Stenotrophomonas maltophilia infection in hematopoietic SCT recipients: high mortality due to pulmonary hemorrhage.

To clarify the clinical features and outcome of Stenotrophomonas maltophilia infection among hematopoietic SCT (HCT) recipients, we retrospectively reviewed the records of 1085 consecutive HCT recipients and identified 42 episodes in 31 HCT recipients with S. maltophilia infection. We compared these recipients with 30 non-HCT patients with S. maltophilia infection. The mortality rate in HCT recipients was significantly higher than that in non-HCT patients (relative risk 5.7, P=0.04), and we identified seven patients with pulmonary hemorrhage due to S. maltophilia, exclusively in the HCT cohort. Six of these latter seven patients died within 1 day from the onset of hemorrhage and the isolate was identified after death in most cases; one patient, who received empiric therapy for S. maltophilia and granulocyte transfusion, survived for more than 2 weeks. The patients with pulmonary hemorrhage had a more severe and longer duration of neutropenia, persistent fever despite of the use of broad-spectrum antibiotics, complication by pneumonia and higher C-reactive protein levels than those without pulmonary hemorrhage. In conclusion, S. maltophilia was associated with fulminant and fatal pulmonary hemorrhage in HCT recipients. Empiric therapy with antibiotics before the onset of pulmonary hemorrhage may be effective in HCT recipients who carry the conditions identified.

Inhibition of cardiac delayed rectifier K+ currents by an antisense oligodeoxynucleotide against IsK (minK) and over-expression of IsK mutant D77N in neonatal mouse hearts.

The IsK (minK or KCNE1) protein is known to co-assemble with the KvLQT1 (KCNQ1) protein to form a channel underlying the slowly activating delayed rectifier K+ current (IKs). Controversy remains as to whether the IsK protein assembles with ERG (the ether-a-go-go-related gene) products to form or modulate the channel-underlying the rapidly activating delayed rectifier K+ current (IKr). We investigated the effects of antisense oligodeoxynucleotides (AS-ODN) against IsK and its mutant D77N [which underlies a form of long QT syndrome (LQT5) in humans] on the delayed rectifier K+ current (IK) of neonatal mouse ventricular myocytes in primary culture. Patch-clamp experiments on these cells showed that IK consists of IKs and IKr. IK was not recorded from ventricular cells transfected with AS-ODN, while it was recorded from cells transfected with the corresponding sense oligodeoxynucleotides (S-ODN). IK was not recorded from cells transfected with the D77N mutant, and the action potential duration was much longer than in cells transfected with wild-type IsK. Furthermore, HERG could not induce currents in COS-1 cells co-expressed with the D77N mutant and HERG (the human form of ERG). These results indicate that the IsK protein associates with both KvLQT1 and ERG products to modulate IKr and IKs in cardiac myocytes.

NK cell and CTL activities can be raised via conditioning of the CNS with unrelated unconditioned stimuli.

Poly I:C, an inducer of interferon alpha and beta, and arecoline, a cholinergic muscarinic agonist, were used as unconditioned stimuli in conjunction with camphor odor to condition the augmentation of natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activities. This observation suggested that two unrelated unconditioned stimuli might be used to associate the signals, and induce mediators at recall that can raise natural killer cell activity. In animals stimulated with an alloantigen to induce CTL, we observed that either poly I:C or arecoline conditioned association, and recall with the same CS raised CTL activity. It appears that conditioning with different substrates can raise either native (NK cell activity) or acquired (CTL activity) immunity. The studies suggest that communication between the CNS and NK cells or CTL appear to take place through a common pathway(s).

Immunotherapy of a murine T cell lymphoma localized to the brain.

Mouse YC8 T cell lymphoma was used as a model to determine whether an effective immunotherapy procedure could be devised for the treatment of lymphoma localized to the brain. Implantation of 5 x 10(4) YC8 cells into the left cerebral hemisphere induced rapid loss of the animal's body weight. Severe loss of weight and early deaths were observed in the untreated control group. Although resistance can be conferred to the brain by immunization of naive BALB/c mice, adoptive chemoimmunotherapy procedures were surprisingly not effective in inducing remissions in animals with lymphoma confined to the brain. Even passive transfer of effector cells from immunized, tumor resistant donor animals combined with systemic IL-2 treatment did not impart resistance to recipients with brain tumors. However, regression of the intracranial tumor and apparent cures could be accomplished, when ex vivo cultured effector cells were transferred intravenously.

Psychoneuroendocrine immunology: perception of stress can alter body temperature and natural killer cell activity.

Psychoimmunology has been credited with using the mind as a way to alter immunity. The problem with this concept is that many of the current psychoimmunology techniques in use are aimed at alleviating stress effects on the immune system rather than at direct augmentation of immunity by the brain. Studies in animals provide a model that permits us to approach the difficulties associated with gaining an understanding of the CNS-immune system connection. A particular advantage of using animals over humans is that psychological and social contributions play a less prominent role for animals than for human subjects, since the animals are all inbred and reared under identical controlled conditions. If the insightful information provided by animal studies is correct, then psychotherapy for the treatment of diseases might be made more effective if some aspect of this knowledge is included in the design of the treatment. We emphasize conditioning as a regimen and an acceptable way to train the brain to remember an output pathway to raise immunity. We propose that a specific drug or perception (mild stress, represented by rotation, total body heating or handling) could substitute and kindle the same output pathway without the need for conditioning. If this view is correct, then instead of using conditioning, it may be possible to use an antigen to activate desired immune cells, and substitute a drug or an external environmental sensory stimulus (perception) to energize the output pathway to these cells. Alternatively, monitoring alterations of body temperature in response to a drug or perception might allow us to follow how effectively the brain is performing in altering immunity. Studies with animals suggest that there are alternative ways to use the mind to raise natural or acquired immunity in man.

Afferent signals to the CNS appear not to condition the modulation of interleukin-1 receptors in the hippocampus.

Conditioned alteration of natural killer (NK) cell activity was used as an indicator of the functional bidirectional communication between the immune and central nervous systems. Poly I:C and lipopolysaccharide (LPS) from Escherichia coli and Salmonella typhimurium were used as unconditioned stimuli and odor of camphor as the conditioned stimulus. An attempt was made to demonstrate the role of central interleukin (IL-1) receptors in this communication process. Brain IL-1 receptors were down-regulated by treatment with 50 microg/mouse of LPS from S. typhimurium, but not with the same dose of LPS from E. coli or poly I:C. A significant level of conditioned augmentation of NK cell activity was observed with poly I:C. Conditioned alteration in NK cell activity was also observed with LPS from E. coli, but at much lower level than poly I:C. NK cell activity was not conditioned with LPS from S. typhimurium at the same dose as E. coli LPS, but conditioned enhancement of NK cell activity was observed with a higher dose (100 microg) of S. typhimurium LPS. These results suggest that modulation of central IL-1 receptors do not seem to play a role in the conditioned augmentation of NK cell activity.

Psychoneuroendocrine immunology: site of recognition, learning and memory in the immune system and the brain.

How the interaction between the brain and immune system takes place has not been clearly defined. Because multiple changes are occurring simultaneously in all organ systems (e.g., cardiovascular, gastrointestinal, reproductive, renal, respiratory, immune, CNS), how many single systems interacts with the brain becomes extraordinarily difficult to understand. The problem boils down to developing an approach that not only allows one to study the whole organism and define the mediators of the interacting systems, but also permit one to establish the connection and physiologic relevance of the responses that are being evaluated. Conditioning, a phenomenon made popular by the work of Pavlov (1906, 1927), may provide insight into the pathways of communication between the brain and possibly any organ system of the body. Conditioning allows one to separate the afferent from the efferent circuits. That is, signals from the immune system to the CNS (IS-->CNS) can be effectively separated from signals from the CNS to immune system (CNS-->IS). This permits one to study each pathway individually. Simple, single association trial models to condition fever, natural killer (NK) cell and cytotoxic lymphocyte (CTL) activities have been developed to evaluate the pathways. Single trial learning is not new. Pavlov has observed that "The electric buzzer set going before administration of food established a conditioned alimentary reflex after only a single combination," whereas the reverse order of presentation failed to condition the animal (Pavlov 1927 p. 27). Thus, conditioning can be used to train the brain to activate the immune system and other organ systems participating in the response. During the course of the conditioned response, presumably the CNS via the hypothalamus integrates in a cohesive orderly fashion all input and output signals and coordinates the responses made by the brain to the organ systems. The odor of camphor, the conditioned stimulus (CS) can be associated with the response produced by an unconditioned stimulus (US). The unconditioned stimuli used are poly I:C to raise fever and nonimmunospecific NK cell activity or alloantigens to raise immunospecific CTL activity. The unconditioned stimulus serves only as a means to activate the immune system and unbalance the homeostasis so that a transient but new bidirectional communication loop can be established between the immune system and the CNS (IS<-->CNS). The expression of the conditioned response (i.e., elevation of fever, NK cell, or CTL activity) induced with the CS (odor stimulus) is an outcome of neural activity (CNS-->IS). This infers that during conditioning, the signals generated by the CS and US imprints a neural pathway located within the central nervous system and leaves behind a CS/US memory of the association. The immune activity (NK cell or CTL activity) which is modulated indicate that the memory pathway was activated in the brain of the animal expressing the conditioned response. The immune cells that are modulated can be considered to be casual bystander cells. These cells however must be in the proper (ready) state of activation to receive salient signals from the brain. Along with changes in the indicator cell population, other complex physiological processes are altered by the brain via sympathetic and neuroendocrine pathways to raise the fever response. These observations suggest that the physiological changes which are being evaluated such as fever, NK cell or CTL activities or perhaps blood pressure, heart rate, fat metabolism, oxygen consumption serve only as indicators (readouts), and infer that the CNS has made a coordinated reply in response to the CS signal.

Conditioning of body temperature and natural killer cell activity with arecoline, a muscarinic cholinergic agonist.

Arecoline, a muscarinic cholinergic agonist, was found to depress body temperature and elevate the activity of preactivated natural killer (NK) cells. To demonstrate that the unconditioned responses produced by arecoline were mediated through central nervous system pathways, we used the drug as an unconditioned stimulus. By pairing camphor odor (conditioned stimulus) with arecoline (unconditioned stimulus), it was possible to simultaneously condition both a decrease in body temperature and augmentation of NK cell activity. The observations suggest that although both the modulation of body temperature and NK cell activities are integrated at the level of the hypothalamus, these pathways of regulation can be differentiated.

Lipopolysaccharide and IL-1 alpha activate CNS pathways as measured by NK cell activity.

The effect of lipopolysaccharide (LPS) on murine unstimulated and prestimulated natural killer (NK) cells and its ability to serve as an unconditioned stimulus was investigated. LPS injection induced a statistically significant increase in NK cell activity when compared with saline-treated control groups. To demonstrate the existence of communication between the peripheral immune system and the central nervous system (CNS), we used a single-trial conditioning paradigm in which camphor served as the conditioned stimulus (CS) and LPS as the unconditioned stimulus (US). Once a CS/US association is made, exposure of animals to the CS alone results in the conditioned response (i.e., increase in NK cell activity). Using 50 micrograms of LPS as the US produced a low but significant increase in NK cell activity when compared to control groups. However, 10 micrograms of LPS did not show a significant increase in NK cell activity. We also observed that interleukin-1 alpha (IL-1 alpha) injected intracisternally can serve as a US to condition a central neuroendocrine pathway. Because the dose of IL-1 alpha employed was too small to raise NK cell activity in the spleen, the NK cells themselves were formally not subjected to conditioning. These observations suggest that LPS and IL-1 alpha conditions the brain and that NK cell activity can be used as an indicator system to detect neuroendocrine signals arising from the activated pathway(s).

The use of conditioning to probe for CNS pathways that regulate fever and NK cell activity.

Immune and central nervous system (CNS) interactions are complicated because afferent signals from the immune system to the CNS in response to antigens or infections may elicit an immediate efferent response to the immune system. This communication loop is required for the homeostatic regulation of the immune system. Conditioning can be used as a tool to take the communication loop apart. In conditioned animals, the conditioned stimulus can be employed later to trigger the site of the association memory located within CNS, and set off the efferent pathway. Conditioning therefore allows one to isolate and identify the potential circuits in the brain that becomes conditioned. We have conditioned a pathway in the brain which can be used to modulate core body temperature (Tc) and natural killer (NK) cell activity. The Tc and NK cell activity are used as readouts to detect the expression of the conditioned response which is taking place in the brain. Since various cytokines (IFN, IL-1 etc) that are produced by antigenic stimulation invariably raise fever, it appears that the immune system could signal the CNS with nonspecific cytokines that activate the hypothalamic-pituitary pathway to modulate core body temperature. These observations infer that the thermoregulatory pathway in the brain becomes conditioned and points to a common pathway of communication in which interferon-beta, prostaglandin E2, CRH and ACTH appear to play a role in modulating both Tc and NK cell activity.

Arecoline a muscarinic cholinergic agent conditions central pathways that modulate natural killer cell activity.

The central nervous system plays an active role in the regulation of the immune system. Modulation of immune activities appears to be in part under the control of the hypothalamic-pituitary-adrenal (HPA) axis. We investigated the effect of a muscarinic cholinergic agonist, arecoline, which stimulates the secretion of corticotropin-releasing hormone (CRF) and adrenocorticotropic hormone (ACTH) on the immune system. In this report we demonstrate that peripherally administered arecoline or ACTH can increase activity of pre-activated NK cells. Second, we show that central administration of arecoline at a dose too low to alter peripheral events is sufficient to induce a significant increase in the activity of pre-activated natural killer (NK) cells. Finally, we demonstrate by using a Pavlovian conditioning paradigm that the pairing of a novel odor (camphor) with administration of arecoline can be used to alter NK cell activity. Subsequent to the conditioning trial, exposure to the odor alone is sufficient to raise NK cell activity. From these observations, we infer that the pathway(s) that are conditioned reside in sites located within the CNS and the conditioned response is evoked in the peripheral compartment (NK cell activity).

Conditioning of the secondary cytotoxic T-lymphocyte response to YC8 tumor.

Studies from our laboratory demonstrated that conditioned resistance to the syngeneic YC8 lymphoma was established by multiple conditioned stimulus (CS)/unconditioned stimulus (US) associations. The conditioned stimulus used was exposure to the odor of camphor for 1 h and the unconditioned stimulus was an injection of DBA/2 spleen cell alloantigen that shares minor histocompatibility determinants with the YC8 lymphoma. To demonstrate a cellular basis for immune resistance to the YC8 tumor, BALB/c mice primed with DBA/2 spleen cell alloantigen were conditioned using a single trial CS/US association paradigm. Conditioned animals showed a measurable conditioned elevation of the cytotoxic T-lymphocyte (CTL) response to the YC8 tumor. Control groups in which the CS and US were not given in the proper sequence were unable to mount a conditioned response. These studies show that a secondary CTL response can be upregulated by the central nervous system (CNS).

Lidocaine interrupts the conditioned natural killer cell response by interfering with the conditioned stimulus.

In the present study, lidocaine, a local anesthetic that inhibits the initiation or conduction of nerve impulses, was used to differentiate between the memory for the conditioned stimulus (CS) and the memory for the CS and unconditioned stimulus (US) association. Lidocaine was used to block memory formation. It was administered into the cisterna magna to localize the inhibition to the central nervous system (CNS) where circuits for the CS and US exist. The results show that lidocaine specifically blocks the ability of the CS to stimulate the circuits responsible for storing the CS/US association, but it does not interfere with the inherent ability of the US to signal the CNS and trigger a peripheral response. The observation that the CS circuit can be interrupted independently of the US circuit suggests that these signals come together to form a new circuit for the memory of the association. The association memory forms later and is independent of the memory for the CS.

Sodium carbonate prevents NK cell conditioning by interfering with the US signal.

The conditioned enhancement of natural killer (NK) cell activity can be blocked by the injection of sodium carbonate solution prior to the association of the camphor odor conditioned stimulus (CS) with the polyinosinic:polycytidylic acid (poly I:C) unconditioned stimulus (US). We have experimentally dissociated the memory which is formed for camphor odor from that developed for the association of camphor with the US. The memory for the odor of camphor can be allowed to develop one day before the administration of the unconditioned stimulus. Sodium carbonate appears to act within the central nervous system to block the association of the unconditioned stimulus with the conditioned stimulus. The data also suggest that in conditioning of NK cell activity, there are two specific sites for memory of the response, one for the conditioned stimulus and the other for the association of the unconditioned stimulus with the conditioned stimulus.

Use of an allogeneic antigen to induce resistance and regression of an autologous tumor.

Studies were initiated to determine if it was possible to use a tumor-bearing animal's own spleen cells to impart resistance to its neoplasm. YC8 T-cell lymphoma-bearing BALB/c mice (TBAs) were immunized with allogeneic DBA/2 spleen cells, which share cross-reacting antigens with the YC8 tumor. Animals immunized with the alloantigen were splenectomized and their spleen cells co-cultured with additional alloantigens for 2 days in media containing 2% polyethylene glycol (PEG) before being returned to the cyclophosphamide (cytoxan) (Ctx) pretreated autologous host. Treatment with Ctx was used to reduce suppressor factors in the TBA. It was found that when cultured spleen cells were returned to the autologous TBA, much greater resistance was imparted to the host and, in many instances, regression of the YC8 tumor was observed.

Role of arcuate nucleus of the hypothalamus in the acquisition of association memory between the CS and US.

A single trial association protocol was used to demonstrate a conditioned increase in natural killer (NK) cell activity. The signals used were odor of camphor as the conditioned stimulus (CS) and polyinosinic-polycytidylic acid (poly I:C) as the unconditioned stimulus (US). This model has been used to dissect the underlying mechanisms of interaction between the central nervous system (CNS) and the immune system (IS) and vice versa. Here, we demonstrate the potential role played by the arcuate nucleus of the hypothalamus in the acquisition of association memory between the CS and the US. Chemical destruction of the arcuate nucleus with monosodium glutamate (MSG) was used for this purpose. Mice with arcuate nucleus lesion prior to the association protocol did not demonstrate a conditioned increase in NK cell activity. However, the lesion has no effect if produced prior to exposure to the CS at recall. These studies demonstrate the significant role played by the hypothalamus (arcuate nucleus) in a conditioned response.

A multiple modality approach combining the effect of conditioning with adoptive chemoimmunotherapy.

It has been demonstrated that significant protection against YC8 lymphoma can be induced in mice preimmunized with normal DBA/2 spleen cells. The DBA/2 spleen cells used as an alloantigen share minor histocompatibility determinants with the YC8 tumor. Our observations showed that once tumor was present in vivo, the use of a potent tumor specific vaccine that can confer 100% protection to preimmunized animals, can help in increasing survival but can no longer produce high incidence of regression and cure. We have used this model to show that adoptive chemoimmunotherapy (ACIT) can be used to regress tumors in mice with large body burden of tumor and that combination of conditioning with ACIT appears to enhance the effectiveness of treatment. The nature of the immunity conferred by conditioned resistance might be due to cytotoxic T-lymphocytes.

Conditioning of the allogeneic cytotoxic lymphocyte response.

Allogeneic cytotoxic T-lymphocyte (CTL) response can be obtained following immunization of BALB/c mice with C57BL/6 spleen cells. We investigated the possibility of behaviorally conditioning this response by associating the C57BL/6 spleen cell immunization [unconditioned stimulus (US)] with camphor odor [conditioned stimulus (CS)]. We reported the possible mechanisms involved in the conditioning of natural killer cell activity. Similar approaches were used to investigate the mechanisms that participate in the conditioned CTL activity. The first mechanism of investigation utilized opioid receptor blockers naltrexone and quaternary naltrexone. Naltrexone, which blocks both the central and peripheral opioid receptors, blocked the recall of the conditioned response, whereas quaternary naltrexone, which does not penetrate the blood-brain barrier, was unable to block the conditioned response, demonstrating that centrally located opioid receptors play a role in the recall of the conditioned response. The studies are of interest because they indicate that resistance or susceptibility to various diseases such as cancer, autoimmunity, and infectious diseases might be influenced by the regulatory network of the CNS.

Identification of specific pathways of communication between the CNS and NK cell system.

The specific signals and pathways utilized by the natural killer (NK) cell system and the central nervous system (CNS) that results in the conditioned response (CR) is not clearly understood. Single trial conditioning of the NK cell activity provides us with a model to probe the mechanisms of communication between two major systems (Immune and CNS) which are involved in the health and disease of the individual. The studies show that the IFN-beta molecules possess the properties attributed to the unconditioned stimulus (US). IFN-beta can penetrate the CNS and evoke the elevation of NK cell activity in the spleen. This unconditioned response (UR) can be linked to a specific conditioned stimulus (CS). Specific odors such as camphor provide a neural pathway for the CS to associate with the US. Evidence is presented that in conditioning there are two locations where memory develops. The CS/US association is made centrally and its memory is stored at a central location, but the memory for the specificity of the odor is presumably stored in the olfactory bulbs. The CS recalls the CR by triggering the olfactory neural pathway which, in turn, signals the hypothalamic-pituitary axis to release mediators that modulate the activity of NK cells in the spleen. These results imply that through conditioning one has direct input into the regulatory hypothalamus that controls the internal environment of the organism and the health and disease of the individual. Consequently, it is not inconceivable that through this approach we might be able to alter the course of a disease process.