Resident enteric microbiota and CD8+ T cells shape the abundance of marginal zone B cells.

Since enteric microbial composition is a distinctive and stable individual trait, microbial heterogeneity may confer lifelong, non-genetic differences between individuals. Here we report that C57BL/6 mice bearing restricted flora microbiota, a distinct but diverse resident enteric microbial community, are numerically and functionally deficient in marginal zone (MZ) B cells. Surprisingly, MZ B-cell levels are minimally affected by germ-free conditions or null mutations of various TLR signaling molecules. In contrast, MZ B-cell depletion is exquisitely dependent on cytolytic CD8(+) T cells, and includes targeting of a cross-reactive microbial/endogenous MHC class 1B antigen. Thus, members of certain enteric microbial communities link with CD8(+) T cells as a previously unappreciated mechanism that shapes innate immunity dependent on innate-like B cells.

Mechanical behaviour of porous hydroxyapatite.

The aim of the study was to investigate the role of microstructure and porosity on the mechanical behaviour of sintered hydroxyapatite. Hydroxyapatite disks with four different porosities were used in this investigation. With a nanoindentation system, elastic modulus, hardness, contact stress-strain relationship, energy absorption and indentation creep behaviour were investigated. The elastic modulus and hardness of hydroxyapatite exhibited an exponential relationship (e(-bP)) with the porosity P, which is similar to Rice's finding with the minimum solid area model. High porosity samples showed more substantial inelastic behaviour, including higher energy absorption, no linear elastic region in the contact stress-strain curve and some indentation creep behaviour. We conclude that porous microstructure endows hydroxyapatite with inelastic deformation properties, which are important in a material for bone substitution usage.

Characterisation of enamel white spot lesions using X-ray micro-tomography.

OBJECTIVES: The aim of this study was to characterise the mineral density (MD) of natural enamel white spot lesions (WSLs) using X-ray micro-tomography calibrated with different density hydroxyapatite phantoms. METHODS: Seven natural WSLs from four extracted non-carious premolar teeth were scanned at a voxel size of 7.6 microm using a desktop X-ray micro-tomography system. Five hydroxyapatite phantoms (sintered pellets of hydroxyapatite powder) with densities ranging from 1.52 to 3.14 g/cm(3) were used as calibration standards for each scan. Three-dimensional image reconstruction enabled MD gradients throughout the lesion to be quantified using an MD calibration equation derived from hydroxyapatite phantoms. Background noise generated during the measurement of MD was reduced using a Gaussian filter. RESULTS: Gaussian filter reduced the signal-to-noise ratio (standard deviation) significantly while the basic MD information (average value) remained intact. The mineral gradients through the WSLs examined were compared and are discussed in terms of existing literature. The MD of sound enamel, apparent intact surface layer of WSL, and lowest level of WSL was found to be 2.65-2.89 g/cm(3), 2.23-2.58 g/cm(3) and 1.48-2.03 g/cm(3), respectively. Our MD results are comparable with other studies. CONCLUSIONS: X-ray micro-tomography is a sensitive in vitro technique capable of characterising and quantifying MD of small non-cavitated WSLs. This method has a promising potential for future carious and quantitative remineralisation studies.

Retroviral replicating vector-mediated gene therapy achieves long-term control of tumor recurrence and leads to durable anticancer immunity.

BACKGROUND: Prodrug-activator gene therapy with Toca 511, a tumor-selective retroviral replicating vector (RRV) encoding yeast cytosine deaminase, is being evaluated in recurrent high-grade glioma patients. Nonlytic retroviral infection leads to permanent integration of RRV into the cancer cell genome, converting infected cancer cell and progeny into stable vector producer cells, enabling ongoing transduction and viral persistence within tumors. Cytosine deaminase in infected tumor cells converts the antifungal prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil, mediating local tumor destruction without significant systemic adverse effects. METHODS: Here we investigated mechanisms underlying the therapeutic efficacy of this approach in orthotopic brain tumor models, employing both human glioma xenografts in immunodeficient hosts and syngeneic murine gliomas in immunocompetent hosts. RESULTS: In both models, a single injection of replicating vector followed by prodrug administration achieved long-term survival benefit. In the immunodeficient model, tumors recurred repeatedly, but bioluminescence imaging of tumors enabled tailored scheduling of multicycle prodrug administration, continued control of disease burden, and long-term survival. In the immunocompetent model, complete loss of tumor signal was observed after only 1-2 cycles of prodrug, followed by long-term survival without recurrence for >300 days despite discontinuation of prodrug. Long-term survivors rejected challenge with uninfected glioma cells, indicating immunological responses against native tumor antigens, and immune cell depletion showed a critical role for CD4+ T cells. CONCLUSION: These results support dual mechanisms of action contributing to the efficacy of RRV-mediated prodrug-activator gene therapy: long-term tumor control by prodrug conversion-mediated cytoreduction, and induction of antitumor immunity.

Toca 511 plus 5-fluorocytosine in combination with lomustine shows chemotoxic and immunotherapeutic activity with no additive toxicity in rodent glioblastoma models.

BACKGROUND: Toca 511, a gamma retroviral replicating vector encoding cytosine deaminase, used in combination with 5-fluorocytosine (5-FC) kills tumor by local production of 5-fluorouracil (5-FU), inducing local and systemic immunotherapeutic response resulting in long-term survival after cessation of 5-FC. Toca 511 and Toca FC (oral extended-release 5-FC) are under investigation in patients with recurrent high-grade glioma. Lomustine is a treatment option for patients with high-grade glioma. METHODS: We investigated the effects of lomustine combined with Toca 511 + 5-FC in syngeneic orthotopic glioma models. Safety and survival were evaluated in immune-competent rat F98 and mouse Tu-2449 models comparing Toca 511 + 5-FC to lomustine + 5-FC or the combination of Toca 511 + 5-FC + lomustine. After intracranial implantation of tumor, Toca 511 was delivered transcranially followed by cycles of intraperitoneal 5-FC with or without lomustine at the first or fourth cycle. RESULTS: Coadministration of 5-FC with lomustine was well tolerated. In F98, combination Toca 511 + 5-FC and lomustine increased median survival, but "cures" were not achieved. In Tu-2449, combination Toca 511 + 5-FC and lomustine increased median survival and resulted in high numbers of cure. Rejection of tumor rechallenge occurred after treatment with Toca 511 + 5-FC or combined with lomustine, but not with lomustine + 5-FC. Mixed lymphocyte-tumor cell reactions using splenocytes from cured animals showed robust killing of target cells in an effector:target ratio-dependent manner with Toca 511 + 5-FC and Toca 511 + 5-FC + lomustine day 10. CONCLUSION: The combination of Toca 511 + 5-FC and lomustine shows promising efficacy with no additive toxicity in murine glioma models. Immunotherapeutic responses resulting in long-term survival were preserved despite lomustine-related myelosuppression.

Intravenous administration of retroviral replicating vector, Toca 511, demonstrates therapeutic efficacy in orthotopic immune-competent mouse glioma model.

Toca 511 (vocimagene amiretrorepvec), a nonlytic, amphotropic retroviral replicating vector (RRV), encodes and delivers a functionally optimized yeast cytosine deaminase (CD) gene to tumors. In orthotopic glioma models treated with Toca 511 and 5-fluorocytosine (5-FC) the CD enzyme within infected cells converts 5-FC to 5-fluorouracil (5-FU), resulting in tumor killing. Toca 511, delivered locally either by intratumoral injection or by injection into the resection bed, in combination with subsequent oral extended-release 5-FC (Toca FC), is under clinical investigation in patients with recurrent high-grade glioma (HGG). If feasible, intravenous administration of vectors is less invasive, can easily be repeated if desired, and may be applicable to other tumor types. Here, we present preclinical data that support the development of an intravenous administration protocol. First we show that intravenous administration of Toca 511 in a preclinical model did not lead to widespread or uncontrolled replication of the RVV. No, or low, viral DNA was found in the blood and most of the tissues examined 180 days after Toca 511 administration. We also show that RRV administered intravenously leads to efficient infection and spread of the vector carrying the green fluorescent protein (GFP)-encoding gene (Toca GFP) through tumors in both immune-competent and immune-compromised animal models. However, initial vector localization within the tumor appeared to depend on the mode of administration. Long-term survival was observed in immune-competent mice when Toca 511 was administered intravenously or intracranially in combination with 5-FC treatment, and this combination was well tolerated in the preclinical models. Enhanced survival could also be achieved in animals with preexisting immune response to vector, supporting the potential for repeated administration. On the basis of these and other supporting data, a clinical trial investigating intravenous administration of Toca 511 in patients with recurrent HGG is currently open and enrolling.

An LXR agonist promotes glioblastoma cell death through inhibition of an EGFR/AKT/SREBP-1/LDLR-dependent pathway.

Glioblastoma (GBM) is the most common malignant primary brain tumor of adults and one of the most lethal of all cancers. Epidermal growth factor receptor (EGFR) mutations (EGFRvIII) and phosphoinositide 3-kinase (PI3K) hyperactivation are common in GBM, promoting tumor growth and survival, including through sterol regulatory element-binding protein 1 (SREBP-1)-dependent lipogenesis. The role of cholesterol metabolism in GBM pathogenesis, its association with EGFR/PI3K signaling, and its potential therapeutic targetability are unknown. In our investigation, studies of GBM cell lines, xenograft models, and GBM clinical samples, including those from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, uncovered an EGFRvIII-activated, PI3K/SREBP-1-dependent tumor survival pathway through the low-density lipoprotein receptor (LDLR). Targeting LDLR with the liver X receptor (LXR) agonist GW3965 caused inducible degrader of LDLR (IDOL)-mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results show that EGFRvIII can promote tumor survival through PI3K/SREBP-1-dependent upregulation of LDLR and suggest a role for LXR agonists in the treatment of GBM patients.

Correlation of mineral density and elastic modulus of natural enamel white spot lesions using X-ray microtomography and nanoindentation.

Our objectives were to correlate the mineral density (MD) and elastic modulus (E) of natural white spot lesions (WSLs) and compare them with analytical and numerical models. Five natural WSLs from four extracted sound premolar teeth were scanned at a voxel size of 7.6µm using a desktop X-ray microtomography (XRMT) system. Five hydroxyapatite phantoms with densities ranging from 1.52 to 3.14gcm⁻³ were used as calibration standards for each scan. MD throughout the WSLs was quantified using an MD calibration equation derived from hydroxyapatite phantoms. Subsequently, teeth were cross-sectioned and the E modulus was measured systematically across the WSLs at intervals of 25 and 50µm using nanoindentation. The MD and E modulus of WSLs correlated well. The relationship may be expressed as E=E°exp(-bP) (R²=0.952) with E° the elastic modulus of the fully dense material, P the porosity and b a constant. The results for sound enamel were compared with Spears model. The limitation of Spears model to the WSLs is discussed and an alternative model developed by Rice for porous materials is proposed. Clinical implications of this work for quantifying de-/remineralization of teeth are pointed out. We conclude that XRMT can be utilized to extrapolate the E modulus of WSLs. This provides a basis for non-destructive, longitudinal analysis of WSLs in de-/remineralization studies of enamel.

Targeted therapy for malignant glioma patients: lessons learned and the road ahead.

Molecularly targeted therapies are transforming the care of patients with malignant gliomas, including glioblastoma, the most common malignant primary brain tumor of adults. With an arsenal of small molecule inhibitors and antibodies that target key components of the signal transduction machinery that are commonly activated in gliomas, neuro-oncologists and neurosurgeons are poised to transform the care of these patients. Nonetheless, successful application of targeted therapies remains a challenge. Strategies are lacking for directing kinase inhibitor or other pathway-specific therapies to individual patients most likely to benefit. In addition, response to targeted agents is determined not only by the presence of the key mutant kinases, but also by other critical changes in the molecular circuitry of cancer cells, such as loss of key tumor suppressor proteins, the selection for kinase-resistant mutants, and the deregulation of feedback loops. Understanding these signaling networks, and studying them in patients, will be critical for developing rational combination therapies to suppress resistance for malignant glioma patients. Here we review the current status of molecular targeted therapies for malignant gliomas. We focus initially on identifying some of the insights gained to date from targeting the EGFR/PI3K/Akt/mTOR signaling pathway in patients and on how this has led toward a reconceptualization of some of the challenges and directions for targeted treatment. We describe how advances from the world of genomics have the potential to transform our approaches toward targeted therapy, and describe how a deeper understanding of the complex nature of cancer, and its adeptness at rewiring molecular circuitry to evade targeted agents, has raised new challenges and identified new leads.

EGFR signaling through an Akt-SREBP-1-dependent, rapamycin-resistant pathway sensitizes glioblastomas to antilipogenic therapy.

Glioblastoma, the most common malignant brain tumor, is among the most lethal and difficult cancers to treat. Although epidermal growth factor receptor (EGFR) mutations are frequent in glioblastoma, their clinical relevance is poorly understood. Studies of tumors from patients treated with the EGFR inhibitor lapatinib revealed that EGFR induces the cleavage and nuclear translocation of the master transcriptional regulator of fatty acid synthesis, sterol regulatory element-binding protein 1 (SREBP-1). This response was mediated by Akt; however, clinical data from rapamycin-treated patients showed that SREBP-1 activation was independent of the mammalian target of rapamycin complex 1, possibly explaining rapamycin's poor efficacy in the treatment of such tumors. Glioblastomas without constitutively active EGFR signaling were resistant to inhibition of fatty acid synthesis, whereas introduction of a constitutively active mutant form of EGFR, EGFRvIII, sensitized tumor xenografts in mice to cell death, which was augmented by the hydroxymethylglutaryl coenzyme A reductase inhibitor atorvastatin. These results identify a previously undescribed EGFR-mediated prosurvival metabolic pathway and suggest new therapeutic approaches to treating EGFR-activated glioblastomas.

Fyn and SRC are effectors of oncogenic epidermal growth factor receptor signaling in glioblastoma patients.

Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. Expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR- and EGFRvIII-dependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies.

Villous B cells of the small intestine are specialized for invariant NK T cell dependence.

B cells are important in mucosal microbial homeostasis through their well-known role in secretory IgA production and their emerging role in mucosal immunoregulation. Several specialized intraintestinal B cell compartments have been characterized, but the nature of conventional B cells in the lamina propria is poorly understood. In this study, we identify a B cell population predominantly composed of surface IgM(+) IgD(+) cells residing in villi of the small intestine and superficial lamina propria of the large intestine, but distinct from the intraepithelial compartment or organized intestinal lymphoid structures. Small intestinal (villous) B cells are diminished in genotypes that alter the strength of BCR signaling (Bruton tyrosine kinase(xid), Galphai2(-/-)), and in mice lacking cognate BCR specificity. They are not dependent on enteric microbial sensing, because they are abundant in mice that are germfree or genetically deficient in TLR signaling. However, villous B cells are reduced in the absence of invariant NK T cells (Jalpha18(-/-) or CD1d(-/-) mice). These findings define a distinct population of conventional B cells in small intestinal villi, and suggest an immunologic link between CD1-restricted invariant NK T cells and this B cell population.

Expression analysis and evolutionary conservation of the mouse germ cell-specific D6Mm5e gene.

During our search for genes required for gonadal development and function in the mouse, we identified D6Mm5e (DNA segment, Chr 6. Miriam Meisler 5, expressed), a gene with an expression pattern highly restricted to the embryonic ovary and the postnatal testis. Based on RT-PCR, Northern blot, and in situ hybridization analyses, we show that D6Mm5e is expressed in the germ cells of the female embryo upon their initial entry into meiosis, and in male germ cells during the last stages of spermatogenesis. Two transcripts are detected in the gonads resulting from the alternative splicing of exon 8. This splicing event does not introduce a frame shift, and creates an mRNA product that uses the same stop codon as the longer transcript. Although D6Mm5e does not belong to any known protein family and does not contain any known protein signature motifs, the high level of evolutionary conservation and the cellular and temporal expression suggest that D6Mm5e may have a role in male and female gametogenesis. Here we report the gonad-restricted mRNA expression profile of D6Mm5e in the mouse, and the evolutionary conservation of its amino acid sequence.

TCR-mediated hyper-responsiveness of autoimmune Galphai2(-/-) mice is an intrinsic naïve CD4(+) T cell disorder selective for the Galphai2 subunit.

Heterotrimeric Gi signaling regulates immune homeostasis, since autoimmunity occurs upon disruption of this pathway. However, the role of the lymphocyte-expressed Galphai subunits (Galphai2 and 3) on T cell activation and cytokine production is poorly understood. To examine this role, we studied T lymphocytes from mice deficient in the Galphai2 or Galphai3 subunits. Galphai2(-/-) but not Galphai3(-/-) splenocytes were hyper-responsive for IFN-gamma and IL-4 production following activation through the TCR. Galphai2(-/-) T cells had a relaxed costimulatory requirement for IL-2 secretion and proliferation compared to wild-type cells. Purified naïve Galphai2(-/-) T cells produced more IL-2 than naïve wild-type T cells following TCR activation, indicating that the hyper-responsive cytokine profile was not due to the expanded Galphai2(-/-) memory T cells, but involved an intrinsic T cell alteration. Cytokine hyper-responsiveness was not seen when purified Galphai2(-/-) T cells were stimulated with phorbol myristic acetate/ionomycin, localizing the alteration to a proximal TCR-specific signaling pathway. Galphai2(-/-) CD4(+) T cells were distinguished from wild-type or Galphai3(-/-) T cells by a globally augmented TCR-induced calcium response. These findings indicate that Galphai2(-/-) mice have an intrinsic CD4(+) T cell abnormality in TCR signaling which may be one cause of augmented T cell effector function and Galphai2(-/-) autoimmune susceptibility.