Chronic treatment with the glucocorticoid receptor antagonist RU486 inhibits diabetes-induced enhancement of experimental periodontitis.

BACKGROUND AND OBJECTIVE: Chronic hypothalamic-pituitary-adrenal (HPA) axis hyperactivity and excessive glucocorticoid hormone release have been associated with diabetes, altered immune responses and increased susceptibility to periodontitis. In the present study we tested the impact of streptozotocin (STZ)-induced diabetes on ligature-induced periodontitis and the effect of subsequent treatment with the glucocorticoid receptor (GR) antagonist, RU486. MATERIAL AND METHODS: A single dose of STZ [45 mg/kg, intraperitoneally (i.p.)] or vehicle was given 10 d before induction of ligature-induced periodontitis and implantation subcutaneously of a drug pellet containing the GR antagonist, RU486, or a placebo pellet. Periodontitis was assessed when the ligatures had been in place for 21 d. Two hours before decapitation all rats received gram-negative bacterial lipopolysaccharide (LPS) (150 µg/kg, i.p.) to induce a robust immune and stress response. RESULTS: Compared with control rats, STZ-treated rats developed significantly more periodontal bone loss, and RU486 treatment of STZ -treated rats significantly inhibited this effect. STZ-treated rats also showed significantly higher levels of the HPA axis-derived hormone, corticosterone, as well as of the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α), but lower levels of the anti-inflammatory cytokines interleukin-10 (IL-10) and transforming growth factor-1beta (TGF-1ß) after LPS stimulation. GR blockade had no statistically significant effects on these measurements in diabetic rats, but tended to enhance the levels of TNF-α and TGF-1ß, and reduce the levels of IL-10 and blood glucose. CONCLUSION: In diabetic subjects, excessive GR activation as a result of chronic high levels of glucocorticoid hormones may alter immune-system responses in a manner that may increase the susceptibility to periodontitis.

Oral treatment with complement factor C5a receptor (CD88) antagonists inhibits experimental periodontitis in rats.

BACKGROUND AND OBJECTIVE: The complement activation product 5a (C5a) is a potent mediator of the innate immune response to infection, and may thus also importantly determine the development of periodontitis. The present study was designed to explore the effect of several novel, potent and orally active C5a receptor (CD88) antagonists (C5aRAs) on the development of ligature-induced periodontitis in an animal model. MATERIAL AND METHODS: Three different cyclic peptide C5aRAs, termed PMX205, PMX218 and PMX273, were investigated. Four groups of Wistar rats (n = 10 in each group) were used. Starting 3 d before induction of experimental periodontitis, rats either received one of the C5aRas (1-2 mg/kg) in the drinking water or received drinking water only. Periodontitis was assessed when the ligatures had been in place for 14 d. RESULTS: Compared with control rats, PMX205- and PMX218-treated rats had significantly reduced periodontal bone loss. CONCLUSION: The findings suggest that complement activation, and particularly C5a generation, may play a significant role in the development and progression of periodontitis. Blockade of the major C5a receptor, CD88, with specific inhibitors such as PMX205, may offer novel treatment options for periodontitis.

Severe gunshot injuries in a porcine model: impact on central markers of innate immunity.

BACKGROUND: the mechanisms behind lipopolysaccharide (LPS) tolerance remain obscure. LPS signals through Toll-like receptor 4 (TLR4) and severe trauma/haemorrhage may influence binding and signalling through this receptor, e.g. by changing membrane expression or by releasing endogenous ligands like High Mobility Group Box 1 (HMGB1). The aim of this study was to examine these relations further in a porcine model with standardized trauma. METHODS: nine anaesthetized pigs sustained one gunshot through the femur and one pistol shot through the upper abdomen. Blood was sampled before and 90 min after shooting. The samples were stimulated for 4 h with LPS 10 ng/ml or an equivalent amount of normal saline. The leucocyte response was evaluated by measuring the tumour necrosis factor-α (TNF-α) and CXC ligand 8 (CXCL8) in the supernatant. Flow cytometry was used to measure the surface expression of TLR4 on CD14+ monocytes. HMGB1 concentrations were measured in the plasma. RESULTS: trauma and treatment caused a significant decline in the LPS-stimulated concentrations of TNF-α [4.53 ± 0.24 pg/ml (ln) at 0 min, 3.54 ± 0.35 pg/ml (ln) at 90 min, P=0.026], but did not modify the release of CXCL8. Monocyte TLR4 expression was unchanged. Plasma HMGB1 increased significantly [<0.92 vs. 3.02 ± 0.19 ng/ml (ln), P<0.001]. The concentrations of TNF-α and CXCL8 did not correlate with TLR4 expression or HMGB1 concentrations. CONCLUSION: the results suggest that trauma-induced LPS tolerance is not primarily regulated by TLR4 expression on circulating CD14+ monocytes or by the release of HMGB1 from damaged tissues.

Nicotinic acetylcholine receptor activation mediates nicotine-induced enhancement of experimental periodontitis.

BACKGROUND AND OBJECTIVE: Smokers have an increased risk of developing periodontitis as well as showing more rapid progression and resistance to treatment of the disease, but the biological mechanisms are poorly understood. This study was designed to investigate putative biological mechanisms by which nicotine may enhance the susceptibility and thus the course of periodontitis in an animal model. MATERIAL AND METHODS: Ligature-induced periodontitis was applied in periodontitis-susceptible Fischer 344 rats. The animals were either given daily intraperitoneal injections of the nicotinic acetylcholine receptor antagonist mecamylamine (1 mg/kg) 45 min before subcutaneous injections in the neck skin of nicotine (0.8 mg/kg), or treated with the same amount of saline intraperitoneally and nicotine subcutaneously, or treated with mecamylamine and saline. Control animals received intraperitoneal and subcutaneous injections of saline only. Periodontal bone loss was assessed when the ligatures had been in place for 3 wk. Two hours before decapitation, all rats received lipopolysaccharide (LPS; 100 microg/kg, intraperitoneally) to induce a robust immune and stress response. RESULTS: Compared with saline/saline-treated control animals, saline/nicotine-treated rats developed significantly more periodontal bone loss, and LPS provoked a significantly smaller increase in circulating levels of the cytokines tumour necrosis factor-alpha, transforming growth factor-1beta and interleukin-10. Mecamylamine pretreatment of nicotine-treated rats abrogated the increased periodontal bone loss and the LPS-induced decrease in tumour necrosis factor-alpha, but had no significant effects on the levels of transforming growth factor-1beta and interleukin-10, or the stress hormone corticosterone. CONCLUSION: The results indicate that nicotine enhances the susceptibility to periodontitis via nicotinic acetylcholine receptors, which may act by suppressing protective immune responses through the cholinergic anti-inflammatory pathway.

Nicotinic acetylcholine receptor activation mediates nicotine-induced enhancement of experimental periodontitis.

BACKGROUND AND OBJECTIVE: Smokers have an increased risk of developing periodontitis as well as showing more rapid progression and resistance to treatment of the disease, but the biological mechanisms are poorly understood. Our objective was to investigate putative biological mechanisms by which nicotine may enhance the susceptibility and thus the course of periodontitis in an animal model. MATERIAL AND METHODS: Ligature-induced periodontitis was applied in periodontitis-susceptible Fischer 344 rats. The animals were given daily intraperiotonal (i.p.) injections of the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine (1 mg/kg) 45 min before subcutaneous (s.c.) injections in the neck skin with nicotine (0.8 mg/kg), or treated with the same amount of saline i.p. and nicotine s.c., or with mecamylamine and saline. Control rats received i.p. and s.c. injections of saline only. Periodontal bone loss was assessed when the ligatures had been in place for 3 weeks. Two hours before decapitation, all rats received lipopolysaccharide (LPS; 100 microg/kg, i.p.) to induce a robust immune and stress response. RESULTS: Compared with saline/saline-treated control rats, saline/nicotine-treated rats developed significantly more periodontal bone loss, and LPS provoked a significantly smaller increase in circulating levels of the cytokines tumour necrosis factor alpha (TNF-alpha), transforming growth factor 1beta (TGF-1beta) and interleukin-10 (IL-10). Mecamylamine pretreatment of nicotine-treated rats abrogated the increased periodontal bone loss and the LPS-induced TNF-alpha decrease, but had no significant effects on the levels of TGF-1beta and IL-10, or the stress hormone corticosterone. CONCLUSION: The results indicate that nicotine enhances susceptibility to periodontitis via nAChRs, which may act via suppressing protective immune responses through the cholinergic anti-inflammatory pathway.

Early time course of altered leukocyte response to lipopolysaccharide and peptidoglycan in porcine gunshot injury.

BACKGROUND: Penetrating injuries are frequently combined with polybacterial soiling. Clearance of the microorganisms depends on the ability to activate immune responses, but post-traumatic hyporeactivity of immune cells is almost universal. The aim of this study was to map the early time course of this altered leukocyte reactivity, and to compare the reactions to subsequent Gram-positive or Gram-negative challenges. METHODS: Twelve juvenile pigs sustained two standardized rounds, one through the right femur and one through the left upper abdomen. First aid treatment and acute surgery were started immediately. Blood samples were drawn before trauma and after 10, 30, 60, and 90 min, and thereafter stimulated in ex vivo whole blood for 3 h with lipopolysaccharide (LPS, 10 ng/ml), peptidoglycan (PepG, 1 microg/ml), or an equivalent amount of normal saline. The leukocyte response was evaluated by measurement of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-6, IL-8, and IL-10 in the supernatant. RESULTS: In the post-traumatic in vivo serum, the concentration of TNF-alpha increased steadily (significant after 60 min). A reduced ex vivo reaction to LPS was evident after 10 min, and was statistically significant after 30 min. The lowest levels were reached after 90 min. The ex vivo synthesis of TNF-alpha after stimulation with PepG remained unaltered. A similar development was seen for IL-6. IL-1 beta levels did not change, while IL-8 increased significantly only after 60 and 90 min. CONCLUSIONS: Trauma almost instantaneously reprogrammed circulating leukocytes. As measured with TNF-alpha, a profound hyporeactivity to LPS, but not to PepG, was induced. In addition, no global down-regulation of leukocyte function was found after stimulation with LPS.

Androgenic hormones during prolonged physical stress, sleep, and energy deficiency.

Androgenic hormones were investigated during two separate 5-day military endurance training courses, with physical activities around the clock corresponding to a daily energy consumption of about 40,000 kilojoules, but with an intake of only 2,000 kilojoules. Altogether, the cadets slept for 1-3 h in the 5 days. Eleven male cadets participated in course I, and 10 in course II. Plasma levels of testosterone, free testosterone, dehydroepiandrosterone, 17 alpha-hydroxyprogesterone, and androstenedione decreased by 60-80% during the course. In contrast, plasma cortisol, aldosterone, progesterone, and dehydroepiandrosterone sulfate increased. LH, FSH, and ACTH decreased to about 50-80% of precourse levels. Weak correlations between plasma levels of hypophyseal and levels of adrenal and testicular hormones indicate a multifactorial regulation. In conclusion, both adrenal and testicular androgens decrease during prolonged physical strain combined with energy and sleep deficiency.

The plasma vasoactive intestinal peptide (VIP) response to exercise is increased after prolonged strain, sleep and energy deficiency and extinguished by glucose infusion.

The VIP response to a 30 minute bicycle exercise test with a workload of 50% of VO2 max was investigated in young men before and after 5 days of continuous physical activities, almost total sleep deprivation and limited amounts of food. It was shown that plasma VIP increased during physical exercise lasting for more than 20 minutes with a workload of more than 50% of VO2 max. A further increase took place in the early recovery period to a maximum level 5-10 minutes after the exercise. This response to exercise is even stronger after prolonged strain. Glucose infusion during the exercise almost abolished the increase of plasma VIP.

Receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes are upregulated during prolonged strain and energy deficiency.

VIP receptors on blood mononuclear leucocytes and plasma VIP concentrations were studied during a ranger training course lasting for five days with almost continuous physical activity, and energy deficiency. The maximum binding capacity (Bmax) for the high affinity receptor increased (p less than 0.0005) from 0.71 (SEM = 0.11, N = 10) fmol/million cells to a maximum of 7.33 (SEM = 1.0) fmol/million cells on Day 4. There was no significant change in the dissociation constant (Kd) for the high affinity receptor, and no effect on Kd nor Bmax for the low affinity VIP receptor was detected. Plasma VIP concentration increased (p less than 0.0005) from 8.8 pmol/l (SEM = 0.6) to a maximum of 23.4 (SEM = 1.9) on the second day of the course. However, the highest plasma concentrations were about one order of magnitude lower than the dissociation constant (Kd) for the high affinity VIP receptor on the mononuclear leucocytes. These data indicate that heterologous upregulation of the high affinity VIP receptor on mononuclear blood cells takes place during combined strenuous physical exercise, and calorie deficiency.

Effect of VIP on the respiratory burst in human monocytes ex vivo during prolonged strain and energy deficiency.

VIP-stimulated cyclic AMP production and VIP effect on the production of reactive oxygen compounds in human monocytes activated by serum opsonized zymosan (respiratory burst) were studied during a ranger training course lasting for five days with almost continuous physical activity, and deficiency of sleep and energy. Respiratory burst was inhibited and cyclic AMP production was stimulated by VIP on all days. Maximum cyclic AMP production stimulated by VIP (0.1 microM) on the day of control was 148.6% of basal, and 255.3%, 213.8%, 218.9% and 198.7% on Days 1, 2, 3 and 5. Maximum inhibition was observed 20 min after addition of the peptide on the day of control, after 5 min on Days 1, 2 and 3, and after 10 min on Day 5. Inhibition at the 5-min time point was 33.1% on the day of control, and 34.7%, 53.6%, 53.3% and 36.2% on the different days during the training course. The observed increment in VIP effect adds to prior reported data about increased VIP secretion during the training course, and may indicate enhanced physiological significance of VIP during stress.

Binding of vasoactive intestinal polypeptide (VIP) by human blood monocytes: demonstration of specific binding sites.

Vasoactive intestinal polypeptide (VIP) interaction with a 94% pure preparation of monocytes isolated from human peripheral blood was studied by direct binding technique using 3-[125I]tyrosyl-VIP as a tracer ligand. Scatchard analysis of binding data was compatible with two classes of binding sites, one with Kd = 0.25 nM and maximal binding capacity of 16 fmol/10(6) cells, and another one with Kd = 25 nM and maximal binding capacity of 180 fmol/10(6) cells. The binding was time-, temperature-, and pH-dependent and was saturable, reversible, and specific. This study has demonstrated that human monocytes have high affinity/low capacity as well as low affinity/high capacity binding sites for VIP. No specific VIP binding was found in pure preparations of human granulocytes, platelets or erythrocytes.

Elevated VIP and endotoxin plasma levels in human gram-negative septic shock.

Vasoactive intestinal polypeptide (VIP) and endotoxin (lipopolysaccharides, LPS) were measured in plasma samples from 11 patients with bacteriologically verified meningococcal disease. Five patients suffered fulminant septicaemia, developed severe septic shock, and 2 died due to circulatory collapse. Initially, all 5 had levels of VIP above 4 pM and plasma endotoxin above 200 ng/liter. Five patients were diagnosed as meningitis and 1 as having meningococcaemia, all with a normal circulatory state. None of these 6 patients had initially levels of VIP above 2.5 pM or endotoxin levels above 25 ng/liter (P less than 0.001). A correlation existed between plasma endotoxin and VIP levels (r = 0.735, P = 0.01). Sequentially collected samples from 3 patients showed rapidly declining VIP levels after initiation of antibiotic and fluid treatment. These results are in agreement with previous animal experiments, suggesting that endotoxin directly or indirectly stimulates the VIP-ergic nervous system in the initial phase of gram-negative septic shock in man.

Secretin--a new stress hormone?

The plasma concentration of secretin was measured during a 5-day military training course comprising prolonged physical exercise (35% of max O2 uptake), severe caloric deficiency (approx. 35700 kJ/24 h) and sleep deprivation (only 2 h of sleep as a total during 5 days). 24 subjects were divided into 3 groups, one group was compensated for the caloric deficiency and another group was partly compensated for the sleep deprivation. The results showed that the fasting plasma secretin increased 3-6-fold (from 1.8-3.7 to 13.3-19.1 pmol/l) during the course with small differences in increase between the groups. Ingestion of a mixed meal reduced the fasting plasma secretin by about 40% during the course, while oral glucose reduced the plasma secretin to the concentrations found in the control experiment. The study shows that plasma secretin is increased when man is exposed to prolonged multifactorial stress. Additional food or sleep appears to have small influence on the fasting plasma secretin, but after giving a meal or oral glucose solution the plasma secretin decreases rapidly.

Basal hyperchlorhydria and its relation to the plasma concentrations of secretin, vasoactive intestinal polypeptide (VIP) and gastrin during prolonged strain.

Twenty young men divided into two groups participated in a five day training course with prolonged and heavy physical exercise, calorie supply deficiency and severe sleep deprivation. Basal acid output (BAO) was measured immediately after the course in seven of ten subjects who were given placebo tablets (placebo group) and in four of ten subjects who had a daily intake of 1 g cimetidine (cimetidine-group) during the course. Median BAO increased 3-fold in the placebo subjects (from 2.7 mmol/h to 8.2 mmol/h) but showed no increase in the cimetidine treated subjects. The median fasting plasma concentrations of secretin increased 2-8-fold during the course. Gastric suction for 1 h or ingestion of cimetidine reduced the plasma concentration of secretin by approx. 50%. Vasoactive intestinal polypeptide (VIP) increased 2-fold and was not influenced by reduction of gastric acid. The placebo group showed a small increase (P less than 0.05) in plasma concentration of gastrin on day two during the course. The study shows a marked hyperchlorhydria which partly explains the fasting hypersecretinemia found during prolonged strain. This strain-induced hyperchlorhydria could be abolished by treatment with the selective H2-receptor antagonist cimetidine.

Biochemical plasticity in the superior colliculus of adult rats after chronic visual cortex ablation.

We have measured the changes of several neurochemical parameters in the adult rat superior colliculus (SC) 1.5-4 months after unilateral visual cortex ablation. High-affinity uptake of D-[3H]Asp was increased by 22.5% and glutamate decarboxylase by 10% in the ipsilateral SC. This may be largely attributed to reactive synaptogenesis of intrinsic glutamatergic and GABAergic neurons, respectively. The noradrenaline content was increased by about 60 and 25% in the ipsi- and contralateral SCs, respectively. The latter probably reflects sprouting of noradrenergic fibres from the locus coeruleus.

Performance, mood, and clinical symptoms in men exposed to prolonged, severe physical work and sleep deprivation.

There were 44 young men who participated in strenuous combat courses of 4 d (course I) or 5 d (course II), almost without sleep. They were tested and examined clinically each morning. Groups 1 and 2 had no organized sleep, whereas groups 3 and 4 got 3 and 6 h, respectively, in the middle of each course. Substantial impairment was observed in all tests, as well as clinical symptoms toward the end of the courses for groups 1 and 2. In the vigilance test, the reaction time task, the code test, and the profile of mood-state, significant impairment was observed even after 24 h. Complaints of symptoms came first. Disturbance of senses and behaviour appeared later. Group 4 had significantly better results than groups 1 and 2 in clinical symptoms and all tests, except the positive score in mood-state. Group 3 occupied an intermediate position. Corresponding results were obtained in the two separate courses. In the morning following the course, recovery after 4 h of sleep was less extensive for courses II than course I participants.

Reduced systolic blood pressure elevations during maximum exercise at simulated altitudes.

Ten healthy female subjects performed maximum exercise on a bicycle in an altitude chamber during normoxia and hypobaric hypoxia simulating altitudes of 2,450, 3,700 and 4,600 m. The increases in systolic blood pressure responses were reduced with the degree of hypobaric hypoxia, whereas heart rate and diastolic pressure responses were unchanged. The increases in blood levels of aldosterone, plasma renin activity, adrenaline, noradrenaline, neuropeptide-Y and vasoactive intestinal peptide were similar at the different simulated altitudes. Angiotensin-converting enzyme and vasoactive intestinal peptide levels were not affected by hypoxia or maximum exercise. The present results suggest that the decreases in systolic blood pressure responses during hypobaric hypoxia could not be explained by altered responses of the measured vasoactive substances from the renin-angiotensin, gastrointestinal, and autonomic nervous systems.

Capacity of glycine to modulate early inflammatory disturbances after serious gunshot injuries in the pig.

OBJECTIVE: Perturbation of immune homeostasis is an important determinant for organ dysfunction following multiple injuries. The aim of this study was to investigate the ability of glycine to influence the immediate post-traumatic inflammatory environment and altered reactivity of circulating leucocytes. MATERIAL AND METHODS: Twenty pigs were subjected to two standardized gunshots to the abdomen and thigh. Treatment was started immediately. The animals were randomized to receive either glycine 180 mg/kg i.v. over 30 min (n=10) or normal saline (n=10). Blood samples were drawn at baseline and 75 min after injury. In a follow-up study 12 pigs were exposed to an identical trauma. Blood was drawn at the same time-points and stimulated with lipopolysaccharide (LPS) or LPS plus glycine for 2 h in an ex vivo whole blood model. RESULTS: Selected physiologic variables and organ injury did not differ between groups 75 min after trauma. Reactive oxygen species decreased to 82.7+/-5.5 % of baseline (p<0.05) in the glycine group (unaltered in the controls). Liver glutathione concentrations decreased in parallel in both groups. In vivo production of TNF-alpha and IL-1-beta increased to the same extent regardless of treatment. Trauma induced a strong LPS tolerance. In whole blood challenged with LPS, glycine inhibited cytokine synthesis, but only in samples drawn at baseline. CONCLUSIONS: Post-traumatic infusion of glycine only modestly influenced the early post-traumatic inflammatory environment. Our ex vivo results confirm previous reports on the anti-inflammatory potential of glycine, but restricted to pre-trauma conditions.