An inferotemporal coding strategy robust to partial object occlusion.

Object coding in primate ventral pathway cortex progresses in sparseness/compression/efficiency, from many orientation signals in V1, to fewer 2D/3D part signals in V4, to still fewer multi-part configuration signals in AIT (anterior inferotemporal cortex). 1-11 This progression could lead to individual neurons exclusively selective for unique objects, the sparsest code for identity, especially for highly familiar, important objects. 12-18 To test this, we trained macaque monkeys to discriminate 8 simple letter-like shapes in a match-to-sample task, a design in which one-to-one coding of letters by neurons could streamline behavior. Performance increased from chance to >80% correct over a period of weeks, after which AIT neurons showed clear learning effects, with increased selectivity for multi-part configurations within the trained alphabet shapes. But these neurons were not exclusively tuned for unique letters based on training, since their responsiveness generalized to different, non-trained shapes containing the same configurations. This multi-part configuration coding limit in AIT is not maximally sparse, but it could explain the robustness of primate vision to partial object occlusion, which is common in the natural world and problematic for computer vision. Multi-part configurations are highly diagnostic of identity, and neural signals for various partial object structures can provide different but equally sufficient evidence for whole object identity across most occlusion conditions.

Multiple adaptable mechanisms early in the primate visual pathway.

We describe experiments that isolate and characterize multiple adaptable mechanisms that influence responses of orientation-selective neurons in primary visual cortex (V1) of anesthetized macaque (Macaca fascicularis). The results suggest that three adaptable stages of machinery shape neural responses in V1: a broadly tuned early stage and a spatio-temporally tuned later stage, both of which provide excitatory input, and a normalization pool that is also broadly tuned. The early stage and the normalization pool are revealed by adapting gratings that themselves fail to evoke a response from the neuron: either low temporal frequency gratings at the null orientation or gratings of any orientation drifting at high temporal frequencies. When effective, adapting stimuli that altered the sensitivity of these two mechanisms caused reductions of contrast gain and often brought about a paradoxical increase in response gain due to a relatively greater desensitization of the normalization pool. The tuned mechanism is desensitized only by stimuli well matched to a neuron's receptive field. We could thus infer desensitization of the tuned mechanism by comparing effects obtained with adapting gratings of preferred and null orientation modulated at low temporal frequencies.

Nonlinear signal summation in magnocellular neurons of the macaque lateral geniculate nucleus.

Magnocellular (M-), but not parvocellular (P-), neurons of the macaque lateral geniculate nucleus (LGN) differ distinctively in their responses to counterphase-modulated and drifting gratings. Relative to stimulation with drifting gratings, counterphase modulation reduces the responses of M- cells in a band around 25 Hz, producing a "notch" in the temporal modulation transfer function (tMTF). The notch is prominent in nearly every M- cell with little variation in the temporal frequency at which it is deepest. The machinery responsible for the notch lies mostly outside the classical linear center. Directly driving the notching mechanism with annular gratings evokes no linear response but elicits a second harmonic (F2) modulation of the discharge accompanied by a drop in the mean discharge (F0). Analysis of the S- potential, which reveals inputs from ganglion cells, shows that 1) tMTFs of the afferent retinal ganglion cells are not notched and 2) during stimulation with annular gratings, the second harmonic component is present, but the drop in the F0 is largely absent from the responses of parasol ganglion cells. These results suggest that the notch is caused by the combined action of the linear response and the second harmonic response, both inherited from retina, and a suppression that originates after the retina. Our results reveal a distinctive signal transformation in the LGN and they show that nearly every M- cell exhibits a spatial nonlinearity like that observed in Y cells of the cat.

A new code for contrast in the primate visual pathway.

We characterize a hitherto undocumented type of neuron present in the regions bordering the principal layers of the macaque lateral geniculate nucleus. Neurons of this type were distinguished by a high and unusually regular maintained discharge that was suppressed by spatiotemporal modulation of luminance or chromaticity within the receptive field. The response to any effective stimulus was a reduction in discharge, reminiscent of the "suppressed-by-contrast" cells of the cat retina. To a counterphase-modulated grating, the response was a phase-insensitive suppression modulated at twice the stimulus frequency, implying a receptive field comprised of multiple mechanisms that generate rectifying responses. This distinctive nonlinearity makes the neurons well suited to computing a measure of contrast energy; such a signal might be important in regulating sensitivity early in visual cortex.

Effects of azithromycin on clinical isolates of Pseudomonas aeruginosa from cystic fibrosis patients.

There is considerable interest in the use of azithromycin for the treatment of lung disease in patients with cystic fibrosis (CF). Although its mechanism of action as an inhibitor of bacterial protein synthesis has been well-established, it is less clear how azithromycin ameliorates the lung disease associated with Pseudomonas aeruginosa, which is considered to be resistant to the drug. We tested the effects of azithromycin on clinical isolates (CIs) from CF patients and compared them with laboratory reference strains to establish how this drug might interfere with the production of bacterial virulence factors that are relevant to the pathogenesis of airway disease in CF patients. Azithromycin inhibited P aeruginosa PAO1 protein synthesis by 80%, inhibiting bacterial growth and the expression of immunostimulatory exoproducts such as pyocyanin, as well as the gene products necessary for biofilm formation. In contrast, the effects of azithromycin on CIs of P aeruginosa were much more variable, due in large part to their slow growth and limited exoproduct expression. Culture supernatants for two of three clinical strains induced appreciable CXCL8 expression from cultured epithelial cells. Azithromycin treatment of the organisms inhibited 65 to 70% of this induction; azithromycin had no direct effect on the ability of either normal cells or CF epithelial cells to produce CXCL8. Azithromycin does decrease the P aeruginosa synthesis of immunostimulatory exoproducts and is likely to be most effective against planktonic, actively growing bacteria. This effect is less predictable against CIs than the prototypic strain PAO1.

Bacterial induction of TNF-alpha converting enzyme expression and IL-6 receptor alpha shedding regulates airway inflammatory signaling.

Airway epithelial cells have a major role in initiating inflammation in response to bacterial pathogens. Through the immediate induction of CXCL8 and cytokine expression, polymorphonuclear cells are mobilized and activated to eradicate the infecting organisms. However, the influx of polymorphonuclear cells and the effects of their toxic exoproducts impede respiratory function. We postulated that respiratory epithelial cells must also participate in the regulation of their own proinflammatory signaling. Both Staphylococcus aureus and Pseudomonas aeruginosa were found to potently activate IL-6 expression immediately upon contact with epithelial cells, and by 1 h induced TNF-alpha converting enzyme (TACE) transcription. By 4 h of bacterial exposure, TACE colocalized with IL-6Ralpha on the apical surface of airway cells, and by 24 h, soluble IL-6Ralpha accumulated in the cell culture supernatant. Epithelial IL-6 and soluble IL-6Ralpha were shown to participate in trans-signaling, interacting with membrane-associated gp130 to activate CCL-2 expression and inhibit additional CXCL8 production. Thus, bacteria are physiological activators of TACE expression, which provides a mechanism to regulate inflammatory signaling that is initiated by airway epithelial cells.

The effect of cyclosporin A on airway cell proinflammatory signaling and pneumonia.

Cyclosporin A (CsA) blocks T cell activation by interfering with the Ca2+-dependent phosphatase, calcineurin. Proinflammatory responses to bacteria that are activated by Ca2+-fluxes in airway cells are a potential target for CsA. Although local immunosuppression may be advantageous to control airway inflammation, it could also increase susceptibility to bacterial pneumonia and invasive infection. As aerosolized CsA is currently under study in lung transplantation, we examined its direct effects on airway cells as well as in a murine model of pneumonia. Epithelial interleukin-6 production was very effectively inhibited by CsA, whereas CXCL8 production, the major PMN chemokine, was only modestly diminished. Responses to a TLR2 agonist Pam3Cys were more sensitive to CsA inhibition than those activated by Pseudomonas aeruginosa. CsA substantially blocked activation of nuclear factor of activated T cells and cAMP-responsive element-binding protein (P<0.001), inhibited CCAAT/enhancer-binding protein by 50% (P<0.05), and minimally blocked activator protein-1 and nuclear factor-kappaB responses to bacteria in epithelial cells. The in vitro effects were confirmed in a mouse model of P. aeruginosa infection with similar rates of PMN recruitment, pneumonia and mortality in CsA treated and control mice. These studies indicate that airway epithelial signaling is a potential target for CsA, and such local immunosuppression may not increase susceptibility to invasive infection.

TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells.

Toll-like receptors (TLRs) mediate host responses to bacterial gene products. As the airway epithelium is potentially exposed to many diverse inhaled bacteria, TLRs involved in defense of the airways must be broadly responsive, available at the exposed apical surface of the cells, and highly regulated to prevent activation following trivial encounters with bacteria. We demonstrate that TLR2 is enriched in caveolin-1-associated lipid raft microdomains presented on the apical surface of airway epithelial cells after bacterial infection. These receptor complexes include myeloid differentiation protein (MyD88), interleukin-1 receptor-activated kinase-1, and TNF receptor-associated factor 6. The signaling capabilities of TLR2 are amplified through its association with the asialoganglioside gangliotetraosylceramide (Gal beta 1,2GalNAc beta 1,4Gal beta 1,4Glc beta 1,1Cer), which has receptor function itself for many pulmonary pathogens. Ligation of either TLR2 or asialoGM1 by ligands with specificity for either receptor, by Pseudomonas aeruginosa, or by Staphylococcus aureus stimulates IL-8 production through activation of NF-kappa B, as mediated by TLR2 and MyD88. Thus, TLR2 in association with asialo-glycolipids presented within the context of lipid rafts provides a broadly responsive signaling complex at the apical surfaces of airway cells to initiate the host response to potential bacterial infection.

Toll-like receptors in normal and cystic fibrosis airway epithelial cells.

Toll-like receptors (TLRs) mediate cellular responses to diverse microbial ligands. The distribution and function of TLRs in airway cells were studied to identify which are available to signal the presence of inhaled pathogens and to establish if differences in TLR expression are associated with the increased proinflammatory responses seen in cystic fibrosis (CF). Isogenic, polarized CF and control bronchial epithelial cell lines, human airway cells in primary culture, and cftr null and wild-type mice were compared. TLRs 1-10, MD2, and MyD88 were expressed in CF and normal cells. Only TLR2 transcription was modestly increased in CF as compared with normal epithelial cells following bacterial stimulation. TLR2 was predominantly at the apical surface of airway cells and was mobilized to cell surface in response to bacteria. TLR4 was present in a more basolateral distribution in airway cells, but appeared to have a limited role in epithelial responses. Lipopolysaccharide failed to activate nuclear factor-kappaB in these cells, and TLR2 dominant negative but not TLR4 dominant negative mutants inhibited activation by both Gram-negative and Gram-positive bacteria. Increased availability of TLR2 at the apical surfaces of CF epithelial cells is consistent with the increased proinflammatory responses seen in CF airways and suggests a selective participation of TLRs in the airway mucosa.

Pseudomonas aeruginosa flagella activate airway epithelial cells through asialoGM1 and toll-like receptor 2 as well as toll-like receptor 5.

The distribution of specific toll-like receptors and components of the signaling pathways activated by Pseudomonas aeruginosa flagella were studied in airway epithelial cells. Initially flagella bound to the apical surface of polarized epithelial cells, where they prominently colocalized with asialoGM1. By 4 h of exposure to flagella, toll-like receptor (TLR)5 expression was induced, mobilized to the apical surface of the cells, and colocalized with superficial flagella. Interleukin-8 expression in airway cells was activated by flagella through induction of Ca(2+) fluxes, Src, Ras, and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase and nuclear factor-kappaB activation, a pathway previously associated with asialoGM1-mediated stimuli. There was evidence for participation of asialoGM1 and TLR2 as well as TLR5 in the response to flagella, and increased asialoGM1 correlated directly with increased signaling. TLR2 DN or TLR5 DN mutations inhibited interleukin-8 induction by 78% and 35%, respectively (P < 0.001 for each). The participation of TLR2 as well as TLR5 was confirmed in Chinese hamster ovary cells transfected with either human TLR2 or TLR5 in which flagella activated a nuclear factor-kappaB-luciferase reporter to the same extent. Flagella signaling in airway cells can be initiated by interactions with asialoGM1 and TLR2 as well as by activation of TLR5. The availability of exposed receptors on the apical surface of polarized airway epithelial cells is a major factor in the activation of signaling pathways by flagella.