the pPorAloop4-FrC DNA fusion plasmid reacted significantly with OMs in enzyme-linked immunosorbent assay and with whole bacteria by immunofluorescence, whereas antisera raised to the pPorAloop4 DNA plasmid and to control plasmid showed little or no reactivity. Significantly, only the pPorALoop4-FrC plasmid induced bactericidal antibodies, demonstrating that the intrinsic immunostimulatory sequence was essential for inducing a protective immune response. The antibodies raised to the P1.16b pPorALoop4-FrC plasmid were serosubtype specific, showing no significant immunofluorescence reactivity or bactericidal activity against other PorA variants. These data provide proof of principle for a DNA fusion plasmid strategy as a novel approach to preparing vaccines based on defined, protective epitopes.

Deglycosylation to obtain stable and homogeneous Pichia pastoris-expressed N-A1 domains of carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is a seven domain membrane glycoprotein widely used as a tumour marker for adenocarcinomas and as a target for antibody-directed therapies. Structural models have proposed that the first two domains of CEA (the N terminal and adjoining A1 domains) bind MFE-23, a single chain Fv antibody in experimental clinical use. We aimed to produce recombinant N-A1 to test this hypothesis. The N-A1 domains were expressed as soluble protein with a C-terminal hexahistidine tag (His6-tag) in the yeast Pichia pastoris. His6-tagged N-A1 was captured from the supernatant by batch purification with copper-loaded Streamline Chelating, an immobilised metal affinity chromatography (IMAC) matrix usually utilised in expanded bed techniques. Purified N-A1 was heterogeneous with a molecular weight range from 38 to 188 kDa. Deglycosylation with endoglycosidase H (Endo H) resulted in three discrete molecular weight forms of N-A1, one partially mannosylated, one fully Endo H-digested and one fully Endo H-digested but lacking the His6-tag. These were separated by concanavalin A chromatography followed by HiTrap IMAC. The procedure resulted in single-band-purity, mannose-free N-A1. The binding interaction of MFE-23 to N-A1 was analysed by surface plasmon resonance. The affinity constants retrieved were KD = 4.49 x 10(-9)M for the P. pastoris expressed, native N-A1, and 5.33 x 10(-9) M for the Endo H-treated N-A1. To our knowledge this is the first time that two consecutive domains of CEA have been stably expressed and purified from P. pastoris. This work confirms that the CEA epitope recognised by MFE-23 resides in N-A1.

Deregulated expression of the Myc cellular oncogene drives development of mouse "Burkitt-like" lymphomas from naive B cells.

Chromosomal translocations juxtaposing immunoglobulin (Ig) and MYC genes are the hallmarks of human Burkitt lymphoma (BL), with deregulated MYC expression being a critical factor in pathogenesis. By inserting an intact mouse Myc gene into the mouse genome, proximal to the Ig enhancer Emu, the effect of a precise mimic of the major t(8;14) translocation of human endemic BL (eBL) could be investigated. Knock-in mice developed IgM-positive B-cell tumors, with most being typical of eBL by histology and immunophenotype, including expression of the germinal center (GC)-associated protein, BCL6. Unlike eBL, however, analysis of Ig V(H) sequences revealed no significant level of somatic mutation. Thus, constitutive expression of Myc in the knock-in mice is apparently able to induce "Burkitt-like" lymphomas before antigen stimulation and formation of a GC. In contrast, human eBL development occurs in a GC or post-GC site with a likely contribution to pathogenesis from Epstein-Barr virus (EBV) and other epigenetic factors.

DNA vaccines to attack cancer.

Delivery of antigens by injection of the encoding DNA allows access to multiple antigen-presenting pathways. Knowledge of immunological processes can therefore be used to modify construct design to induce selected effector functions. Expression can be directed to specific intracellular sites, and additional genes can be fused or codelivered to amplify responses. Therapeutic vaccination against cancer adds a requirement to overcome tolerance and to activate a weakened immune repertoire. Induction of CD4(+) T helper cells is critical for both antibody and T cell effector responses. To activate immunity against tumor antigens, we fused the tumor-derived sequences to genes encoding microbial proteins. This strategy engages T helper cells from the large antimicrobial repertoire for linked help for inducing antibody against cell-surface tumor antigens. The principle of linked T cell help also holds for induction of epitope-specific antitumor CD8(+) T cells, but the microbial sequence has to be minimized to avoid competition with tumor antigens. Epitope-specific DNA vaccination leads to powerful antitumor attack and can activate immunity from a profoundly tolerized repertoire. Vaccine designs validated in preclinical models are now in clinical trial with immune responses detected against both tumor antigens and fused microbial antigens. DNA priming is highly efficient, but boosting may benefit from increased antigen expression. Physical methods including electroporation provide increased expression without introducing additional competing antigens. A wide range of cancers can be targeted, and objective assays of response will determine efficacy.