High-throughput analysis of T cell-monocyte interaction in human tuberculosis.

The lack of efficient tools for identifying immunological correlates of tuberculosis (TB) protection or risk of disease progression impedes the development of improved control strategies. To more clearly understand the host response in TB, we recently established an imaging flow cytometer-based in-vitro assay, which assesses multiple aspects of T cell-monocyte interaction. Here, we extended our previous work and characterized communication between T cells and monocytes using clinical samples from individuals with different TB infection status and healthy controls from a TB endemic setting. To identify T cell-monocyte conjugates, peripheral blood mononuclear cells (PBMC) were stimulated with ds-Red-expressing Mycobacterium bovis bacille Calmette-Guérin or 6-kDa early secreted antigenic target (ESAT 6) peptides for 6 h, and analyzed by imaging flow cytometer (IFC). We then enumerated T cell-monocyte conjugates using polarization of T cell receptor (TCR) and F-actin as markers for synapse formation, and nuclear factor kappa B (NF-κB) nuclear translocation in the T cells. We observed a reduced frequency of T cell-monocyte conjugates in cells from patients with active pulmonary tuberculosis (pTB) compared to latent TB-infected (LTBI) and healthy controls. When we monitored NF-κB nuclear translocation in T cells interacting with monocytes, the proportion of responding cells was significantly higher in active pTB compared with LTBI and controls. Overall, these data underscore the need to consider multiple immunological parameters against TB, where IFC could be a valuable tool.

Reduced MCMV Δm157 viral clearance in the absence of TSAd.

The T cell specific adapter protein (TSAd) is expressed in activated T cells and NK cells. While TSAd is beginning to emerge as a critical regulator of Lck and Itk activity in T cells, its role in NK cells has not yet been explored. Here we have examined susceptibility to virus infections in a murine model using various viral infection models. We report that TSAd-deficient mice display reduced clearance of murine cytomegalovirus (MCMV) that lack the viral MHC class I homologue m157, which is critical for Ly49H-mediated NK cell recognition of infected cells. In this infection model, NK cells contribute in the early stages of the disease, whereas CD8+ T cells are critical for viral clearance. We found that mice infected with MCMV Δm157 displayed reduced viral clearance in the spleen as well as reduced proliferation in spleen NK cells and CD8+ T cells in the absence of TSAd. Though no other immunophenotype was detected in the infection models tested, these data suggests that in the absence of the Ly49H ligand activation, NK cell and CD8+ T cell responses may be compromised in TSAd-deficient mice.

Leaching from organic matter-rich soils by rain of different qualities: I. Concentrations.

Soil monoliths from an area exposed to acid precipitation and from an unpolluted area were used in a lysimeter experiment to study effects of different rain qualities on the chemical composition of the leachate from shallow soils rich in organic matter. The vegetation was either dominated by moorgrass [Molinia caerulea (L.) Moench] or heather [Calluna vulgaris (L.) Hull]. The lysimeters received either "acid rain" (pH 4.3) or "normal rain" (pH 5.3). High concentrations of dissolved organic carbon (DOC) were characteristic of the leachate. The different "rain" qualities had no significant influence on the DOC concentration. More DOC was, however, leached from lysimeters with heather vegetation. Roughly 50% of the aluminum (Al) was in complex with organic material and the Al charge was calculated to be between +1.4 and +2.0. Sulfate (SO4(2-)) was the only component that was significantly influenced by the treatment, as more was leached from lysimeters receiving "acid rain." Sulfate was poorly correlated with pH, suggesting that reduced SO4(2-) input would not necessarily lead to reduced acidity. Differences in the pH of the leachate due to the treatments were less than 0.15 pH units. Nitrate (NO3-) was only leached in very low concentrations and of little consequence for the leachate acidity. Some observations do, however, suggest that NO3- may contribute to acidification in episodes with high precipitation. High concentrations of Cl- in the leachate and a significant positive correlation between Cl-, H+, and base cations indicate that sea salt episodes may be important for soil acidification and acidity of the leachate.

Sex differences in relation to conditioned fear-induced enhancement of morphine analgesia.

A number of studies have reported that both the immediate and proactive effects of exposure to a shock stressor are less pronounced in female than in male rats. A separate area of research has demonstrated that female rats are less sensitive to the analgesic effects of morphine than males. Experiments from our laboratory, as well as others, have found that exposure to a context associated with shock (i.e., conditioned fear context) at the time of morphine administration, enhances the analgesic effects of morphine. Since previous studies have exclusively employed male rats, the purpose of Experiment 1 was to determine if a sex difference exists to this context conditioned fear-induced enhancement of morphine-induced analgesia. The findings of Experiment 1 showed that females do not appear to exhibit conditioned fear-induced enhancement of morphine analgesia as compared to males. Experiment 2 demonstrated that females exhibited higher levels of conditioned fear-induced enhancement of morphine analgesia during diestrus I than estrous. Experiment 3 demonstrated that females exhibited lower levels of conditioned analgesia compared to males, while both groups exhibited similar freezing levels. The findings of the present experiments suggest that the sex difference observed in Experiment 1 may be due to differences in conditioned analgesia.

Effect of adrenalectomy on cocaine facilitation of medial prefrontal cortex self-stimulation.

Adrenalectomy (ADX) is known to block the acquisition of intravenous cocaine self-administration. A previous study therefore examined whether ADX decreases sensitivity of the 'brain reward system' in general, or its response to cocaine in particular, by measuring thresholds for intracranial self-stimulation with and without concurrent cocaine administration. ADX had no effect on thresholds for lateral hypothalamic self-stimulation (LHSS) and did not alter the cocaine dose-response curve for lowering the LHSS threshold. This result suggested that ADX does not affect sensitivity of the brain reward system. However, medial prefrontal cortex (MPFC) appears to be an important site in the mediation of cocaine reinforcing effects, and MPFC self-stimulation (MPFCSS) is mediated by a neural substrate that is largely independent of that which mediates LHSS. The present study therefore assessed whether ADX diminishes cocaine facilitation of MPFCSS. It was found that the threshold-lowering effect of cocaine (5.0, 10.0 and 20.0 mg/kg, i.p. ) did not differ between ADX rats maintained on 0.7% saline, ADX rats maintained on corticosterone (50 microg/ml) in 0.7% saline, and sham-operated controls. However, there was a trend toward desensitization of MPFCSS, itself, following ADX in the group that did not receive corticosterone supplementation. Based on this observation, and the similar responses of MPFCSS and cocaine self-administration to noncontingent priming stimulation, stress, and NMDA receptor antagonism, it is speculated that acquisition of MPFCSS and cocaine self-administration may be dependent upon a common sensitization process that is regulated by corticosterone.

Enhancement of morphine analgesia in rats following removal from contextual conditioned fear cues.

1. Previous studies have shown that morphine analgesia is enhanced when analgesia testing is conducted in an environment that has been previously paired with shock, but not in a novel or neutral environment. 2. Two experiments were conducted to assess if enhanced morphine analgesia could be demonstrated in a neutral context if rats were first exposed to conditioned fear cues. This was done by pre-exposing rats to a context previously paired with shock and testing for enhanced morphine analgesia in a neutral context immediately following removal from the conditioned fear context. To determine if conditioned analgesia contributed to the enhanced morphine analgesia, rats were tested for analgesic responsiveness immediately following removal from conditioned fear cues, prior to morphine administration. 3. In Experiment 1, although conditioned analgesia was not observed, a small enhancement of morphine analgesia was demonstrated in an neutral context in rats pre-exposed to conditioned fear cues, compared to non-conditioned controls. 4. In Experiment 2, which employed more sensitive test procedures, a strong enhancement of morphine analgesia was observed in a neutral context only in those rats that demonstrated conditioned analgesia.

Effect of adrenalectomy on cocaine facilitation of lateral hypothalamic self-stimulation.

An emerging body of evidence indicates that the adrenal hormone corticosterone modulates behavioral effects of abused drugs. Recently, it was reported that the self-administration and locomotor stimulatory effect of cocaine are blocked by adrenalectomy (ADX). In order to evaluate the effect of ADX on the brain reward system in general, and cocaine reward in particular, the effect of ADX on lateral hypothalamic self-stimulation (LHSS) and its facilitation by cocaine were investigated. Using curve-shift methodology, effects of cocaine (1.0, 3.0 and 10.0 mg/kg, i.p.) on the rewarding efficacy of brain stimulation were determined in ADX rats, with and without corticosterone supplementation, and compared with sham-operated controls. Results indicate that ADX does not affect LHSS or the facilitatory effect of cocaine. The divergence between these results and the results of cocaine self-administration studies is discussed in terms of the neuroanatomical and psychological processing of reward.

Aminoglutethimide, a corticosteroid synthesis inhibitor, facilitates brain stimulation reward in food-restricted rats: an investigation of underlying mechanisms.

It was previously observed that the corticosteroid synthesis inhibitor, aminoglutethimide (AG), markedly facilitates lateral hypothalamide (AG), markedly facilitates lateral hypothalamic self-stimulation (LHSS) in food-restricted rats. This effect is not present 30 min after injection when plasma corticosterone levels are suppressed, but rather at 2 h when corticosterone has recovered from suppression. In experiment 1, it was confirmed that AG (50.0 mg/kg, s.c.) lowers the threshold for LHSS in food-restricted rats but not in control rats that have ad libitum access to food. This effect occurred independently of whether food restriction, by itself, lowered threshold. Experiment 2 examined whether the facilitation of LHSS coincides with biosynthetic rebound of corticosteroid precursors. While a pregnenolone surge was demonstrated by radioimmunoassay, dose-response testing with exogenous pregnenolone and progesterone (0.1, 1.0 and 10.0 mg/kg, s.c.) failed to confirm the prediction that one of these precursors facilitates reward. Therefore, a general test of the involvement of adrenocortical biosynthetic events was conducted in experiment 3 where rats were adrenalectomized (ADX) or sham-operated prior to food restriction. Surprisingly, ADX did not diminish the effect of AG. This finding raises the possibility of a CNS, rather than adrenal, site of action. AG is known to penetrate the blood-brain barrier and exert weak anticonvulsant effects. The facilitation of reward may result from central inhibitory effects of the drug and share a common basis with the enhanced reinforcing potency of other CNS depressants in food-restricted rats.

Diabetes alters mu and kappa opioid binding in rat brain regions: comparison with effects of food restriction.

Diabetic rats display changes in opioid pharmacology and brain regional levels of opioid peptides and prodynorphin mRNA. Previous investigations of opioid receptor binding, carried out in whole-brain homogenates, have, however, failed to detect changes. In the present study, quantitative autoradiography was used to measure mu and kappa opioid receptor binding in discrete brain regions of streptozotocin-treated diabetic rats. Measurement was limited to regions that previously displayed opioid binding changes in chronically food-restricted rats, since our primary aim is to identify brain mechanisms that mediate adaptive responses to persistent metabolic need and adipose depletion. Diabetics displayed strong trends or statistically significant changes which matched seven of the thirteen binding changes observed in food-restricted rats. In no case did diabetics display changes in the opposite direction. The two statistically significant changes common to food-restricted and diabetic rats are increased kappa binding in the medial preoptic area and decreased mu binding in the lateral habenula. The possible functional significance of these changes is discussed.

Effects of corticosteroid synthesis inhibitors on the sensitization of reward by food restriction.

Chronic food restriction sensitizes animals to the rewarding effects of food, drugs and lateral hypothalamic electrical stimulation. The present study employed a curve-shift analysis of lateral hypothalamic self-stimulation (LHSS) to evaluate whether the elevated plasma corticosterone levels that accompany food restriction mediate the sensitization of reward. In Experiment 1, two adrenocorticoid synthesis inhibitors, aminoglutethimide and metyrapone, were administered to food-restricted rats and the magnitude of plasma corticosterone suppression was determined at two post-administration time points. In Experiment 2, these compounds were administered to ad libitum fed and food-restricted rats whose LHSS behavior was evaluated at a time coincident with suppression of corticosterone. It was found that neither compound reversed the sensitizing effect of food-restriction on the rewarding efficacy of brain stimulation. However, aminoglutethimide (50 mg/kg) produced an increase in maximal response rates (a performance factor) across groups while metyrapone (100 mg/kg) produced a decrease. The most interesting result of this study was that 2 h after aminoglutethimide administration, when corticosterone levels had recovered from suppression, the rewarding efficacy of LHSS increased markedly in food-restricted rats. Possible explanations for this effect, including adrenocortical rebound, alterations in neurosteroid synthesis, and exacerbation of metabolic need are discussed.

Pavlovian aversive context conditioning using carbon dioxide as the unconditional stimulus.

Four experiments were conducted to examine the utility of carbon dioxide (CO2) as an aversive unconditioned stimulus (US) in a Pavlovian context conditioning paradigm. Experiment 1 demonstrated that rats exposed to CO2 in a distinctive context showed elevated levels of freezing relative to controls. Experiment 2 replicated this basic effect with a modified conditioning procedure and additionally demonstrated conditioned analgesia. Experiment 3 demonstrated a positive monotonic relationship between US duration and resistance to extinction of freezing behavior as well as conditioned analgesia. Experiment 4 demonstrated extinction and an extinction-related phenomenon, renewal. These studies clearly demonstrate the utility of CO2 as a Pavlovian US.

Methotrexate does not interfere with an appetitive Pavlovian conditioning task in Sprague-Dawley rats.

There has been considerable interest in developing an animal model of the neuropsychological toxicity of chemotherapeutic agents used in the treatment of patients with cancer, especially children, since these agents often cause significant, long-term neuropsychological deficits. Yanovski, Packer, Levine, Davidson, Micalizzi, D'Angio (13) recently proposed such a model based on their finding that methotrexate retarded the formation of aversive Pavlovian excitatory associations. The present experiment examined the generality of methotrexate induced cognitive impairments by testing rats in Appetitive Pavlovian Conditioning tasks and a Conditioned Taste Aversion paradigm. The results of our study revealed no impairment following methotrexate exposure on the Appetitive Pavlovian tasks or on the Taste Aversion task, relative to two control conditions. While there were a number of methodological differences between the present experiment and those conducted by Yanovski et al. (13), the present results question the robustness and generality of Yanovski's et al. (13) animal model.

Curve-shift analysis of self-stimulation in food-restricted rats: relationship between daily meal, plasma corticosterone and reward sensitization.

Chronic food restriction lowers the threshold for lateral hypothalamic electrical self-stimulation (LHSS). This effect has previously been interpreted to reflect a sensitization of reward. In the present study a curve-shift method was used to explicitly differentiate effects of food restriction on brain stimulation rewarding efficacy and performance. Food restriction consistently shifted rate-frequency curves to the left, lowering the M-50 and Theta-0 parameters of rewarding efficacy. Asymptotic rates of reinforcement and slopes of rate-frequency functions were unaffected, confirming that food restriction does not facilitate LHSS by enhancing performance. In this and previous studies, LHSS in food-restricted rats was measured in the period immediately preceding the daily meal when hunger (i.e., period since last meal) and plasma corticosterone are at peak levels. In the light of evidence that corticosterone may regulate sensitivity of the mesolimbic dopamine pathway and account for the sensitizing effect of stress on psychomotor effects of opiates and stimulants, LHSS and corticosterone were measured in the immediate pre-and post-meal periods. While all food-restricted rats displayed elevated corticosterone levels in the pre-meal period and generally displayed a decline to control levels in the post-meal period, the sensitization of reward was not reversed in the post-meal period. These results indicate that chronic food restriction produces a sensitization of reward that does not depend upon the acute state of hunger that precedes the daily meal and does not vary with dynamic changes in plasma corticosterone level.

Conditioned fear exacerbates acute morphine dependence.

A variety of physical stressors have been shown to enhance reactivity to opioid drugs. Few studies have examined the effects of nonphysical stressors on opioid drug reactivity. In this regard, it has previously been shown that animals administered morphine in the presence of shock-associated cues demonstrate increases in hypoalgesia relative to nonshock control animals. These findings have typically been viewed as being mediated by the activation of endogenous pain inhibition systems via conditioned fear. In this series, we further examined the nature of these effects by assessing the effects of conditioned fear on acute morphine dependence. Experiment 1 revealed that animals administered 3 mg/kg morphine in the presence of context fear cues demonstrated an enhanced withdrawal response when removed and administered 3 mg/kg naloxone. Because it is known that conditioning effects do not diminish over time, a second experiment examined whether the enhancement of acute dependence by context fear would still be evident 72 h postconditioning. As in Experiment 1, animals administered morphine in a context associated with shock demonstrated an enhancement of acute dependence. Experiment 2b revealed that the shock parameters used in these studies can induce a hypoalgesic response on the test that is opioid mediated. These findings are discussed with regard to the neuroanatomy of fear systems as they relate to the neuropharmacological study of opioid withdrawal.

Modulation of hypoalgesia by morphine and number of shock trials: covariation of a measure of context fear and hypoalgesia.

In a recent series of studies, we observed that exposure to prolonged foot shock increased hypoalgesia induced by morphine. This increase was observed only when testing was conducted in the presence of shock-associated cues, suggesting that it resulted from context-conditioned fear. However, we do not know whether the extended stressor parameters employed in that study are necessary for an observance of the effect. Therefore, in the present study, we assessed the effect of the number of shock trials (either 0, 20, 100, or 200) on the hypoalgesia observed following morphine administration. In addition, we measured activity as an independent index of context-conditioned fear, because in prior studies there had been no independent behavioral assessment of the conditioning of fear to the context. Although others have shown a covariation of conditioned fear and context-induced hypoalgesia using shock parameters and test paradigms different from our own, we sought to assess whether the same covariation would hold for conditioned fear and the hypoalgesia observed following the administration of morphine. The results showed increased hypoalgesia in all groups exposed to foot shock, demonstrating that prolonged exposure to foot shock is not necessary for an observance of this effect. In addition, the results revealed a linear relationship between number of trials of shock and hypoalgesia, but a U-shaped relationship between trials and activity. The pattern of results is considered in light of Fanselow's Perceptual-Defensive-Recuperative model.

Learned helplessness inducing foot shock can exacerbate morphine responsiveness.

Exposure to inescapable tail shock or foot shock has been shown to produce effects on a number of learning tasks. Tail-shock exposure is also known to influence nociception and morphine reactivity. The present series of experiments investigated the effects of foot shock known to induce learned helplessness effects in our laboratory on the subsequent reactivity to morphine. A first set of experiments investigated the hypoalgesic response to a 4 mg/kg dose morphine over 4 consecutive days following exposure to foot shock. Experiment 1A did not reveal an effect of foot shock on morphine-induced hypoalgesia when testing was conducted in a novel context. In Experiment 1B, we observed an increased hypoalgesic response to morphine when testing was conducted in the shock context. The findings of Experiment 1B were replicated in Experiment 2 and extended to assess the contribution of conditioned fear hypoalgesia to these effects. The possible mechanisms responsible for these findings are discussed.

Environmental enrichment: the influences of restricted daily exposure and subsequent exposure to uncontrollable stress.

Environmental enrichment has been proposed to enhance an animal's subsequent ability to learn. While this proposal has received considerable support from experiments involving maze tasks, it has received equivocal support from experiments employing operant and pavlovian tasks. The purpose of the present study is two-fold. The first is to demonstrate that a regimen of restricted daily exposure to environmental enrichment is capable of producing effects similar to those using more standard exposure regimens when compared to the most appropriate control, a group given social exposure. The second is to examine the proposed learning enhancement of environmental enrichment on an operant task both before and following exposure to uncontrollable stress. Uncontrollable stress, as interpreted by learned-helplessness theory, results in the formation of an expectancy of response-reinforcer independence which proactively interferes with the subsequent acquisition of response-outcome associations. It may be possible, then, that environmental enrichment and uncontrollable stress may interact in such a way as to allow the potential learning effects of environmental enrichment to be assessed on an operant task. Rats were exposed to differential environments; one group exposed to an enriched environment and another exposed to a social environment 2 hours daily for 30 days. Each group was then tested on the object-exploration test. Following the acquisition of an appetitive-operant response, a subset of these two groups was exposed to either controllable, uncontrollable, or no stress using parameters known to induce learned helplessness. Animals were then tested on an appetitive-noncontingent test. It was found that, while the enrichment procedure was effective in producing effects on the object-exploration test, environmental enrichment did not modify the acquisition of the operant or the effect produced by uncontrollable stress on the appetitive-noncontingent test.