SI neuron response variability is stimulus tuned and NMDA receptor dependent.

Skin brushing stimuli were used to evoke spike discharge activity in single skin mechanoreceptive afferents (sMRAs) and anterior parietal cortical (SI) neurons of anesthetized monkeys (Macaca fascicularis). In the initial experiments 10-50 presentations of each of 8 different stimulus velocities were delivered to the linear skin path from which maximal spike discharge activity could be evoked. Mean rate of spike firing evoked by each velocity (MFR) was computed for the time period during which spike discharge activity exceeded background, and an across-presentations estimate of mean firing rate (MFR) was generated for each velocity. The magnitude of the trial-by-trial variation in the response (estimated as CV; where CV = standard deviation in MFR/MFR) was determined for each unit at each velocity. MFR for both sMRAs and SI neurons (MFRsMRA and MFRSI, respectively) increased monotonically with velocity over the range 1-100 cm/s. At all velocities the average estimate of intertrial response variation for SI neurons (CVSI) was substantially larger than the corresponding average for sMRAs (CVsMRA). Whereas CVsMRA increased monotonically over the range 1-100 cm/s, CVSI decreased progressively with velocity over the range 1-10 cm/s, and then increased with velocity over the range 10-100 cm/s. The position of the skin brushing stimulus in the receptive field (RF) was varied in the second series of experiments. It was found that the magnitude of CVSI varied systematically with stimulus position in the RF: that is, CVSI was lowest for a particular velocity and direction of stimulus motion when the skin brushing stimulus traversed the RF center, and CVSI increased progressively as the distance between the stimulus path and the RF center increased. In the third series of experiments, either phencylidine (PCP; 100-500 microg/kg) or ketamine (KET; 0.5-7.5 mg/kg) was administered intravenously (iv) to assess the effect of block of N-methyl-D-aspartate (NMDA) receptors on SI neuron intertrial response variation. The effects of PCP on both CVSI and MFRSI were transient, typically with full recovery occurring in 1-2 h after drug injection. The effects of KET on CVSI and MFRSI were similar to those of PCP, but were shorter in duration (15-30 min). PCP and KET administration consistently was accompanied by a reduction of CVSI. The magnitude of the reduction of CVSI by PCP or KET was associated with the magnitude of CVSI before drug administration: that is, the larger the predrug CVSI, the larger the reduction in CVSI caused by PCP or KET. PCP and KET exerted variable effects on SI neuron mean firing rate that could differ greatly from one neuron to the next. The results are interpreted to indicate that SI neuron intertrial response variation is 1) stimulus tuned (intertrial response variation is lowest when the skin stimulus moves at 10 cm/s and traverses the neuron's RF center) and 2) NMDA receptor dependent (intertrial response variation is least when NMDA receptor activity contributes minimally to the response, and increases as the contribution of NMDA receptors to the response increases).     

Both the kind and magnitude of stimulus are important in overcoming the negative regulation of macrophage activation by PGE2

Macrophages activated for tumor cell killing by bacterial lipopolysaccharide (LPS) were shown to lose their cytolytic activity if exposed to physiological levels of prostaglandin E2 (PGE2). Increasing the LPS stimulus more than 100-fold over the amount needed to activate the cells did not substantially increase their resistance to the negative regulatory effect of PGE2. By contrast, killing mediated by macrophages activated by a mixture of LPS and gamma interferon was maintained. The degree of resistance conferred was directly related to the magnitude of the stimulus employed, reaching the point where not even 10(-5) M PGE2 would diminish killing. Killing by both activated resident and inflammatory peritoneal macrophages could be maintained, but it was easier to do so if the cells had been elicited by an inflammatory stimulus. A preparation of type I interferons produced by cells of the macrophage cell line J774A. 1 behaved similarly, but was over 500 times less efficient at helping to maintain killing than gamma (type II) interferon was. Alpha interferon alone, i.e., without LPS, was capable both of activating macrophages and of maintaining the activated state in the presence of PGE2. The capacity for both activation and maintenance could be strikingly enhanced, however, by mixing alpha and gamma interferons together under conditions that were free of detectable LPS. The data reported here collectively suggest that induction and maintenance of macrophage activation may be separable mechanistically, and that the interferons are important to host defense not only because they participate in the induction of macrophage activation for tumor cell killing but also because they help to maintain the activated state once it has been induced.     

In vivo activation of neutrophil function in hamsters by recombinant human granulocyte colony-stimulating factor.

The in vivo effect of Escherichia coli-derived recombinant human granulocyte colony-stimulating factor on neutrophil function was studied in golden Syrian hamsters. Significant increases in superoxide generation and specific binding of N-formylmethionyl-leucyl-phenylalanine were observed in neutrophils isolated 4 h following a single subcutaneous injection of the factor (30 micrograms/kg). However, phagocytotic activity was not significantly stimulated in hamsters treated with the factor. Recombinant human granulocyte colony-stimulating factor hastened the recovery of peripheral neutrophil counts in animals made leukopenic by prior treatment with cyclophosphamide. Beginning several hours after infection, resistance to lethal infection following intraperitoneal injection of Staphylococcus aureus was increased when neutropenic animals were treated daily with the factor. This protective effect was associated with increased peritoneal neutrophil counts and a decreased incidence of positive peritoneal bacterial cultures at 24 h after the start of treatment. These results suggest that recombinant human granulocyte colony-stimulating factor may be a useful adjunct in the treatment of bacterial infections in neutropenic patients.     

Interleukin-2 production in response to phytohemagglutinin is not necessarily dependent upon the T3-mediated pathway of T-cell activation

The present study was designed to investigate whether heterogeneity exists in the requirement for T3 molecules in the mechanism of T-cell activation and interleukin-2 (IL-2) release by IL-2-producing T-cell clones. Clones were derived from peripheral blood by a culture system which allows clonal expansion of essentially all T lymphocytes or from mixed lymphocyte culture (MLC) activated T-cell population. IL-2-producing clones were selected and cell aliquots treated with anti-T3 monoclonal antibodies (mAb) to induce modulation of T3 surface molecules. Although stimulation of modulated clones with different anti-T3 mAb did not lead to IL-2 production, 4/39 of these clones produced IL-2 after stimulation with phytohemagglutinin (PHA). The ability of these clones to be activated by PHA could not be explained by incomplete T3 modulation. In addition, two alloreactive clones were isolated from MLC population in which modulation of T3 antigens abrogated the IL-2 production induced by either anti-T2 mAb or allogeneic stimulation but had no effect on PHA-induced IL-2 release. These data further support the concept that PHA may trigger some T cells via surface molecules that are independent from the T3-Ti cell receptor molecular complex.     

The cortical response to sensory deprivation in adult rats is affected by gonadectomy

The present study investigated the effects of adult-onset sensory deprivation and gonadectomy. Adult male and female rats underwent unilateral transection of the infraorbital nerve. Half of the subjects had been gonadectomized 1 week prior to the nerve injury. We found that the areas of deprived barrels were significantly reduced when compared to barrels in the contralateral control hemisphere, and that this shrinkage was independent of sex and gonadectomy. We also found significant reductions in cytochrome oxidase staining intensity in the deprived barrels. While there were no differences in the magnitude of this effect between males and females, this effect was substantially more pronounced in the gonadectomized subjects. That is, gonadal hormones appeared to play a significant neuroprotective role in the metabolic response of the barrel cortex to deprivation. Thus, either males and females have a common neuroprotective hormonal pathway, or each has a sex-specific hormone pathway that serves an equivalent neuroprotective function.     

Expression and function of AIM, an activation inducer molecule of human lymphocytes, is dependent on the activation of protein kinase C

AIM is an activation inducer molecule selectively expressed by activated lymphocytes through which agonistic proliferative signals can be triggered. The relationship between the expression of AIM with the activation of protein kinase C (PKC) has been studied. Different activators of PKC such as the active phorbol esters, phorbol myristate acetate and phorbol dibutyrate, or the phorbol-related ester mezerein were able to induce AIM expression on peripheral blood lymphocytes as assessed by immunofluorescence flow cytometry. Moreover, the expression of this activation antigen was also induced by treatment of peripheral blood lymphocytes either with dioctanoyl-rac-glycerol, a synthetic analogue of diacylglycerol, the physiological mediator of PKC activation. Further indirect evidence that AIM expression was dependent on the activation of PKC was obtained by blockade of the induction of its expression in cells treated with H7, an inhibitor of PKC. The AIM expression can be detected as early as 3 h after addition of phorbol esters and it requires active RNA and protein synthesis. The activation of PKC appears to be also required in the proliferative response induced by anti-AIM monoclonal antibody (mAb) in conjunction with phorbol esters. Agents such as phorbol myristate acetate, phorbol dibutyrate or mezerein but not the inactive phorbol ester methyl-phorbol myristate acetate induced a high proliferation of peripheral blood lymphocytes in the presence of anti-AIM mAb. In addition, we have demonstrated that the anti-AIM mAb is not sufficient by itself to induce cellular proliferation once the AIM antigen is expressed at the cell surface, requiring the simultaneous stimulation of the PKC to trigger high proliferative responses. Furthermore, the anti-AIM mAb did not appear to exert its effect on proliferation by rapidly increasing the intracytoplasmic Ca2+ levels. Taken together all these results indicate that the expression and function of AIM antigen is dependent on the activation of PKC.     

Effects of attention, activation and stimulus regularity on short-term 'habituation' of the averaged evoked response.

Three studies are reported in which the effects of direction of attention, level of activation and regularity of stimulation on the rate of amplitude decrement over time of the auditory evoked vertex responses in humans were examined. Short-term, stimulus-by-stimulus changes were assessed by averaging across trains each of 10 click stimuli. The effect of directing attentions towards the stimuli was to enhance the N1 - P2 component, but usually only under conditions of high activation and with irregular stimulus presentation. Habituation rate was hardly affected by the experimental manipulations. The most clear-cut relationship between psychological influences and the AER was that between level of activation and the P2 - N2 component.     

The effect of NOD2 activation on TLR2-mediated cytokine responses is dependent on activation dose and NOD2 genotype

The mechanism by which mutations in NOD2 predispose to Crohn's disease (CD) is incompletely understood. In mice, NOD2 has been found to function as a negative regulator of Toll-like receptor 2 (TLR2) signaling. In contrast, studies in humans so far showed no negative regulatory interaction between NOD2 and TLR2, and in fact suggest a synergistic effect between the two. Here, we show that this interaction is dose dependent. Adding low doses of muramyl dipeptide (MDP) to TLR2 primed monocytes results in a significant increase in cytokine production, whereas adding higher doses of MDP led to a striking downregulation of the responses. This downregulation by high-dose MDP does not occur in monocytes from NOD2-deficient patients. The inhibitory role of NOD2 at high concentrations of MDP implicates a safety mechanism to prevent exaggerated antibacterial immune responses in the gut to high or perpetuating bacterial load. This regulatory mechanism is lost in NOD2-deficient CD patients.     

Plasticity of dendritic cell function in response to prostaglandin E2 (PGE2) and interferon-gamma (IFN-gamma)

Current evidence suggests that maturing dendritic cells (DCs) acquire a migratory phenotype to induce T cell responses in lymph nodes or a proinflammatory phenotype to condition the microenvironment at peripheral sites. We show that the interplay of PGE(2) and IFN-gamma generates a more complex pattern of mixed DC phenotypes in response to TLR stimulation. DCs activated by the TLR ligand R-848 in the presence of IFN-gamma and PGE(2) produced high levels of IL-12p70 and IL-23, started migration toward CCL19 within only 10 h, and still continued to secrete IL-12p70 without further restimulation following the migration step. The accelerated onset of migration was a result of PGE(2) and was associated with reduced plastic adherence and lower amounts of activated CD29. In contrast, IFN-gamma by itself enhanced cell adhesion and strongly hindered CCR7-mediated migration in the absence of PGE(2). This suggests a new role for IFN-gamma in the direct regulation of DC migration through enhanced cell adhesion, perhaps to support the development of T cell effector functions at peripheral sites. Together, our data are relevant to the development of DC vaccines, as they demonstrate the existence of dual-functional DCs, which as a result of the simultaneous effects of PGE(2) and IFN-gamma, can migrate rapidly toward lymph node chemokines and carry with them a wave of primary cytokines.     

The innate immune response to Salmonella enterica serovar Typhimurium by macrophages is dependent on TREM2-DAP12

Macrophage recognition of Salmonella enterica serovar Typhimurium leads to a cascade of signaling events, including the activation of Src family and Syk kinases and the production of reactive oxygen species (ROS), which are critical for host innate defense during early stages of bacterial infection. ROS production depends on the NADPH oxidase, but little is known about the innate immune receptors and proximal adapters that regulate Salmonella-induced ROS. Herein, we demonstrate that serovar Typhimurium induces ROS through a pathway that requires both triggering receptor expressed on myeloid cells 2 (TREM2) and DAP12. This pathway is highly analogous to the pathways utilized by Fc receptors and integrins to regulate ROS production. Oral infection of mice with serovar Typhimurium demonstrates that the DAP12-dependent pathway regulates cecal colonization during early stages of Salmonella infection. Thus, DAP12 is an important regulator of Salmonella-induced ROS production in macrophages, and TREM2 is essential for linking DAP12 to the innate response to serovar Typhimurium.     

When pentobarbital is the conditioned stimulus and amphetamine is the unconditioned stimulus, conditioning depends on the type of conditioned response

Injections of drugs into rats were used as conditioned stimuli (CSs) and as unconditioned stimuli (USs). With heart rate (HR) conditioning, the pentobarbital CS produces a higher HR than under control conditions. With avfail (aversion failure) conditioning, the pentobarbital CS loses much of its capacity to induce a conditioned taste aversion. HR conditioning was obtained with forward delays of up to 30 min and backward delays of up to 270 min, where the delays are defined by the interinjection interval. Avfail was obtained with forward delays of up to 270 min but not with backward delays. Neither HR conditioning nor avfail were context specific but could be demonstrated in a test apparatus after pairings that occurred in the home cage. This indicated that the external environment was not an important part of the effective stimulus complex. When HR conditioning was obtained, its latency and duration was not related to the delay between the CS and US injections or whether they were forward or backward.     

Macrophage activation in falciparum malaria as measured by neopterin and interferon-gamma.

Macrophage activation during acute falciparum malaria in 71 Thai adults was investigated by measuring urinary neopterin and serum interferon-gamma (IFN-gamma). Neopterin, a product of IFN-gamma-activated macrophages, was elevated in 94% of patients upon admission (day 0, prior to treatment) and in all at some time during the period of study. Neopterin levels tended to rise further (days 1-5) before falling back towards the normal range as patients recovered following effective chemotherapy (days 6-8). IFN-gamma was measured in 32 patients and found to be directly related to neopterin concentration. Both neopterin and IFN-gamma values were highest in patients experiencing a first malaria infection. Among those with histories of prior malaria, neopterin and IFN-gamma levels were inversely related to the number of previous infections. Morbidity, as assessed by degree and duration of fever, was directly related to neopterin concentration. This longitudinal study quantitatively describes the extent and duration of macrophage activation in falciparum malaria. The data also suggest that with repeated malaria infection and antigen exposure, there is a progressive decrease or possibly suppression of the T cell-macrophage interaction mediated by IFN-gamma.     

The THC-induced suppression of Th1 polarization in response to Legionella pneumophila infection is not mediated by increases in corticosterone and PGE2

T helper cell type 1 (Th1)-polarizing cytokines are induced by Legionella pneumophila infection and are suppressed by pretreatment with marijuana cannabinoids (CB). Glucocorticoids and prostaglandin E2(PGE2) are also reported to suppress Th1 polarization and are induced by Delta9-tetrahydrocannabinol (THC), so their role in the suppression of polarizing cytokines was examined. Injection of L. pneumophila or THC alone into BALB/c mice induced a rapid and transient rise in serum corticosterone (CS), and the injection of both agents significantly augmented the CS response, demonstrating that THC increased CS in Legionella-infected mice. Pretreatment with the CB receptor 1 (CB1) antagonist SR141716A had no effect on the THC-induced CS response, but CB2 antagonist (SR144528) treatment increased the CS response. To see if increased CS contributed to the down-regulation of Th1 cytokines, mice were pretreated with the steroid antagonist RU486 before THC injection and Legionella infection. The results showed that RU486 did not attenuate the THC-induced suppression of serum interleukin (IL)-12 or interferon-gamma (IFN-gamma). In addition to CS, THC injection increased urinary PGE2 metabolites, and the CB1 antagonist attenuated this increase. Although L. pneumophila infection increased urinary PGE2, THC pretreatment did not enhance this response; in addition, treatment with the cyclooxygenase inhibitor, indomethacin, did not block the THC-induced suppression of IL-12 and IFN-gamma. These results suggest that the elevation of CS and PGE2 does not account for the THC-induced attenuation of the Th1 cytokine response, and it is concluded that other suppressive mediators are induced by THC or that the drug acts directly on immune cells to suppress cytokine production.     

Conditioning using a cerebral cortical conditioned stimulus is dependent on the cerebellum and brain stem circuitry.

Electrical stimulation of the auditory cortex (AC) was used as a conditioned stimulus (CS) in the rabbit conditioned eyeblink preparation to trace the functional anatomical connections between the AC and the circuitry underlying this conditioned response. Conditioning was shown to be dependent on the cerebellar interpositus nucleus and the pontine nuclei (PN), structures that are essential for conditioning using a peripheral CS. The results suggest that the cerebellum and associated brain stem circuitry are a necessary part of the memory trace circuit for the conditioned eyeblink response, even when the cerebral cortex is artifically engaged as a CS by electrical stimulation. The results also suggest that the PN are a site of convergence between the CS circuit subserving classical conditioning for peripheral stimuli and the AC, and may therefore be a site where the AC can modulate more elaborate forms of conditioning.     

The chemotactic response of granulocytes to the low molecular weight chemoattractants f-MLP, C5f, and LTB4 is dependent on chemokinetic factors

The dependence of the low molecular weight chemoattractants (LMCs) formylmethionyl-leucylphenylalanine (f-MLP), C5f, and leukotriene B4 (LTB4) on albumin to express their chemotactic activity towards granulocytes (PMNs) was investigated in order to study the required qualities of albumin and if albumin could be replaced by any other proteins. The results demonstrated that the supporting effect of isolated albumin was dependent on the method of purification. Only isolated albumin exposed to ethanol precipitation during the purification procedure supported the chemotactic effect of LMCs. The albumin preparation that supported the effect of LMCs also mediated a chemokinetic effect on PMN migration. Albumin isolated by methods other than ethanol precipitation neither exerted a chemokinetic effect nor supported the chemotactic effect of LMCs. Heated, normal serum and isolated alpha 1-antitrypsin supported the chemotactic activity of LMCs and also mediated a chemokinetic effect on PMN migration. The present investigation suggests an important role for the chemokinetic factors, since it is indicated that their presence is necessary for the chemotactic response of PMNs to the low molecular weight chemoattractants C5f; LTB4, and f-MLP.     

The Candida Th17 response is dependent on mannan- and beta-glucan-induced prostaglandin E2

The fungus Candida albicans is a potent inducer of the T(h)17 response. Prostaglandin E2 (PGE2) is a strong pro-inflammatory mediator, which has proven to be essential for the T(h)17 response. In this study, we have investigated the role of PGE2 in the T(h)17 response induced by C. albicans in humans. PBMC were stimulated with C. albicans in the absence or presence of a non-steroidal anti-inflammatory drug (NSAID). In separate experiments, PGE2 or the prostlaglandin receptors agonists butaprost or misoprostol were added to the cells. PBMC were also stimulated with fungal components and small interfering RNA for mannose receptor (MR) was performed. PGE2 and cytokines were measured by ELISA or luminex, and the source of IL-17 production was determined using FACS analysis. Blocking Candida-induced PGE2 production by an NSAID resulted in decreased IL-17 and IL-22 production and inhibited expression of RAR-related orphan receptor gamma T mRNA. Furthermore, when PGE2 production was blocked, IL-6, IL-23 and IL-10 were decreased, while tumor necrosis factor α increased. Stimulation with PGE2 or E prostanoid (EP)2/EP4 agonists restored IL-17 production. Candida albicans mannan was the only fungal component that was able to directly induce PGE2 and silencing of the MR resulted in a reduction of Candida-induced PGE2. β-Glucan engagement of dectin-1 synergistically increased Toll-like receptor 2 (TLR2)-induced PGE2 production. These data provide evidence that PGE2 pathway is important for the T(h)17 response induced by C. albicans and that PGE2 is induced by the fungal components mannan and β-glucan that are recognized by the MR and the dectin-1/TLR2 pathway, respectively.