IntroductionThe role of CD4 ϩ cells in antitumor immunity remains controversial and poorly understood. 1,2 They are known to mediate potent therapeutic effect in the setting of hematopoietic stem cell allotransplantation and donor lymphocyte infusion in hematologic malignancy, 3,4 but antigen-specific T helper (Th) cells have been studied to much lesser extent. A lack of clarity regarding CD4 ϩ cells is due, in no small part, to the complexity of their biology. CD4 ϩ T cells can differentiate into diverse subsets with specific phenotypes that can have self-reinforcing and opposing functions, but these T-cell subsets have not been comprehensively studied in tumor-bearing mice.Historically, CD4 ϩ T lymphocytes have been thought of as mere providers of stimuli to help the putatively more important CD8 ϩ effectors, which eliminate cancer by direct cytotoxicity. [5][6][7] There are several studies showing that CD4 ϩ T helper (Th) cells are capable of protecting the host against tumor challenge and even of mediating tumor regression on their own in the setting of either solid or hematopoietic disease. [8][9][10][11][12][13] Furthermore, protection was maintained against MHC class II-negative multiple myeloma model and involved cross-presentation by professional antigenpresenting cells (APCs) and activation of tumoricidal activity mediated by macrophages secreting IFN-␥. 14 A similar IFN-␥-dependent mechanism was involved in the rejection of MHC class II-negative tumor in severe combined immunodeficient (SCID) mice. 15 In some cases, the ability to reject antigen-expressing tumor by specific naive Th cells was thought to be substantially better than the ability of CD8 ϩ cells. 16 Classically, effector CD4 ϩ T cells have been categorized into T helper 1 (Th1) and T helper 2 (Th2) subsets. 17,18 Limited studies indicate that both subtypes elicit antitumor effects, 19-21 but the Th1-polarized cells, secreting IFN-␥ and capable of enhancing activity of cytotoxic CD8 ϩ lymphocytes, have traditionally been regarded as more efficient. [22][23][24][25] However, it is also clear that CD4 ϩ T regulatory cells (T regs ) can efficiently suppress the function of antitumor CD8 ϩ T cells. 5,[26][27][28] Recently, the novel Th17 lineage, generated in the presence of TGF-␤ and IL-6 and expanded under the influence of IL-23, [29][30][31] has been associated with responses against certain infections and implicated in the development of autoimmunity in animal models that had been previously linked to Th1-type responses (experimental autoimmune encephalitis, collagen-induced arthritis). 32,33 They also seem to play an important role in the pathogenesis of graft-versus-host disease (GVHD). 34,35 Th17 cells have been found in various tumors, including mycosis fungoides, Sézary syndrome, and prostate cancer. 36,37 Kryczek et al reported the presence of naturally occurring Th17 cells and T regs in the tumor microenvironment and tumor-draining lymph nodes in both human and mice tumors. 38 Proinflammatory cytokines including IL-17A, IL-6, and I...

Brainstem serotonin (5-HT) neurons modulate activity of many neural circuits in the mammalian brain, but in many cases endogenous mechanisms have not been resolved. Here, we analyzed actions of raphé 5-HT neurons on respiratory network activity including at the level of the pre-Bötzinger complex (pre-BötC) in neonatal rat medullary slices in vitro, and in the more intact nervous system of juvenile rats in arterially perfused brainstem-spinal cord preparations in situ. At basal levels of activity, excitation of the respiratory network via simultaneous release of 5-HT and substance P (SP), acting at 5-HT 2A/2C , 5-HT 4 , and/or neurokinin-1 receptors, was required to maintain inspiratory motor output in both the neonatal and juvenile systems. The midline raphé obscurus contained spontaneously active 5-HT neurons, some of which projected to the pre-BötC and hypoglossal motoneurons, colocalized 5-HT and SP, and received reciprocal excitatory connections from the pre-BötC. Experimentally augmenting raphé obscurus activity increased motor output by simultaneously exciting pre-BötC and motor neurons. Biophysical analyses in vitro demonstrated that 5-HT and SP modulated background cation conductances in pre-BötC and motor neurons, including a nonselective cation leak current that contributed to the resting potential, which explains the neuronal depolarization that augmented motor output. Furthermore, we found that 5-HT, but not SP, can transform the electrophysiological phenotype of some pre-BötC neurons to intrinsic bursters, providing 5-HT with an additional role in promoting rhythm generation. We conclude that raphé 5-HT neurons excite key circuit components required for generation of respiratory motor output.

Action potential firing rates are generally limited by the refractory period, which depends on the recovery from inactivation of voltage-gated Na channels. In cerebellar Purkinje neurons, the kinetics of Na channels appear specialized for rapid firing. Upon depolarization, an endogenous open-channel blocker rapidly terminates current flow but prevents binding of the “fast” inactivation gate. Upon repolarization, unbinding of the blocker produces “resurgent” Na current while allowing channels to recover rapidly. Because other cerebellar neurons, including granule cells, unipolar brush cells, and neurons of the cerebellar nuclei, also fire rapidly, we tested whether these cells might also express Na channels with resurgent kinetics. Neurons were acutely isolated from mice and rats, and TTX-sensitive Na currents were recorded under voltage clamp. Unlike Purkinje cells, the other cerebellar neurons produced only tiny resurgent currents in solutions optimized for voltage-clamping Na currents (50 mM Na+; Co2+ substitution for Ca2+). Under more physiological ionic conditions (155 mM Na+; 2 mM Ca2+ with 0.03 mM Cd2+), however, granule cells, unipolar brush cells, and cerebellar nuclear cells all produced robust resurgent currents. The increase in resurgent current, which was greater than predicted by the Goldman-Hodgkin-Katz equation, appeared to result from a combination of knock-off of open-channel blockers by permeating ions as well as relief of divalent block at negative potentials. These results indicate that resurgent current is typical of many cerebellar neurons and suggest that rapid open-channel block and unblock may be a widespread mechanism for restoration of Na channel availability in rapidly firing neurons.

We examined the kinetic properties of voltage-gated Na ϩ channels and their contribution to the repetitive spiking activity of medullary raphé neurons, which exhibit slow pacemaking and strong spiking adaptation. The study is based on a combination of whole-cell patch-clamp, modeling and real-time computation. Na ϩ currents were recorded from neurons in brain slices obtained from male and female neonatal rats, using voltage-clamp protocols designed to reduce space-clamp artifacts and to emphasize functionally relevant kinetic features. A detailed kinetic model was formulated to explain the broad range of transient and stationary voltage-dependent properties exhibited by Na ϩ currents. The model was tested by injecting via dynamic clamp a model-based current as a substitute for the native TTX-sensitive Na ϩ currents, which were pharmacologically blocked. The model-based current reproduced well the native spike shape and spiking frequency. The dynamics of Na ϩ channels during repetitive spiking were indirectly examined through this model. By comparing the spiking activities generated with different kinetic models in dynamic-clamp experiments, we determined that statedependent slow inactivation contributes significantly to spiking adaptation. Through real-time manipulation of the model-based current, we established that suprathreshold Na ϩ current mainly controls spike shape, whereas subthreshold Na ϩ current modulates spiking frequency and contributes to the pacemaking mechanism. Since the model-based current was injected in the soma, the results also suggest that somatic Na ϩ channels are sufficient to establish the essential spiking properties of raphé neurons in vitro.

Summary Recent advances in molecular imaging and nanotechnology are providing new opportunities for biomedical imaging with great promise for the development of novel imaging agents. The unique optical, magnetic, and chemical properties of materials at the scale of nanometers allow the creation of imaging probes with better contrast enhancement, increased sensitivity, controlled biodistribution, better spatial and temporal information, multi-functionality and multi-modal imaging across MRI, PET, SPECT, and ultrasound. These features could ultimately translate to clinical advantages such as earlier detection, real time assessment of disease progression and personalized medicine. However, several years of investigation into the application of these materials to cancer research has revealed challenges that have delayed the successful application of these agents to the field of biomedical imaging. Understanding these challenges is critical to take full advantage of the benefits offered by nano-sized imaging agents. Therefore, this article presents the lessons learned and challenges encountered by a group of leading researchers in this field, and suggests ways forward to develop nanoparticle probes for cancer imaging. Published by Elsevier Ltd.