CpG oligodeoxynucleotides promote the host protective response against infection with Cryptococcus neoformans through induction of interferon-gamma production by CD4+ T cells.

In the present study, we elucidated the effect of synthetic CpG-containing oligodeoxynucleotides (ODN) on pulmonary and disseminated infection caused by Cryptococcus neoformans. CDF-1 mice were inoculated intratracheally with a highly virulent strain of this pathogen, which resulted in massive bacterial growth in the lung, dissemination to the brain and death. Administration of CpG-ODN promoted the clearance of C. neoformans in the lungs, decreased their dissemination to brain and prolonged the survival of infected mice. These effects correlated well with the enhanced production of interleukin (IL)-12 and interferon (IFN)-gamma and attenuated secretion of IL-4 in bronchoalveolar lavage fluids (BALF) and promoted development of Th1 cells, as indicated by the increased production of IFN-gamma by paratracheal lymph node cells upon restimulation with cryptococcal antigens. The IFN-gamma synthesis in BALF was inhibited by depletion of CD8(+) and CD4(+) T cells on days 7 and 14 after infection, respectively, but not by depletion of NK and gammadelta T cells. Consistent with these data, intracellular expression of IFN-gamma was detected predominantly in CD8(+) and CD4(+) T cells in the lung on days 7 and 14, respectively. The protective effect of CpG-ODN, as shown by the prolonged survival, was completely and partially inhibited by depletion of CD4(+) or CD8(+) T cells, respectively, but not by depletion of other cells. Finally, TNF-alpha was markedly induced by CpG-ODN, and the protective effect of this agent was strongly inhibited by neutralizing anti-TNF-alpha MoAb. Our results indicate that CpG-ODN alters the Th1-Th2 cytokine balance and promotes host resistance against infection with C. neoformans.

Lower expression of Th1-related cytokines and inducible nitric oxide synthase in mice with streptozotocin-induced diabetes mellitus infected with Mycobacterium tuberculosis.

Diabetes mellitus is an important predisposing factor for tuberculosis. The aim of this study was to investigate the mechanism underlying this association using a murine model. Mice with streptozotocin-induced diabetes mellitus were prone to Mycobacterium tuberculosis infection, as indicated by increased numbers of live bacteria in lung, liver and spleen. In diabetic mice, the levels of IL-12 and IFN-gamma in the lung, liver and spleen were lower than those in control animals on day 14 postinfection, while the opposite was true for IL-4 levels in the lung and liver. The expression pattern of inducible nitric oxide synthase (iNOS), in the two mice types was as for IL-12 and IFN-gamma. In addition, peritoneal exudate cells obtained from diabetic mice produced lower amounts of IL-12 and NO than those from control mice, when stimulated in vitro with M. bovis BCG. Spleen cells from diabetic mice infected with M. tuberculosis produced a significantly lower amount of IFN-gamma upon restimulation with purified protein derivatives (PPD) than those from infected nondiabetic mice. Interestingly, addition of high glucose levels (33 mM) to the cultures of PPD-restimulated spleen cells reduced the synthesis of IFN-gamma only in diabetic mice, and not in nondiabetic mice. Finally, control of blood glucose levels by insulin therapy resulted in improvement of the impaired host protection and Th1-related cytokine synthesis. Our results suggest that the reduced production of Th1-related cytokines and NO account for the hampered host defense against M. tuberculosis infection under diabetic conditions.

Monocyte chemoattractant protein-1-dependent increase of V alpha 14 NKT cells in lungs and their roles in Th1 response and host defense in cryptococcal infection.

To elucidate the role of NKT cells in the host defense to cryptococcal infection, we examined the proportion of these cells, identified by the expression of CD3 and NK1.1, in lungs after intratracheal infection with Cryptococcus neoformans. This population increased on day 3 after infection, reached a peak level on days 6-7, and decreased thereafter. In Valpha14 NKT cell-deficient mice, such increase was significantly attenuated. The proportion of Valpha14 NKT cells, detected by binding to alpha-galactosylceramide-loaded CD1d tetramer, and the expression of Valpha14 mRNA increased after infection with a similar kinetics. The delayed-type hypersensitivity response and differentiation of the fungus-specific Th1 cells was reduced in Valpha14 NKT cell-deficient mice, compared with control mice. Additionally, elimination of this fungal pathogen from lungs was significantly delayed in Valpha14 NKT cell-deficient mice. Production of monocyte chemoattractant protein (MCP)-1 in lungs, detected at both mRNA and protein levels, increased on day 1, reached a peak level on day 3, and decreased thereafter, which preceded the increase in NKT cells. Finally, the increase of total and Valpha14(+) subset of NKT cells after infection was significantly reduced in MCP-1-deficient mice. Our results demonstrated that NKT cells, especially Valpha14(+) subset, accumulated in a MCP-1-dependent manner in the lungs after infection with C. neoformans and played an important role in the development of Th1 response and host resistance to this fungal pathogen.

Enhanced gamma interferon production through activation of Valpha14(+) natural killer T cells by alpha-galactosylceramide in interleukin-18-deficient mice with systemic cryptococcosis.

We showed recently that activation of Valpha14(+) natural killer T cells (NKT cells) by alpha-galactosylceramide (alpha-GalCer) resulted in increased gamma interferon (IFN-gamma) production and host resistance to intravenous infection with Cryptococcus neoformans. In other studies, interleukin-18 (IL-18) activated NKT cells in collaboration with IL-12, suggesting the possible contribution of this cytokine to alpha-GalCer-induced IFN-gamma synthesis. Here we examined the role of IL-18 in alpha-GalCer-induced Th1 response by using IL-18KO mice with this infection. In these mice, levels of IFN-gamma in serum and its synthesis in vitro by spleen cells stimulated with live organisms were not reduced, but rather enhanced, compared to those in wild-type (WT) mice, while such production was completely absent in IL-12KO mice. The enhanced production of IFN-gamma correlated with increased IL-12 synthesis but not with reduced production of IL-4, which was rather increased. IFN-gamma synthesis in IL-18KO mice was abolished by neutralizing anti-IL-12 antibody and significantly inhibited by neutralization of endogenous IL-4 with a specific monoclonal antibody. In addition, administration of recombinant IL-4 significantly enhanced the production of IFN-gamma in WT mice. Finally, the enhanced production of IFN-gamma in IL-18KO mice correlated with increased host defense against cryptococcal infection, as indicated by enhancement in alpha-GalCer-related clearance of microorganisms. Our results indicated that in IL-18KO mice, IFN-gamma synthesis was enhanced through overproduction of IL-12 and IL-4 after intravenous infection with C. neoformans and a ligand-specific activation of Valpha14(+) NKT cells.

Activation of Valpha14(+) natural killer T cells by alpha-galactosylceramide results in development of Th1 response and local host resistance in mice infected with Cryptococcus neoformans.

We examined the effect of alpha-galactosylceramide (alpha-GalCer) on the synthesis of gamma interferon (IFN-gamma) and local resistance in mice infected intravenously with Cryptococcus neoformans. The level of IFN-gamma in serum increased on day 3, reached a peak level on day 7, and decreased to the basal level on day 14 postinfection in mice treated with alpha-GalCer, while in vehicle-treated mice, no increase was detected at any time points except for a small increase on day 7. Such effects were not observed in NKT-KO mice. In CD4KO mice, minor synthesis of IFN-gamma was detected on day 3 in sera but was completely abolished by day 7. The alpha-GalCer-induced IFN-gamma production on day 3 was partially reduced in mice depleted of NK cells by treatment with anti-asialo-GM(1) antibody (Ab). Spleen cells obtained from infected and alpha-GalCer-treated mice on day 7 produced a large amount of IFN-gamma upon restimulation with live organisms, while only a marginal level of production was detected in splenocytes from infected and vehicle-treated mice. Such effects were abolished in CD4KO and NKT-KO mice. Finally, the fungal loads in the lungs and spleen on days 7 and 14 were significantly reduced in alpha-GalCer-treated mice compared to those in control mice. In NKT-KO mice, local resistance elicited by alpha-GalCer was completely abolished, although no obvious exacerbation of infection was detected. Furthermore, treatment with anti-IFN-gamma monoclonal Ab mostly abrogated the protective effect of this agent. Thus, our results indicated that activation of Valpha14(+) NKT cells resulted in an increased Th1 response and local resistance to C. neoformans through production of IFN-gamma.

Surface imprinting polymers for the recognition of nucleotides.

A highly selective polymer has been prepared for the selective separation of nucleotides by the surface imprinting polymerization. A dialkyl quaternary ammonium chloride was effective as the functional molecule for recognizing the difference in the structure of nucleotides. Adsorptive behavior of the ionic species of the structural analogues, inosine-5'-monophosphoric acid (IMP) and guanosine-5'-monophosphoric acid (GMP), could be controlled by changing the pH condition. Surface imprinting polymers were prepared under different pH conditions; pH 9.0 and pH 8.5. The IMP-imprinted polymers exhibited higher template effect for IMP than for a structural analogue, GMP. A reference polymer prepared without the imprint molecule neither exhibit any selectivity to IMP nor to GMP. The adsorption behavior was quantitatively evaluated by the binding constants for the IMP-imprinted polymer. The imprinting polymer was found to recognize a small structural difference in nucleotides.

Modulation of memory processing in the cingulate cortex of mice.

To evaluate the possible role of the cingulate cortex in memory processing for training using a noxious stimulus, we trained mice on foot shock avoidance in a T-maze. Cholinergic, GABAergic, serotonergic, and glutamatergic agonists and antagonists were administered into the cingulate cortex immediately after training. Retention for the foot shock avoidance training was tested 1 week later. The results indicate that muscarinic and nicotinic agonists improved retention, while antagonists impaired it. GABA and serotonin agonists impaired retention, while antagonists improved it. Drugs acting on GABA(A) and GABA(B) receptors had similar effects on retention, as did drugs acting on serotonin 1 and 2 receptor subtypes. Glutamate improved retention, and AP5, an antagonist of the excitatory amino acid site of the NMDA receptor, impaired retention. The cingulate cortex, like other parts of the limbic system, is involved in memory processing that occurs shortly after training.

NK cells eliminate Cryptococcus neoformans by potentiating the fungicidal activity of macrophages rather than by directly killing them upon stimulation with IL-12 and IL-18.

In the present study, we examined whether natural killer (NK) cells have direct fungicidal activity against Cryptococcus neoformans. Splenic NK cells were obtained from SCID mice and stimulated with a combination of interleukin (IL)-12 and IL-18 in flat culture plates or round tubes. They were then or at the same time cultured with the yeast cells and the number of viable yeast cells was examined. We could not detect direct fungicidal activity by NK cells under any culture condition, although they produced a large amount of IFN-gamma and exerted marked cytotoxic activity against YAC-1 cells. On the other hand, NK cells significantly potentiated the nitric oxide-mediated cryptococcocidal activity of thioglycolate-elicited peritoneal macrophages obtained from SCID mice upon stimulation with IL-12 and IL-18. The culture supernatants of NK cells stimulated with IL-12 and IL-18 provided similar results when used in place of NK cells. The induction of macrophage anticryptococcal activity by NK cells and NK cell culture supernatants were both mediated by IFN-gamma because the specific mAb almost completely abrogated such effect. Considered collectively, our results suggested that NK cells may play a regulatory role in potentiating macrophage-mediated fungicidal mechanisms in host resistance to infection with C. neoformans rather than exerting a direct killing activity against the fungal pathogen.

Beta-amyloid precursor polypeptide in SAMP8 mice affects learning and memory.

Senescence accelerated (SAMP8 [P8]) mice develop age-related deficits in memory and learning. We show that increased expression of amyloid precursor protein (APP) and its mRNA in the hippocampus are also age-related. Immunocytochemical data suggest that a critical amount of APP expression may be needed to generate amyloid (Abeta) protein plaques in the hippocampus. Deficits in acquisition and retention test performance were alleviated by administration of antibody to Abeta protein into the cerebral ventricles. This reversal of cognitive deficits provides a link between increased expression of both APP and Abeta protein and learning and memory loss in these mice.

Septo-hippocampal drug interactions in post-trial memory processing.

To determine if serotonin and GABA regulate post-trial memory processing of the cholinergic projection from the septum to the hippocampus, mice were trained on footshock avoidance in a T-maze. Immediately after training, drugs were injected into the septum, hippocampus or both. Retention was tested 1 week after training and drug administration. Ketanserin, a serotonin type 2 receptor antagonist at a dose of 0.5 ng, had no measurable effect on retention, but it reduced the dose of bicuculline, in the septum, or arecoline in the hippocampus that was needed to improve retention. DOI, a serotonin type 2 receptor agonist at a dose of 2.5 ng, had the opposite effect of increasing the doses of bicuculline and arecoline needed to improve retention. Bicuculline, a GABA(A) receptor antagonist at a dose of 0.1 pg, did not affect retention when injected alone into the septum, but it reduced the dose of arecoline needed to improve retention in the hippocampus. Muscimol, a GABA(A) receptor agonist at a dose of 5 ng, injected into the septum, increased the dose of arecoline needed to improve retention. The results of this study are compatible with models that propose that serotonin innervation from the median raphe drives GABA interneurons in the medial septum that synapse on cholinergic neurons projecting to the hippocampus.

The pharmacology of post-trial memory processing in septum.

The septum is recognized as important in learning and memory, but relatively little is known about the role of specific neurotransmitter receptors in memory processing in the septum. We evaluated the role of the classical neurotransmitters in mice that were prepared for intraseptal microinfusion of drug solution after footshock avoidance training in T-maze. Retention for the footshock training was determined 1 week after training and drug administration. The results indicated that receptor agonists of dopamine, norepinephrine, glutamate and acetylcholine improved retention, while the antagonists impaired retention. Receptor agonists of serotonin, gamma-amino butyric acid (GABA) and opioids impaired retention, while antagonists improved retention.

Age-related changes in septal serotonergic, GABAergic and glutamatergic facilitation of retention in SAMP8 mice.

SAMP8/TaJf(P8) mouse strain has an inherited age-related impairment of learning and memory with its onset relatively early in its lifespan. Previously, it was reported that cholinergic and glutamatergic drugs injected into the hippocampus after behavioral training showed considerable shifts in the dose that improved retention in mice at 12 compared to 4 months of age. Cholinergic neurons in the septum supply most of the acetylcholine released in the hippocampus. In the present study, we determined if altered functional status of neurotransmission in the septum might account for the decrease in cholinergic and glutamatergic activity in the hippocampus of older SAMP8 mice. After training on footshock avoidance, P8 mice received a drug injection into the septum. Retention was tested 1 week later. The results indicate that bicuculline, GABA-A, and saclofen, GABA-B, receptor antagonist had to be injected at a higher dose in 12- than in 4-month-old mice to improve retention. The serotonergic antagonists, ketanserin and methiothepin, both showed dose response shifts such that less drug was needed to improve retention in 12- as compared to 4-month-old mice. It required four times more L-glutamate to improve retention in 12- than in 4-month-old mice. Agonists for acetylcholine, dopamine and norepinephrine receptors or an opiate antagonist required little or no change in the dose needed to improve retention in older P8 mice. SAMP8 mice may show an age-related impairment of septohippocampal functioning.

Effect of histamine H2 and H3 receptor modulation in the septum on post-training memory processing.

We compared the effects of modulating the postsynaptic histamine receptor subtype 2 (H2) and inhibitory presynaptic autoreceptor subtype 3 (H3) on memory processing in the septum. Mice were partially trained on footshock avoidance in a T-maze. Immediately after training, saline or a drug solution was infused into the septum. One week later, retention was tested by continuing training until the mice made five avoidance responses in six consecutive trials. The results indicate that dimaprit, an H2 agonist, facilitated retention (25 and 50 pg) with a U-shaped dose-response curve typical of drugs acting at postsynaptic receptors. Cimetidine, an H2 antagonist, impaired retention (15-50 ng). The H3 agonist. imetit, impaired retention (25-200 ng), while the H3 antagonist, thioperamide, facilitated retention (10-400 ng). An unusual feature of the dose-response curve for thioperamide was that it did not appeal to yield a U-shaped curve as occurs with drugs acting postsynaptically, but facilitated retention to approximately the same degree from 50 to 400 ng. As histamine neurons project to various limbic system structures involved in memory processing, it may play an important role in regulating the activity of structures such as the septum, hippocampus and amygdala.

The cortical neuropeptide, cortistatin-14, impairs post-training memory processing.

Cortistatin-14, a neuropeptide, is present primarily in the cortex and hippocampus. In the hippocampus, cortistatin-14 inhibits pyramidal cell firing and co-exists with GABA. To determine if cortistatin-14 would impair retention, saline or cortistatin-14 were injected intracerebroventricularly after footshock avoidance training in CD-1 mice. After 1 week, training was resumed to determine the effect of cortistatin-14 on retention. Cortistatin-14 was found to impair retention relative to the control group at doses of 0.5-5.0 micrograms.

Effects of dietary phosphatidylcholine on memory in memory deficient mice with low brain acetylcholine concentration.

Data concerning the effect of phosphatidylcholine (PCh) administration on the improvement of memory in senile dementia of Alzheimer type are inconsistent, probably due to the different conditions under which studies were conducted. Animal studies provide a good model, but data on this is limited. We studied the effect of PCh on memory in memory deficient mice (Dull mice) with low brain acetylcholine (ACh) concentration and normal mice. Mice were fed 24% casein diet (control) or this diet supplemented with 2 or 8% egg yolk PCh from gestation (Experiment 1) and after weaning (Experiment 2). Memory acquisition and retention were studied by step-down type passive avoidance performance at 8 and 10 weeks old, respectively. Control group of Dull mice had poorer memories than that of the normal mice in Experiments 1 and 2. On the 2% PCh diet, Dull mice improved memory acquisition and retention in Experiment 1 and retention in Experiment 2. On the 8% PCh diet in Dull mice there was improvement of memory and retention in Experiment 1, but no effect was observed in Experiment 2 (P > 0.05). In the normal mice, the 2% PCh diet did not affect memory acquisition and retention, however on the 8% PCh diet, there was no or adverse effect. These results suggest that dietary supplementation of egg yolk PCh improves memory of Dull mice, particularly when given from gestation and that the 2% PCh diet elicits better response than the 8% PCh diet.

Effects of dietary nucleoside-nucleotide mixture on memory in aged and young memory deficient mice.

Intestinal mucosa, bone marrow hematopoietic cells and brain have limited capacity for the de novo synthesis of nucleosides (NSs) and nucleotides (NTs). Whereas the role of dietary NS and NT in the former two tissues is known, it is not known in the brain. Therefore we studied the effect of dietary NS and NT mixture on memory in aged mice (Experiment 1) and young memory deficient mice (Experiment 2). Memory retention was studied by step-through type passive avoidance performance (maximum 180 seconds). In Experiment 1 aged (7 month old) senescence accelerated mice (SAM) were fed 20% casein diet (control) or this diet supplemented with 0.5% NS/NT mixture for 12 weeks. Memory was studied 1, 2 and 3 days after the electric shock (punishment). In Experiment 2, young (1 month old) memory deficient mice (Dull mice) and normal mice (ddY mice) were fed the same diets as those in Experiment 1 for 12 weeks. Memory retention was studied 1 and 3 days after the punishment. In the aged SAM the average time of avoidance and also the percentages of successful memory 2 and 3 days after the punishment were significantly higher in the NS/NT diet group than the control diet group (P < 0.05). In the Dull mice percentage of successful memory was higher in the NS/NT diet group than in the control group 3 days after the punishment, however, such an effect was not observed in the normal mice. These results suggest that insufficient endogenous supply of NSs and NTs may be responsible for the factor of memory deficiency with aging or of genetical memory deficiency, which can be improved by the dietary administration of NSs and NTs.

Administration of phosphatidylcholine increases brain acetylcholine concentration and improves memory in mice with dementia.

Studies on the effect of phosphatidylcholine administration on memory are limited. We administered egg phosphatidylcholine to mice with dementia and to normal mice and compared the differences in memory and serum choline concentration, and choline and acetylcholine concentrations and choline acetyltransferase activities of three forebrain regions (cortex, hippocampus and the remaining forebrain). Mice with dementia were produced by mating sibling mice who had impaired memory for > 20 generations. These mice had poor memory and low brain acetylcholine concentration. We administered 100 mg of egg phosphatidylcholine (phosphatidylcholine group) or water (control group) by gavage to each mouse daily for about 45 d. Control mice with dementia had poorer memory in passive avoidance performance and lower brain choline (cortex and hippocampus) and acetylcholine (hippocampus and forebrain excluding cortex and hippocampus) concentrations and lower cortex choline acetyltransferase activity than the control normal mice (P < 0.05). The administration of phosphatidylcholine to mice with dementia improved memory and generally increased brain choline and acetylcholine concentrations to or above the levels of the control normal mice. In normal mice, phosphatidylcholine treatment did not affect memory or acetylcholine concentrations in spite of the great increase in choline concentrations in the three brain regions. Serum choline concentration in mice treated with phosphatidylcholine increased to a similar level in both strains of mice, indicating that the absorption of phosphatidylcholine was not impaired in mice with dementia. The results suggest that administration of egg phosphatidylcholine to mice with dementia increases brain acetylcholine concentration and improves memory.

Ex vivo effect of insulin on normal and diabetic rat hearts hypoperfused with norepinephrine.

The effect of ex vivo insulin on contractile and energy metabolism dysfunctions was examined during hypoperfusion (0.6 ml/min per g heart weight) with 10(-6) M norepinephrine in isolated non-diabetic and streptozotocin-diabetic rats hearts. Insulin (2 mU/min per g heart weight) was infused for 15 min before as well as during 60-min hypoperfusion. Insulin significantly reduced the elevated diastolic tension in diabetic hearts (from 3.8 to 0.7 delta g), but not in non-diabetic hearts (from 1.4 to 1.2 delta g). Insulin partly improved the ATP decrease in the subendocardium of the left ventricle of the diabetic hearts (from 3.5 to 10.2 mumol/g dry weight) but did not affect non-diabetic hearts (from 6.9 to 6.8 mumol/g dry weight). Insulin also partly improved the creatine phosphate decrease and the inorganic phosphate increase in diabetic hearts only. Lactate accumulation was greater in non-diabetic than in diabetic hearts, even in the presence of insulin (77 vs. 45 mumol/g dry weight). The results indicate that acute intracoronary application of insulin in diabetic hearts improves hypoperfusion with norepinephrine injury to a level above that of non-diabetic hearts, but does not improve a less severe injury in non-diabetic hearts.

Effects of the new thromboxane A2 antagonist vapiprost on isolated canine blood vessels.

Effects of the new thromboxane A2 antagonist vapiprost (SN-309, GR-32191B, CAS 85505-64-2) on isolated canine blood vessels were investigated. U46619 ((15S)-hydroxy-11a, 9a-(epoxymethano) prosta-5Z, 13E-dienoic acid) 10(-10)-10(-6) mol/l, a thromboxane A2 analogue, produced concentration-dependent contractions of oblong or ring preparations isolated from basilar, coronary, mesenteric and femoral arteries. Vapiprost 10(-8) and 10(-7) mol/l significantly and concentration-dependently shifted the concentration-contraction curves for U46619 of these arteries to the right. The pA2 values were 8.80 +/- 0.09 in basilar arteries, 8.67 +/- 0.12 in coronary arteries, 8.86 +/- 0.05 in mesenteric arteries and 9.01 +/- 0.07 in femoral arteries. On the other hand, oblong or ring preparations of basilar, coronary, mesenteric and femoral arteries showed sustained contractile responses to KCl 3 x 10(-2) mol/l, U46619 10(-7) mol/l or prostaglandin (PG) F2 alpha 10(-5) mol/l. Norepinephrine (NE) 3 x 10(-5) mol/l also produced sustained contractions in mesenteric and femoral arterial preparations, but not in basilar and coronary arterial preparations. Vapiprost 10(-10)-3 x 10(-6) mol/l relaxed these four arterial preparations constricted with U46619 10(-7) mol/l and PGF 2 alpha 10(-5) mol/l in a concentration-dependent fashion, but hardly affected them constricted with KCl 3 x 10(-2) mol/l. NE 3 x 10(-5) mol/l-induced contractures of mesenteric and femoral arterial preparations were not influenced by any concentrations of vapiprost. Results indicate that vapiprost has an antagonistic action on a so-called TP-receptor and/or a vasoconstrictive prostaglandin(s)-receptor and thus produces vasorelaxation.