Delineating the mechanism(s) of BDNF/TrkB mediated proliferation in Neuroblastoma

Delineating the mechanism(s) of BDNF/TrkB mediated proliferation in Neuroblastoma Timothy Christopher Graham, B.S. Supervisory Professor: Patrick Zweidler-McKay, MD/PhD Neuroblastoma is the most common extra-cranial solid tumor in children, arising from neural crest precursor cells. The neurotrophin receptors (TrkA/B/C) have been implicated as important prognostic markers, linking the biology of the tumor to patient outcome. High expression of TrkA and TrkC receptors have been linked to favorable biological features and high patient survival, while TrkB is expressed in unfavorable, aggressive tumors. Several studies suggest that high levels and activation of TrkB by its ligand brain-derived neurotrophic factor (BDNF) stimulates tumor cell survival, proliferation, and chemoresistance. However, little is known about the molecular mechanisms that regulate proliferation. The TrkB signaling pathway in neuroblastoma cells has been difficult to evaluate due to the loss of TrkB expression when the cells are used in vitro. Here we determined the role of proximal signaling pathways downstream of TrkB on neuroblastoma proliferation. By analyzing a panel of neuroblastoma cell lines, we found that the SMS-KCN cells express detectable levels of protein and mRNA levels of TrkB as analyzed by western, RT-PCR, and surface expression by flow cytometry. By the addition of exogenous human recombinant BDNF, we showed that activation of TrkB is important in the proliferation of the cells and can be repressed by inhibiting TrkB kinase function. By BDNF stimulation and use of specific kinase inhibitors, the common pathways involving PLCg, PI3K/AKT, and MAPK were initially investigated in addition to PI3K/MTOR and FYN pathways. We demonstrate for the first time that Fyn plays a critical role in TrkB mediated proliferation in neuroblastoma. Constitutively active and over-expressed Fyn reduced neuroblastoma proliferation, as measured by PCNA expression. Knockdown of Fyn by shRNA was shown to cooperate with activated TrkB for an enhanced proliferative response. Although TrkB activation has been implicated in the proliferation of neuroblastoma cells, little is known about its effects on cell cycle regulation. Protein levels of pRB, CDK2, CDK4, CDC25A, cyclin D1, and cyclin E were analyzed following BDNF stimulation. We found that BDNF mediated activation of TrkB induces multiple common proximal signaling pathways including the anti-proliferative Fyn pathway and drives cell cycle machinery to enhance the proliferation of neuroblastoma cells.

Nonclassical mechanisms of progesterone action in the brain: I. Protein kinase C activation in the hypothalamus of female rats.

The modulation of gene regulation by progesterone (P) and its classical intracellular regulation by progestin receptors in the brain, resulting in alterations in physiology and behavior has been well studied. The mechanisms mediating the short latency effects of P are less well understood. Recent studies have revealed rapid nonclassical signaling action of P involving the activation of intracellular signaling pathways. We explored the involvement of protein kinase C (PKC) in P-induced rapid signaling in the ventromedial nucleus of the hypothalamus (VMN) and preoptic area (POA) of the rat brain. Both the Ca2+-independent (basal) PKC activity representing the activation of PKC by the in vivo treatments and the Ca+2-dependent (total) PKC activity assayed in the presence of exogenous cofactors in vitro were determined. A comparison of the two activities demonstrated the strength and temporal status of PKC regulation by steroid hormones in vivo. P treatment resulted in a rapid increase in basal PKC activity in the VMN but not the POA. Estradiol benzoate priming augmented P-initiated increase in PKC basal activity in both the VMN and POA. These increases were inhibited by intracerebroventricular administration of a PKC inhibitor administered 30 min prior to P. The total PKC activity remained unchanged demonstrating maximal PKC activation within 30 min in the VMN. In contrast, P regulation in the POA significantly attenuated total PKC activity +/- estradiol benzoate priming. These rapid changes in P-initiated PKC activity were not due to changes in PKC protein levels or phosphorylation status.

The effects of ultraviolet B radiation on immune responses to {\it Borrelia burgdorferi}, the causative agent of Lyme disease

Ultraviolet B (UVB) radiation, in addition to being carcinogenic, is also immunosuppressive. Immunologically, UVB induces suppression locally, at the site of irradiation, or systemically, by inducing the production of a variety of immunosuppressive cytokines. Systemic effects include suppression of delayed-type hypersensitivity (DTH) responses to a variety of antigens (e.g. haptens, proteins, bacterial antigens, or alloantigens). One of the principal mediators of UV-induced immune suppression is the T helper-2 (Th2) cytokine interleukin-10 (IL-10); this suggests that UV irradiation induces suppression by shifting the immune response from a Th1 (cellular) to a Th2 (humoral) response. These "opposing" T helper responses are usually mutually exclusive, and polarized Th1 or Th2 responses may lead to either protection from infection or increased susceptibility to disease, depending on the infectious agent and the route of infection.^ This study examines the effects of UVB irradiation on cellular and humoral responses to Borrelia burgdorferi (Bb), the causative agent of Lyme disease (LD) in both immunization and infectious disease models; in addition, it examines the role of T cells in protection from and pathology of Bb infection. Particular emphasis is placed on the Bb-specific antibody responses following irradiation since UVB effects on humoral immunity are not fully understood. Mice were irradiated with a single dose of UV and then immunized (in complete Freund's adjuvant) or infected with Bb (intradermally at the base of the tail) in order to examine both DTH and antibody responses in both systems. UVB suppressed the Th1-associated antibodies IgG2a and IgG2b in both systems, as well as the DTH response to Bb in a dose dependent manner. Injection of anti-IL-10 antibody into UV-irradiated mice within 24 h after UV exposure restored the DTH response, as well as the Th1 antibody (IgG2a and IgG2b) response. In addition, injecting recombinant IL-10 mimicked some of the effects of UV radiation.^ Bb-specific Th1 T cell lines (BAT2.1-2.3) were generated to examine the role of T cells in Lyme borreliosis. All lines were CD4$\sp+,$ $\alpha\beta\sp+$ and proliferated specifically in response to Bb. The BAT2 cell lines not only conferred a DTH response to naive C3H recipients, but reduced the number of organisms recovered from the blood and tissues of mice infected with Bb. Furthermore, BAT2 cell lines protected mice from Bb-induced periarthritis. ^

Developmental effects of estrogen and PCBs in the reproductive tract of BALB/C mice

Many of the tumorigenic effects that result from neonatal exposure to both natural and synthetic estrogens resemble those found in humans exposed to diethylstilbestrol (DES) in utero. Using this established DES neonatal mouse model, my goal was to investigate long-term molecular and morphological effects of certain polychlorinated biphenyls (PCBs) that are weakly estrogenic in adult mice. Focusing on the cervicovaginal (CV) tract, since this is where tumors develop in the BALB/c mouse, I first assessed the 17β-estradiol (E2) dose-response for expression of lactoferrin (LTF). LTF is a highly inducible estrogen biomarker that is permanently altered in uteri from neonatally treated mice. Treatments were administered via 5 subcutaneous injections beginning within 16 hrs after birth, days 1–5. ^ The ontogeny of LTF expression from mouse CV tracts was determined by examining three different stages of life: pups, immature, and mature mice. Northern RNA analysis and immunohistochemistry showed that neonatal E 2 treatment both increases and decreases LTF expression. Early expression of LTF in the CV tract at all doses occurred in pups. In both immature and adult mice, increased LTF expression was dependent on whether E2 induced ovary-dependent or ovary-independent persistent vaginal cornification. ^ Next, I studied biological responses from neonatally PCB exposed adult mice. As expected, using a neonatal uterine bioassay I showed that 2 ′4′6′-trichloro-4-biphenylol (OH-PCB-30), 2′3′4′ 5-tetrachloro-4-biphenyloI (OH-PCB-61), and OH-PCB-30/61 (50/50 mixture), were estrogenic causing a dose-dependent increase in uterine weight. ^ Long-term effects of OH-PCB 30 [200 μg/pup/day] were most similar to E2 as seen by an increased uterine wet weight in day 50 mice similar to E2 [5 μg/pup/day] (141% and 140% of control, respectively). Another similarity between OH-PCB 30 and E2 neonatally treated mice was found in those sacrificed at 20 months of age. At these same doses CV tract squamous cell carcinoma induction was 43% of E2 treated mice and 47% of OH-PCB 30 treated mice. Differences were noted in adenoaquamous; cell carcinoma development, where 16% of OH-PCB-30 neonatally treated mice developed tumors versus 8% for E2. Based on these results using the neonatal mouse model, I conclude that the OH-PCBs tested are strongly estrogenic and tumorigenic showing dose-response relationships when exposure occurs during development of the reproductive tract in mice. These results may have important implications for risk assessment in determining the effects of xenoestrogens exposure early versus later in life. ^

Bovine mastitis: the diagnostic properties of a PCR-based assay to monitor the Staphylococcus aureus genotype B status of a herd, using bulk tank milk

Staphylococcus aureus genotype B (GTB) is a contagious mastitis pathogen in cattle, occurring in up to 87% of individuals. Because treatment is generally insufficient, culling is often required, leading to large economic loss in the Swiss dairy industry. As the detection of this pathogen in bulk tank milk (BTM) would greatly facilitate its control, a novel real-time quantitative PCR-based assay for BTM has previously been developed and is now being evaluated for its diagnostic properties at the herd level. Herds were initially classified as to their Staph. aureus GTB status by a reference method. Using BTM and herd pools of single-quarter and 4-quarter milk, the herds were then grouped by the novel assay, and the resulting classifications were compared. A total of 54 dairy herds were evaluated. Using the reference method, 21 herds were found to be GTB positive, whereas 33 were found to be negative. Considering the novel assay using both herd pools, all herds were grouped correctly, resulting in maximal diagnostic sensitivities (100%) and specificities (100%). For BTM samples, diagnostic sensitivities and specificities were 90 and 100%, respectively. Two herds were false negative in BTM, because cows with clinical signs of mastitis were not milked into the tank. Besides its excellent diagnostic properties, the assay is characterized by its low detection level, high efficiency, and its suitability for automation. Using the novel knowledge and assay, eradication of Staph. aureus GTB from a dairy herd may be considered as a realistic goal.

Parasite- and predator-induced maternal effects in the great tit (Parus major)

Both predators and parasites can elicit behavioral and physiological responses in prey and hosts, respectively. These responses may involve the reallocation of resources and may thus limit each other. We investigated the effects of concurrent pre-laying exposure of great tit females (Parus major) to both a simulated predation risk and a nest-based ectoparasite, the hen flea (Ceratophyllus gallinae), on nestling growth and development. We manipulated perceived predation risk using models and vocalizations of sparrowhawks (Accipiter nisus). At the start of incubation, we swapped whole clutches between treated and untreated nests to separate pre-laying maternal effects from posthatching effects. Since costs and benefits of maternal responses to parasites need to be assessed under parasite pressure, we infested half of the rearing nests with hen fleas. Parasites had negative effects on mass gain and wing growth, both via maternal effects and via direct exposure of nestlings, whereas maternal predation risk had no significant effect. The interaction between predator and parasite treatments was not significant and, thus, suggests the absence of a trade-off between the 2 stressors operating at the level of maternal effects. Alternatively, the complexity of the design, despite a relatively large sample size, may have limited the power for detection of this expected trade-off.

Judaism defended against the attacks of T. J. C. of Oxford : a reply to 2 letters on the Damascus question, in "The Times" newspaper of the 20th and 27th oct., 1840

by Morris J. Raphall

Zionistisches A-B-C-Buch

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