The periaqueductal gray as a critical site to mediate reward seeking during predatory hunting

Previous studies using morphine-treated dams reported a role for the rostral lateral periaqueductal gray (rIPAG) in the behavioral switching between nursing and insect hunting, likely to depend on an enhanced seeking response to the presence of an appetitive rewarding cue (i.e., the roach). To elucidate the neural mechanisms mediating such responses, in the present study, we first observed how the rIPAG influences predatory hunting in male rats. Our behavioral observations indicated that bilateral rIPAG NMDA lesions dramatically interfere with prey hunting, leaving the animal without chasing or attacking the prey, but do not seem to affect the general levels of arousal, locomotor activity and regular feeding. Next, using Phaseolus vulgaris-leucoagglutinin (PHA-L), we have reviewed the rIPAG connection pattern, and pointed out a particularly dense projection to the hypothalamic orexinergic cell group. Double labeled PHA-L and orexin sections showed an extensive overlap between PHA-L labeled fibers and orexin cells, revealing that both the medial/perifornical and lateral hypothalamic orexinergic cell groups receive a substantial innervation from the rIPAG. We have further observed that both the medial/perifornical and lateral hypothalamic orexinergic cell groups up-regulate Fos expression during prey hunting, and that rIPAG lesions blunted this Fos increase only in the lateral hypothalamic, but not in the medial/perifornical, orexinergic group, a finding supposedly associated with the lack of motivational drive to actively pursue the prey. Overall, the present results suggest that the rIPAG should exert a critical influence on reward seeking by activating the lateral hypothalamic orexinergic cell group. (C) 2011 Elsevier B.V. All rights reserved.

Antibody responses in Plasmodium falciparum malaria and their relation to protection against the disease

Protective immunity against Plasmodium falciparum may be obtained after repeated exposure to infection. Several studies indicate that immunity against the blood stages of the P. Falciparum infection is mainly antibody mediated. Protective antibodies may act either on their own, mediate antibody-dependent phagocytosis and/or cell-mediated neutralization of parasites. This thesis describes several aspects of humoral immune responses to P. falciparum infection in individuals of different age groups, different genetic background and with different degrees of malaria exposure. Several target antigens for antibody-mediated inhibition of parasite growth or invasion have been identified. One such antigen is Pf332, which appears on the surface of parasitized erythrocytes at late trophozoite and schizont stage. This surface exposure makes the antigen a possible target for opsonizing antibodies. We optimized an in vitro assay for studying cellmediated parasite neutralization in the presence of Pf332-reactive antibodies. Our data demonstrate that, Pf332 specific antibodies are able to inhibit parasite growth on their own and in cooperation with human monocytes. The P. falciparum parasites have evolved several mechanisms to evade the host neutralizing immune responses. In this thesis, we show that freshly isolated P. falciparum parasites from children living in a malaria endemic area of Burkina Faso were less sensitive for growth inhibition in vitro by autologous immunoglobulins (Ig) compared with heterologous ones. Analyses of two consecutive isolates taken 14 days apart, with regard to genotypes and sensitivity to growth inhibition in vitro, did not give any clear-cut indications on possible mechanisms leading to a reduced inhibitory activity in autologous parasite/antibody combinations. The frequent presence of persisting parasite clones in asymptomatic children indicates that the parasite possesses as yet undefined mechanisms to evade neutralizing immune responses. Transmission reducing measures such insecticide treated nets (ITNs) have been shown to be effective in reducing morbidity and mortality from malaria. However, concerns have been raised that ITNs usage could affect the acquisition of malaria immunity. We studied the effect of the use of insecticide treated curtains (ITC) on anti-malarial immune responses of children living in villages with ITC since birth. The use of ITC did neither affect the levels of parasite neutralizing immune responses nor the multiplicity of infection. These results indicate that the use of ITC does not interfere with the acquisition of anti-malarial immunity in children living in a malaria hyperendemic area. There is substantial evidence that the African Fulani tribe is markedly less susceptible to malaria infection compared to other sympatrically living ethnic tribes. We investigated the isotypic humoral responses against P. falciparum asexual blood stages in different ethnic groups living in sympatry in two countries exhibiting different malaria transmission intensities, Burkina Faso and Mali. We observed higher levels of the total malaria-specific-IgG and its cytophilic subclasses in individuals of the Fulani tribe as compared to non-Fulani individuals. Fulani individuals also showed higher levels of antibodies to measles antigen, indicating that the intertribal differences are not specific for malaria and might reflect a generally activated immune system in the Fulani.

Influences of porcine circo-, arteriviruses and cathelicidins in plasmacytoid dendritic cell-derived interferon-alpha responses

Type I interferons (IFNs), mainly IFN-α/β play a crucial role in innate defense against viruses. In addition to their direct antiviral activity, type I IFNs have antitumoral and immunomodulatory effects. Although all cells are virtually able to induce IFN-α, the plasmacytoid dendritic cell (pDC) subset represents the ultimate producers of IFN-α as well as other proinflammatory cytokines. Due to the specific expression of TLR7 and TLR9 recognizing single-stranded (ss) RNA and unmethylated CpG motifs respectively, pDCs can secrete up to 1000 times more IFN-α than any cellular types. Additionally, it is well known that several cytokines including type I and II IFNs, Flt3-L, IL-4 and GM-CSF favor pDC-derived IFN-α responses to unmethylated CpG motifs. In a first step, we aimed to characterize and clarify the interactions of two porcine viruses with pDCs. The double-stranded DNA replicative forms of porcine circovirus type 2 (PCV2) were demonstrated to inhibit CpG-induced IFN- α by pDCs. Our study showed that none of the cytokines known to enhance pDC responsiveness can counter-regulate the PCV2-mediated inhibition of IFN-α induced by CpG, albeit IFN-γ significantly reduced the level of inhibition. Interestingly, the presence of IFN-γ enabled pDCs to induce IFN-α to low doses of PCV2. We also noted that after DNase treatment, PCV2 preparations were still able to stimulate pDCs. These data suggest that encapsulated viral ssDNA promotes the induction of IFN-α in pDCs treated with IFN-γ whereas free DNA, presumably as double-stranded forms, was responsible for inhibiting pDC responses. Regarding PRRSV, it has been reported that North American isolates did not induce and even inhibited IFN-α response in pDCs. However, PRRSV infection was also shown to lead to an induction of IFN-α in the serum and in the lungs suggesting that certain cells are responsive to the virus. Contrasting to previous reports we found that numerous PRRSV isolates directly induced IFN-α in pDCs. This response was still observed after UV-inactivation of viruses and required TLR7 signaling. The inhibition of CpG-induced IFN-α was weak and strain dependent, again contrasting with a previous report. We also observed that IFN-γ and IL-4 enhanced IFN-α response to two prototype strains, VR-2332 and LVP23. In summary, we demonstrated that both PCV2 and PRRSV promote IFN-α secretion in pDCs in vitro suggesting that IFN-α detected in PCV2- or PRRSV-infected animal might originate from pDCs. On the other hand, PRRSV replication is restricted to the macrophage (MΦ) lineage. These innate immune cells represent a heterogeneous population which can be induce to “classical” (M1) and “alternative” (M2) activated MΦ acquiring inflammatory or “wound-healing” functional properties, respectively. Nonetheless, little is known about the effect of polarization into M1 or M2 and the susceptibility of these cells to PRRSV. Thus, we examined the impact of cytokine on MΦ polarization into M1 or M2. Infections of these cells by several PRRSV isolates enabled the discrimination of PRRSV isolate in a genotype- and irulencedependent manner in M1 and IFN-β-activated MΦ. In contrast, the expression of PRRSV nucleocapsid in M2 or inactivated MΦ was indistinguishable among the PRRSV isolates tested. In the last part of my Thesis, we investigated the influence of three synthetic porcine cathelicidin peptides for their ability to deliver nucleic acid to pDCs. We reported that all cathelicidins tested can complex and quickly deliver nucleic acids resulting in IFN-α induction. Moreover, we show that the typical α- helical amphipathic conformation is required to mediate killing of bacteria but not for inducing IFN-α secretion by pDCs. Furthermore, we found that E.coli treated with one of these cathelicidins is able to induce significantly higher levels of IFN-α compared to a non-sense version of the peptide. These data suggest that cathelicidins could influence the immune response in a two-step process. First, these peptides target bacteria leading to cell lysis. In turn, cathelicidins form complexes and deliver extracellular microbial nucleic acids released into pDCs. These pDC-derived IFN-α responses could be of particular relevance in driving the adaptive immune responses against microbial infections.

Tropomyosin implicated in host protective responses to microfilariae in onchocerciasis

A cDNA from adult female Onchocerca volvulus encoding the C-terminal portion of a tropomyosin isoform (termed MOv-14) has been shown previously to confer protective immunity in rodent models of onchocerciasis. The full-length sequence (designated Ov-tmy-1) obtained by PCR amplification, codes for a protein of 33 kDa and shares 91% identity with tropomyosins from other nematodes, falling to 57% identity with human α-tropomyosin. Ov-TMY-1 migrates with an apparent molecular mass of 42 kDa on SDS/PAGE and is present in all life-cycle stages, as determined by immunoblotting. Immunogold electron microscopy identified antigenic sites within muscle blocks and the cuticle of microfilariae and infective larvae. Anti-MOv14 antibodies were abundant in mice exhibiting serum-transferable protection against microfilariae conferred by vaccination with a PBS-soluble parasite extract. In contrast, little or no MOv14-specific antibody was present in mice inoculated with live microfilariae, in which resistance is mediated by antibody-independent mechanisms. In human infections, there was an inverse correlation between anti-tropomyosin IgG levels and densities of microfilariae in the skin. Seropositivity varied with the relative endemicity of infection. An immunodominant B cell epitope within Ov-TMY-1 (AQLLAEEADRKYD) was mapped to the N terminus of the MOv14 protein by using sera from protectively vaccinated mice. Intriguingly, the sequence coincides with an IgE-binding epitope within shrimp tropomyosin, believed to be responsible for hypersensitivity in individuals exhibiting allergy to shellfish. IgG and IgE antibodies reacting with the O. volvulus epitope were detected in human infections. It is concluded that antibody responses to tropomyosin may be important in limiting microfilarial densities in a proportion of individuals with onchocerciasis and have the potential to mediate hypersensitivity reactions to dead microfilariae, raising the possibility of a link with the immunopathology of infection.

A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens

Experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) residues 139–151 (HSLGKWLGHPDKF) can be prevented by treatment with a T cell receptor (TCR) antagonist peptide (L144/R147) generated by substituting at the two principal TCR contact residues in the encephalitogenic peptide. The TCR antagonist peptide blocks activation of encephalitogenic Th1 helper cells in vitro, but the mechanisms by which the antagonist peptide blocks EAE in vivo are not clear. Immunization with L144/R147 did not inhibit generation of PLP-(139–151)-specific T cells in vivo. Furthermore, preimmunization with L144/R147 protected mice from EAE induced with the encephalitogenic peptides PLP-(178–191) and myelin oligodendrocyte protein (MOG) residues 92–106 and with mouse myelin basic protein (MBP). These data suggest that the L144/R147 peptide does not act as an antagonist in vivo but mediates bystander suppression, probably by the generation of regulatory T cells. To confirm this we generated T cell lines and clones from animals immunized with PLP-(139–151) plus L144/R147. T cells specific for L144/R147 peptide were crossreactive with the native PLP-(139–151) peptide, produced Th2/Th0 cytokines, and suppressed EAE upon adoptive transfer. These studies demonstrate that TCR antagonist peptides may have multiple biological effects in vivo. One of the principal mechanisms by which these peptides inhibit autoimmunity is by the induction of regulatory T cells, leading to bystander suppression of EAE. These results have important implications for the treatment of autoimmune diseases where there are autopathogenic responses to multiple antigens in the target organ.

Elevated anxiety and antidepressant-like responses in serotonin 5-HT1A receptor mutant mice

The brain serotonin (5-hydroxytryptamine; 5-HT) system is a powerful modulator of emotional processes and a target of medications used in the treatment of psychiatric disorders. To evaluate the contribution of serotonin 5-HT1A receptors to the regulation of these processes, we have used gene-targeting technology to generate 5-HT1A receptor-mutant mice. These animals lack functional 5-HT1A receptors as indicated by receptor autoradiography and by resistance to the hypothermic effects of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). Homozygous mutants display a consistent pattern of responses indicative of elevated anxiety levels in open-field, elevated-zero maze, and novel-object assays. Moreover, they exhibit antidepressant-like responses in a tail-suspension assay. These results indicate that the targeted disruption of the 5-HT1A receptor gene leads to heritable perturbations in the serotonergic regulation of emotional state. 5-HT1A receptor-null mutant mice have potential as a model for investigating mechanisms through which serotonergic systems modulate affective state and mediate the actions of psychiatric drugs.

Selection of Gβ Subunits with Point Mutations That Fail to Activate Specific Signaling Pathways In Vivo: Dissecting Cellular Responses Mediated by a Heterotrimeric G Protein in Dictyostelium discoideum

In Dictyostelium discoideum, a unique Gβ subunit is required for a G protein–coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2, triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization, and chemotaxis. Null mutations of the cAR1, Gα2, and Gβ genes completely impair all these responses. To dissect specificity in Gβγ signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gβ genes and isolated Gβ alleles that lacked the capacity to activate some effectors but retained the ability to regulate others. These mutant Gβ subunits were able to link cAR1 to G2, to support gene expression, and to mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None was able to activate adenylyl cyclase, and some did not support chemotaxis. Thus, we separated in vivo functions of Gβγ by making point mutations on Gβ. Using the structure of the heterotrimeric G protein displayed in the computer program CHAIN, we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gβs and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase. These residues formed an area that overlaps but is not identical to regions where bovine Gtβγ interacts with its regulators, Gα and phosducin.

The role of antigen and IL-12 in sustaining Th1 memory cells in vivo: IL-12 is required to maintain memory/effector Th1 cells sufficient to mediate protection to an infectious parasite challenge

IL-12 plays a central role in both the induction and magnitude of a primary Th1 response. A critical question in designing vaccines for diseases requiring Th1 immunity such as Mycobacterium tuberculosis and Leishmania major is the requirements to sustain memory/effector Th1 cells in vivo. This report examines the role of IL-12 and antigen in sustaining Th1 responses sufficient for protective immunity to L. major after vaccination with LACK protein (LP) plus rIL-12 and LACK DNA. It shows that, after initial vaccination with LP plus rIL-12, supplemental boosting with either LP or rIL-12 is necessary but not sufficient to fully sustain long-term Th1 immunity. Moreover, endogenous IL-12 is also shown to be required for the induction, maintenance, and effector phase of the Th1 response after LACK DNA vaccination. Finally, IL-12 is required to sustain Th1 cells and control parasite growth in susceptible and resistant strains of mice during primary and secondary infection. Taken together, these data show that IL-12 is essential to sustain a sufficient number of memory/effector Th1 cells generated in vivo to mediate long-term protection to an intracellular pathogen.

Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs

Heterotrimeric G proteins mediate the earliest step in cell responses to external events by linking cell surface receptors to intracellular signaling pathways. Gz is a member of the Gi family of G proteins that is prominently expressed in platelets and brain. Here, we show that deletion of the α subunit of Gz in mice: (i) impairs platelet aggregation by preventing the inhibition of cAMP formation normally seen at physiologic concentrations of epinephrine, and (ii) causes the mice to be more resistant to fatal thromboembolism. Loss of Gzα also results in greatly exaggerated responses to cocaine, reduces the analgesic effects of morphine, and abolishes the effects of widely used antidepressant drugs that act as catecholamine reuptake inhibitors. These changes occur despite the presence of other Giα family members in the same cells and are not accompanied by detectable compensatory changes in the level of expression of other G protein subunits. Therefore, these results provide insights into receptor selectivity among G proteins and a model for understanding platelet function and the effects of psychoactive drugs.

Arabidopsis nph1 and npl1: Blue light receptors that mediate both phototropism and chloroplast relocation

UV-A/blue light acts to regulate a number of physiological processes in higher plants. These include light-driven chloroplast movement and phototropism. The NPH1 gene of Arabidopsis encodes an autophosphorylating protein kinase that functions as a photoreceptor for phototropism in response to low-intensity blue light. However, nph1 mutants have been reported to exhibit normal phototropic curvature under high-intensity blue light, indicating the presence of an additional phototropic receptor. A likely candidate is the nph1 homologue, npl1, which has recently been shown to mediate the avoidance response of chloroplasts to high-intensity blue light in Arabidopsis. Here we demonstrate that npl1, like nph1, noncovalently binds the chromophore flavin mononucleotide (FMN) within two specialized PAS domains, termed LOV domains. Furthermore, when expressed in insect cells, npl1, like nph1, undergoes light-dependent autophosphorylation, indicating that npl1 also functions as a light receptor kinase. Consistent with this conclusion, we show that a nph1npl1 double mutant exhibits an impaired phototropic response under both low- and high-intensity blue light. Hence, npl1 functions as a second phototropic receptor under high fluence rate conditions and is, in part, functionally redundant to nph1. We also demonstrate that both chloroplast accumulation in response to low-intensity light and chloroplast avoidance movement in response to high-intensity light are lacking in the nph1npl1 double mutant. Our findings therefore indicate that nph1 and npl1 show partially overlapping functions in two different responses, phototropism and chloroplast relocation, in a fluence rate-dependent manner.

Neurotensin-deficient mice show altered responses to antipsychotic drugs

The peptide transmitter neurotensin (NT) exerts diverse neurochemical effects that resemble those seen after acute administration of antipsychotic drugs (APDs). These drugs also induce NT expression in the striatum; this and other convergent findings have led to the suggestion that NT may mediate some APD effects. Here, we demonstrate that the ability of the typical APD haloperidol to induce Fos expression in the dorsolateral striatum is markedly attenuated in NT-null mutant mice. The induction of Fos and NT in the dorsolateral striatum in response to typical, but not atypical, APDs has led to the hypothesis that the increased expression of these proteins is mechanistically related to the production of extrapyramidal side effects (EPS). However, we found that catalepsy, which is thought to reflect the EPS of typical APDs, is unaffected in NT-null mutant mice, suggesting that NT does not contribute to the generation of EPS. We conclude that NT is required for haloperidol-elicited activation of a specific population of striatal neurons but not haloperidol-induced catalepsy. These results are consistent with the hypothesis that endogenous NT mediates a specific subset of APD actions.

Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach.

The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation.

Intracellular polyamines mediate inward rectification of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that lack the glutamate receptor GluR2 subunit are Ca(2+)-permeable and exhibit inwardly rectifying current responses to kainate and AMPA. A proportion of cultured rat hippocampal neurons show similar Ca(2+)-permeable inwardly rectifying AMPA receptor currents. Inward rectification in these neurons was lost with intracellular dialysis and was not present in excised outside-out patches but was maintained in perforated-patch whole-cell recordings, suggesting that a diffusible cytoplasmic factor may be responsible for rectification. Inclusion of the naturally occurring polyamines spermine and spermidine in the recording pipette prevented loss of rectification in both whole-cell and excised-patch recordings; Mg2+ and putrescine were without effect. Inward rectification of Ca(2+)-permeable AMPA receptors may reflect voltage-dependent channel block by intracellular polyamines.

Integration of autonomic and local mechanisms in regulating cardiovascular responses to heating and cooling in a reptile (Crocodylus porosus)

Reptiles change heart rate and blood flow patterns in response to heating and cooling, thereby decreasing the behavioural cost of thermoregulation. We tested the hypothesis that locally produced vasoactive substances, nitric oxide and prostaglandins, mediate the cardiovascular response of reptiles to heat. Heart rate and blood pressure were measured in eight crocodiles (Crocodylus porosus) during heating and cooling and while sequentially inhibiting nitric-oxide synthase and cyclooxygenase enzymes. Heart rate and blood pressure were significantly higher during heating than during cooling in all treatments. Power spectral density of heart rate and blood pressure increased significantly during heating and cooling compared to the preceding period of thermal equilibrium. Spectral density of heart rate in the high frequency band (0.19-0.70 Hz) was significantly greater during cooling in the saline treatment compared to when nitric-oxide synthase and cyclooxygenase enzymes were inhibited. Cross spectral analysis showed that changes in blood pressure preceded heart rate changes at low frequencies (

Consumers' responses to sponsorship by foreign companies

Purpose: The study aims to explore the novel issue of how consumers perceive sponsorship initiatives by foreign companies and how the sponsors' country-of-origin (COO) cue may influence sponsorship outcomes. Design/methodology/approach: The measurement model and proposed relationships were tested based on a sample of 811 Chinese consumers using confirmatory factor analysis and structural path analysis. Findings: The results reveal that attitudes toward the sponsor completely mediate the effect of event involvement on willingness to buy from the sponsor (WBS). Attitudes toward the sponsor also partially mediate the relationship between economic animosity towards a specific sponsor's COO and WBS. Additionally, the level of a country's economic competitiveness found to moderate the negative relationship between economic animosity, attitudes toward the sponsor and WBS. Research limitations/implications: The nature of the product type and the desire of the consumer to own such a product may have influenced the measurement of willingness to buy. Practical implications: The research adds to the existing knowledge by identifying the opportunities and potential biases that a foreign company may encounter when considering sponsoring a mega sport event in a different cultural context. The study helps managers to understand how sports sponsorship could be used effectively in emerging markets. Originality/value: The proposed conceptual model advances the application of classical conditioning theory, the consumer animosity model of foreign product purchase and the belief-attitude-intention hierarchy in the sponsorship arena. It is the first investigation of the role of event involvement and economic animosity in understanding sponsorship responses. © Emerald Group Publishing Limited.