Correlation of 6-18F-fluoro-L-dopa PET uptake with proliferation and tumor grade in newly diagnosed and recurrent gliomas

6-(18)F-fluoro-l-dopa ((18)F-FDOPA) measured with PET as a biomarker of amino acid uptake has been investigated in brain tumor imaging. The aims of the current study were to determine whether the degree of (18)F-FDOPA uptake in brain tumors predicted tumor grade and was associated with tumor proliferative activity in newly diagnosed and recurrent gliomas.

Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression

Background Inappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Epithelial cells constitute the interface between gut microbiota and host tissue, and may regulate host responses to commensal enteric bacteria. Gnotobiotic animals represent a powerful approach to study bacterial-host interaction but are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete the cultivable intestinal microbiota of conventionally raised mice and that would prove to have significant biologic validity. Methodology/Principal Findings Previously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by 400 fold while ensuring the animals' health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer's patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors to a level similar to that of germ-free mice and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium. Conclusion We present a robust protocol for depleting conventionally raised mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion phenocopies physiological characteristics of germ-free mice.

In pediatric lymphoblastic leukemia of B-cell origin, a small population of primitive blast cells is noncycling, suggesting them to be leukemia stem cell candidates

Kinetic investigations in pediatric acute lymphoblastic leukemia (ALL) are based on all blast cells and, therefore, reflect the proliferative characteristics of the predominant immunophenotype of leukemic cells. Nothing is known about proliferation of immunologically defined rare subpopulations of leukemic cells. In this study, mononuclear cells from the bone marrow of 15 children with untreated CD19 B-cell precursor ALL were examined for proliferative features according to the immunophenotype. After exclusion of highly proliferating residual normal hematopoietic cells, ∼ 3% of blast cells were CD19 and showed a low percentage of cells in S-phase assessed by the bromodeoxyuridine labeling index (BrdU-LI): median BrdU-LI, 0.19% [interquartile range (IQR), 0.15-0.40%]. In contrast, a median BrdU-LI of 7.2% (IQR, 5.7-8.8%) was found for the major CD19 blast cell compartment. Staining smears of sorted CD19 cells for CD10 or CD34 revealed a small fraction of CD19CD10 or CD19CD34 blast cells. These cells were almost nonproliferating with a median BrdU-LI of <0.1% (IQR, 0-0.2%). This proliferative behavior is suggestive of a stem/progenitor cell function and, in addition, the low proliferative activity might render them more resistant to an antiproliferation-based chemotherapy. However, xenotransplantation experiments will be necessary to demonstrate a possible stem cell function.

Morphofunctional adaptations of the olfactory mucosa in postnatally developing rabbits

Rabbits are born blind and deaf and receive unusually limited maternal care. Consequently, their suckling young heavily rely on the olfactory cue for nipple attachment. However, the postnatal morphofunctional adaptations of olfactory mucosa (OM) are not fully elucidated. To clarify on the extent and the pattern of refinement of the OM following birth in the rabbit, morphologic and morphometric analysis of the mucosa were done at neonatal (0-1 days), suckling (2 weeks), weanling (4 weeks), and adult (6-8 months) stages of postnatal development. In all the age groups, the basic components of the OM were present. However, proliferative activity of cells of the mucosal epithelium decreased with increasing age as revealed by Ki-67 immunostaining. Diameters of axon bundles, packing densities of olfactory cells, and cilia numbers per olfactory cell knob increased progressively with age being 5.5, 2.1, and 2.6 times, respectively, in the adult as compared with the neonate. Volume fraction values for the bundles increased by 5.3% from birth to suckling age and by 7.4% from weaning to adulthood and the bundle cores were infiltrated with blood capillaries in all ages except in the adult where such vessels were lacking. The pattern of cilia projection from olfactory cell knobs also showed age-related variations, that is, arose as a tuft from the tips of the knobs in neonates and sucklings and in a radial pattern from the knob bases in weanlings and adults. These morphological changes may be attributed to the high olfactory functional demand associated with postnatal development in the rabbit.

Absence of the calcium-binding protein calretinin, not of calbindin D-28k, causes a permanent impairment of murine adult hippocampal neurogenesis

Calretinin (CR) and calbindin D-28k (CB) are cytosolic EF-hand Ca(2+)-binding proteins and function as Ca(2+) buffers affecting the spatiotemporal aspects of Ca(2+) transients and possibly also as Ca(2+) sensors modulating signaling cascades. In the adult hippocampal circuitry, CR and CB are expressed in specific principal neurons and subsets of interneurons. In addition, CR is transiently expressed within the neurogenic dentate gyrus (DG) niche. CR and CB expression during adult neurogenesis mark critical transition stages, onset of differentiation for CR, and the switch to adult-like connectivity for CB. Absence of either protein during these stages in null-mutant mice may have functional consequences and contribute to some aspects of the identified phenotypes. We report the impact of CR- and CB-deficiency on the proliferation and differentiation of progenitor cells within the subgranular zone (SGZ) neurogenic niche of the DG. Effects were evaluated (1) two and four weeks postnatally, during the transition period of the proliferative matrix to the adult state, and (2) in adult animals (3 months) to trace possible permanent changes in adult neurogenesis. The absence of CB from differentiated DG granule cells has no retrograde effect on the proliferative activity of progenitor cells, nor affects survival or migration/differentiation of newborn neurons in the adult DG including the SGZ. On the contrary, lack of CR from immature early postmitotic granule cells causes an early loss in proliferative capacity of the SGZ that is maintained into adult age, when it has a further impact on the migration/survival of newborn granule cells. The transient CR expression at the onset of adult neurogenesis differentiation may thus have two functions: (1) to serve as a self-maintenance signal for the pool of cells at the same stage of neurogenesis contributing to their survival/differentiation, and (2) it may contribute to retrograde signaling required for maintenance of the progenitor pool.

The structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development

OBJECTIVE: During postnatal development, mammalian articular cartilage acts as a surface growth plate for the underlying epiphyseal bone. Concomitantly, it undergoes a fundamental process of structural reorganization from an immature isotropic to a mature (adult) anisotropic architecture. However, the mechanism underlying this structural transformation is unknown. It could involve either an internal remodelling process, or complete resorption followed by tissue neoformation. The aim of this study was to establish which of these two alternative tissue reorganization mechanisms is physiologically operative. We also wished to pinpoint the articular cartilage source of the stem cells for clonal expansion and the zonal location of the chondrocyte pool with high proliferative activity. METHODS: The New Zealand white rabbit served as our animal model. The analysis was confined to the high-weight-bearing (central) areas of the medial and lateral femoral condyles. After birth, the articular cartilage layer was evaluated morphologically at monthly intervals from the first to the eighth postnatal month, when this species attains skeletal maturity. The overall height of the articular cartilage layer at each juncture was measured. The growth performance of the articular cartilage layer was assessed by calcein labelling, which permitted an estimation of the daily growth rate of the epiphyseal bone and its monthly length-gain. The slowly proliferating stem-cell pool was identified immunohistochemically (after labelling with bromodeoxyuridine), and the rapidly proliferating chondrocyte population by autoradiography (after labelling with (3)H-thymidine). RESULTS: The growth activity of the articular cartilage layer was highest 1 month after birth. It declined precipitously between the first and third months, and ceased between the third and fourth months, when the animal enters puberty. The structural maturation of the articular cartilage layer followed a corresponding temporal trend. During the first 3 months, when the articular cartilage layer is undergoing structural reorganization, the net length-gain in the epiphyseal bone exceeded the height of the articular cartilage layer. This finding indicates that the postnatal reorganization of articular cartilage from an immature isotropic to a mature anisotropic structure is not achieved by a process of internal remodelling, but by the resorption and neoformation of all zones except the most superficial (stem-cell) one. The superficial zone was found to consist of slowly dividing stem cells with bidirectional mitotic activity. In the horizontal direction, this zone furnishes new stem cells that replenish the pool and effect a lateral expansion of the articular cartilage layer. In the vertical direction, the superficial zone supplies the rapidly dividing, transit-amplifying daughter-cell pool that feeds the transitional and upper radial zones during the postnatal growth phase of the articular cartilage layer. CONCLUSIONS: During postnatal development, mammalian articular cartilage fulfils a dual function, viz., it acts not only as an articulating layer but also as a surface growth plate. In the lapine model, this growth activity ceases at puberty (3-4 months of age), whereas that of the true (metaphyseal) growth plate continues until the time of skeletal maturity (8 months). Hence, the two structures are regulated independently. The structural maturation of the articular cartilage layer coincides temporally with the cessation of its growth activity - for the radial expansion and remodelling of the epiphyseal bone - and with sexual maturation. That articular cartilage is physiologically reorganized by a process of tissue resorption and neoformation, rather than by one of internal remodelling, has important implications for the functional engineering and repair of articular cartilage tissue.

Ex vivo assessment of chemotherapy-induced apoptosis and associated molecular changes in patient tumor samples

BACKGROUND: There are inherent conceptual problems in investigating the pharmacodynamics of cancer drugs in vivo. One of the few possible approaches is serial biopsies in patients. However, this type of research is severely limited by methodological and ethical constraints. MATERIALS AND METHODS: A modified 3-dimensional tissue culture technique was used to culture human tumor samples, which had been collected during routine cancer operations. Twenty tumor samples of patients with non-small cell lung cancer (NSCLC) were cultured ex vivo for 120 h and treated with mitomycin C, taxotere and cisplatin. The cytotoxic activity of the anticancer agents was quantified by assessing the metabolic activity of treated tumor cultures and various assays of apoptosis and gene expression were performed. RESULTS: The proliferative activity of the tissue was maintained in culture as assessed by Ki-67 staining. Mitomycin C, cisplatin and taxotere reduced the metabolic activity of the tumor tissue cultures by 51%, 29% and 20%, respectively, at 120 h. The decrease in metabolic activity corresponded to the induction of apoptosis as demonstrated by the typical morphological changes, such as chromatin condensation and nuclear fragmentation. In addition, activated caspase-3 could be verified in apoptotic cells by immunohistochemistry. To verify functional aspects of apoptosis, the induction of chemotherapy-induced cell death was inhibited with the caspase inhibitor z-VAD.fmk. RNA was extracted from the tissue cultures after 120 h of ex vivo drug treatment and was of sufficient quality to allow quantitative PCR. CONCLUSION: The 3-dimensional ex vivo culture technique is a useful method to assess the molecular effects of pharmacological interventions in human cancer samples in vitro. This culture technique could become an important tool for drug development and for the prediction of in vivo drug efficacy.

Pathology: Classification and Immunoprofile

The classification of neuroendocrine neoplasms (NENs) has been evolving steadily over the last decades. Important prognostic factors of NENs are their proliferative activity and presence/absence of necrosis. These factors are reported in NENs of all body sites; however, the terminology as well as the exact rules of classification differ according to the location of the primary tumor. Only in gastroenteropancreatic (GEP) NENs a formal grading is performed. This grading is based on proliferation assessed by the mitotic count and/or Ki-67 proliferation index. In the lung, NEN grading is an intrinsic part of the tumor designation with typical carcinoids corresponding to neuroendocrine tumor (NET) G1 and atypical carcinoids to NET G2; however, the presence or absence of necrotic foci is as important as proliferation for the differentiation between typical and atypical carcinoids. Immunohistochemical markers can be used to demonstrate neuroendocrine differentiation. Synaptophysin and chromogranin A are, to date, the most reliable and most commonly used for this purpose. Beyond this, other markers can be helpful, for example in the situation of a NET metastasis of unknown primary, where a hormonal profile or a panel of transcription factors can give hints to the primary site. Many immunohistochemical markers have been shown to correlate with prognosis but are not used in clinical practice, for example cytokeratin 19 and KIT expression in pancreatic NETs. There is no predictive biomarker in use, with the exception of somatostatin receptor (SSTR) 2 expression for predicting the amenability of a tumor to in vivo SSTR targeting for imaging or therapy.

Synthesis and cytotoxic activity of non-naturally substituted 4-oxycoumarin derivatives

Coumarins are a large family of natural and synthetic compounds exerting different pharmacological effects, including cytotoxic, anti-inflammatory or antimicrobial. In the present communication we report the synthesis of a series of 12 diversely substituted 4-oxycoumarin derivatives including methoxy substituted 4-hydroxycoumarins, methyl, methoxy or unsubstituted 3-aryl-4-hydroxycoumarins and 4-benzyloxycoumarins and their anti-proliferative effects on breast adenocarcinoma cells (MCF-7), human promyelocytic leukemia cells (HL-60), human histiocytic lymphoma cells (U937) and mouse neuroblastoma cells (Neuro2a). The most potent bioactive molecule was the 4-hydroxy-5,7-dimethoxycoumarin (compound 1) which showed similar potency (IC(50) 0.2-2 μM) in all cancer cell lines tested. This non-natural product reveals a simple bioactive scaffold which may be exploited in further studies.

Impact of culture conditions on the proliferative lifespan of human T cells in vitro

BACKGROUND: In human T cells, telomerase is transiently expressed upon activation and stimulation and, as shown previously, telomerase levels are able to control the lifespan of T cells. To improve T-cell expansion it is of critical importance to understand the effects of culture parameters on telomerase activity and lifespan. METHODS: We investigated the influence of culture condition (FCS, human AB serum and autologous serum) and stimulation (PHA/feeder cells, anti-CD3/CD28 beads) on the lifespan, clonogenicity (number of positive wells), cell cycle, telomerase activity and telomere length of T cells in vitro. RESULTS: The proliferative lifespan of T cells expanded with PHA/feeder cells and autologous serum from different donors was doubled compared with stimulation with PHA/feeder cells and AB serum. No or only a small difference was found for T cells expanded with anti-CD3/CD28 beads and autologous or AB serum. The use of autologous serum also increased the clonogenicity to about three-fold compared with the use of AB serum or FCS, without any signs of differences in the fractions of cycling cells. Interestingly, T cells cultured with autologous serum exhibited a significantly higher telomerase activity at day 6 after stimulation and a reduced decline of telomerase activity compared with cultures with AB serum. DISCUSSION: The use of autologous serum combined with PHA stimulation and feeder cells remarkably extends the proliferative lifespan and clonogenicity and increases the telomerase activity of human T cells in vitro. This might be useful for applications where large numbers of specific T cells are required.

The absence of angiopoietin-2 leads to abnormal vascular maturation and persistent proliferative retinopathy

Angiopoietin-2 (Ang-2) antagonises the maturing effect of angiopoietin-1 (Ang-1) on blood vessels, and cooperates with VEGF to induce neovascularisation. In knockout mice, Ang-2 displayed a specific role in postnatal angiogenic remodelling. Here, we demonstrate that mice deficient in Ang-2 fail to form a proper spatial retinal vascular network. The retinal vasculature was characterised by reduced large vessel numbers and defects forming the superficial periphery mostly on the arteriolar site, and the secondary and tertiary deep capillary network. Hypoxia in the retinal periphery induced a four-fold VEGF upregulation and active endothelial proliferation for up to 60 days. Concomitantly, retinal digest preparations showed increased arteriolar (+33%) and capillary diameters (+90%), and fluorescein angiograms revealed leakiness of neovascular front. At one year of age, persistent preretinal vessels were non-leaky in accordance with a relative increase in the ratio of Ang-1 to VEGF. Taken together, the data suggest that Ang-2 has an important function in the spatial configuration of the three-dimensional retinal vasculature. Secondarily, prolonged VEGF activity results in a model of persistent proliferative retinopathy.

Decreased hypoxia-induced neovascularization in angiopoietin-2 heterozygous knockout mouse through reduced MMP activity

BACKGROUND/AIMS: Proliferative diabetic retinopathy is characterized by the formation of retinal neovascularization. Angiopoietin-2 (Ang-2) and matrix metalloproteinase (MMP) play a critical role in angiogenesis. However, the precise location and function of Ang-2 during formation of retinal neovascularizations driven by hypoxia in relation to MMP activity have not been elucidated. In this study, we investigated the response of Ang-2 heterozygous knockout retinas (Ang2(+/-) mouse) to hypoxia and its link to MMP activity in an oxygen-induced retinopathy (OIR) model. METHODS: Pre-retinal neovascularizations were quantitated in vertical sections. Intra-retinal angiogenesis was assessed by whole mount immunofluorescence staining of retinas. MMP activity was examined in retinal protein lysate and whole mount retinal in situ zymography. RESULTS: Ang2(+/-) retinas subjected to the OIR model showed 33% reduced neovascularization and 271% increased avascular zones at postnatal day 17. In the OIR model, Ang-2 was modestly expressed in pre-retinal neovascularizations and venules, but strongly in arterioles and capillary sprouts. MMPs were activated in close association to where Ang-2 is expressed. MMP activity was substantially decreased in Ang2(+/-) retinas. CONCLUSIONS: Our present data suggest the spatially concomitant expression of Ang2 and MMPs, and that Ang2 modulates hypoxia-induced neovascularization by regulating MMP activity.