Increased oxygen exposure alters collagen expression and tissue architecture during ligature-induced periodontitis.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the effects of increased oxygen availability on gene expression and on collagen deposition/maturation in the periodontium following disease. MATERIAL AND METHODS: Male Wistar rats had ligatures placed around their molars to induce periodontal disease, and a subset of animals underwent hyperbaric oxygen (HBO) treatment for 2 h twice per day. At 15 and 28 d, tissue gene expression of COL1A1, transforming growth factor-ß1 and alkaline phosphatase was determined; other histological samples were stained with Picrosirius red to evaluate levels of collagen deposition, maturation and thickness. RESULTS: In animals that underwent HBO treatment, type I collagen expression was higher and collagen deposition, maturation and thickness were more robust. Reduced mRNA levels of transforming growth factor-beta1 and alkaline phosphatase in HBO-treated rats on day 28 suggested that a quicker resolution in both soft tissue and bone remodeling occurred following oxygen treatment. No differences in inflammation were observed between groups. CONCLUSIONS: The extracellular matrix regenerated more quickly in the HBO-treated group as evidenced by higher collagen expression, deposition and maturation.

Psychological distress and salivary secretory immunity.

Stress-induced impairments of mucosal immunity may increase susceptibility to infectious diseases. The present study investigated the association of perceived stress, depressive symptoms, and loneliness with salivary levels of secretory immunoglobulin A (S-IgA), the subclasses S-IgA1, S-IgA2, and their transporter molecule Secretory Component (SC). S-IgA/SC, IgA1/SC and IgA2/SC ratios were calculated to assess the differential effects of stress on immunoglobulin transport versus availability. This study involved 113 university students, in part selected on high scores on the UCLA Loneliness Scale and/or the Beck Depression Inventory. Stress levels were assessed using the Perceived Stress Scale. Unstimulated saliva was collected and analysed for total S-IgA and its subclasses, as well as SC and total salivary protein. Multiple linear regression analyses, adjusted for gender, age, health behaviours, and concentration effects (total protein) revealed that higher perceived stress was associated with lower levels of IgA1 but not IgA2. Perceived stress, loneliness and depressive symptoms were all associated with lower IgA1/SC ratios. Surprisingly, higher SC levels were associated with loneliness and depressive symptoms, indicative of enhanced transport activity, which explained a lower IgA1/SC ratio (loneliness and depression) and IgA2/SC ratio (depression). This is the first study to investigate the effects of protracted psychological stress across S-IgA subclasses and its transporter SC. Psychological stress was negatively associated with secretory immunity, specifically IgA1. The lower immunoglobulin/transporter ratio that was associated with higher loneliness and depression suggested a relative immunoglobulin depletion, whereby availability was not keeping up with enhanced transport demand.

Hypnosis as a modulator of cellular immune dysregulation during acute stress.

To assess the influence of a hypnotic intervention on cellular immune function during a commonplace stressful event, the authors selected 33 medical and dental students on the basis of hypnotic susceptibility. Initial blood samples were obtained during a lower stress period, and a second sample was drawn 3 days before the first major exam of the term. Half of the participants were randomly assigned to hypnotic-relaxation training in the interval between samples. Participants in the hypnotic group were, on average, protected from the stress-related decrements that were observed in control participants' proliferative responses to 2 mitogens, percentages of CD3+ and CD4+ T-lymphocytes, and interleukin 1 production by peripheral blood leukocytes. More frequent hypnotic-relaxation practice was associated with higher percentages of CD3+ and CD4+ T-lymphocytes. These data provide encouraging evidence that interventions may reduce the immunological dysregulation associated with acute stressors.

Social stress increases the susceptibility to endotoxic shock.

The influence of social disruption stress (SDR) on the susceptibility to endotoxic shock was investigated. SDR was found to increase the mortality of mice when they were challenged with the bacterial endotoxin lipopolysaccharide (LPS). Histological examination of SDR animals after LPS injection revealed widespread disseminated intravascular coagulation in the brain and lung, extensive meningitis in the brain, severe hemorrhage in the lung, necrosis in the liver, and lymphoid hyperplasia in the spleen, indicating inflammatory organ damage. In situ hybridization histochemical analysis showed that the expression of the glucocorticoid receptor mRNA was down-regulated in the brain and spleen of SDR animals while the ratio of expression of AVP/CRH-the two adrenocorticotropic hormone secretagogue, increased. After LPS injection, the expression of pro-inflammatory cytokines, IL-1beta and TNF-alpha, was found significantly higher in the lung, liver, spleen, and brain of the SDR mice as compared with the LPS-injected home cage control animals. Taken together, these results show that SDR stress increases the susceptibility to endotoxic shock and suggest that the development of glucocorticoid resistance and increased production of pro-inflammatory cytokines are the mechanisms for this behavior-induced susceptibility to endotoxic shock.

The role of protein kinase C and calcium in induction of human polymorphonuclear leukocyte IL-1 beta gene expression by GM-CSF.

At infection sites, synthesis of interleukin (IL-)1beta by polymorphonuclear leukocytes (PMNs) facilitates the recruitment of inflammatory cells and enhances the inflammatory response. We investigated the role of protein kinase C (PKC) and Ca2+ in the induction of PMN IL-1beta gene expression by GM-CSF. The PKC inhibitors chelerythrine and H7 blocked induction of IL-1beta mRNA expression in human PMNs. HA1004, an H7 analogue with little activity towards PKC, had no inhibitory effect. Similarly, H7 blocked IL-1beta transcription in nuclear run-on analysis, while HA1004 had little effect. The intracellular Ca2+ chelator BAPTA/AM inhibited induction of IL1beta mRNA accumulation and transcription by GM-CSF. At concentrations similar to those used to inhibit IL-1beta gene expression, H7, chelerythrine, and BAPTA all inhibited substrate phosphorylation by PKC isolated from PMN lysates. Thus, PKC and Ca2+ are potential targets for modulating an important PMN immunoregulatory function.

Stress and immunity: implications for viral disease and wound healing.

It is now well established that psychological stress can downregulate the cellular immune response. Communication between the central nervous system and the immune system occurs via a complex network of bidirectional signals linking the nervous, endocrine, and immune systems. Stress disrupts the homeostasis of this network, which in turn, alters immune function. In this review, we discuss the role of stress in modulating cellular immune function and the potential health implications of this downregulation.

Stress-related changes in proinflammatory cytokine production in wounds.

BACKGROUND: Several recent studies have shown that stress markedly delays wound healing. This study assessed the relationship between psychological stress and the secretion of proinflammatory cytokines at an actual wound site, providing in vivo data on the development of local immune responses that are central in the early stages of wound repair. METHODS: To study the dynamics of inflammation, skin blisters were induced on the forearm of 36 women (mean age, 57 years) by suction. After the blister roofs were removed, a plastic template was taped to the arm, and wells were filled with 70% autologous serum in buffer. Specimens were aspirated from blister chamber wells 5 and 24 hours after wounding. RESULTS: Women with higher perceived stress scores demonstrated significantly lower levels of 2 key cytokines--interleukin 1alpha and interleukin 8--at wound sites. In addition, subjects who had low levels of both cytokines after 24 hours reported more stress and negative affect, and they had higher levels of salivary cortisol than those who had high cytokine levels. CONCLUSION: Consistent with the evidence that stress delays wound healing, these data suggest a possible mechanism: psychological stress has measurable effects on proinflammatory cytokine production in the local wound environment.

Psychological influences on surgical recovery. Perspectives from psychoneuroimmunology.

Greater fear or distress prior to surgery is associated with a slower and more complicated postoperative recovery. Although anxiety presumably interferes with recuperation through both behavioral and physiological mechanisms, the pathways have been unclear. Recent work in psychoneuroimmunology (PNI) has demonstrated that stress delays wound healing. In addition, a second line of research has illustrated the adverse effects of pain on endocrine and immune function. A biobehavioral model is described that is based on these and other data; it suggests a number of routes through which psychological and behavioral responses can influence surgery and post-surgical outcomes. Clinical and research implications are highlighted.

Mucosal wound healing is impaired by examination stress.

OBJECTIVE: Impairment of wound healing is a well-recognized sequelae of conditions that alter immune function, including diabetes, jaundice, and advanced age. There is also growing evidence that psychological stress has adverse consequences for immune function. This study addressed the effects of a commonplace stressor on wound healing. METHOD: Two punch biopsy wounds were placed on the hard palate of 11 dental students. The first wound was timed during summer vacation, whereas the second was placed on the contralateral side 3 days before the first major examination of the term; thus, each student served as her or his own control. Two independent methods assessed healing (daily photographs and a foaming response to hydrogen peroxide). RESULTS: Students took an average of 3 days longer to completely heal the 3.5-mm wound during examinations, ie, 40% longer to heal a small, standardized wound. Production of interleukin 1beta (IL-1beta) messenger RNA (mRNA) declined by 68% during examinations, providing evidence of one possible immunological mechanism. These differences were quite reliable: No student healed as rapidly or produced as much IL-1beta mRNA during examinations as during vacation. CONCLUSIONS: These data suggest that even something as transient, predictable, and relatively benign as examination stress can have significant consequences for wound healing.

Restraint stress slows cutaneous wound healing in mice.

The impact of stress on cutaneous wound healing was assessed in a murine model. Female, hairless SKH-1 mice, 6-8 weeks of age were subjected to restraint stress (RST) 3 days before and for 5 days following dorsal application of a 3.5-mm sterile punch wound. Control mice were wounded, but not restrained. Using photography and image analysis, the rate of wound healing was compared between the two groups. Wounds on control mice healed on average 3.10 days sooner than RST-treated mice. In addition, cross-sectional, morphometric analysis of the dermal and epidermal layers revealed reduced inflammation surrounding wounds from RST mice at 1, 3, and 5 days after wounding. In the RST group, serum corticosterone levels averaged 162.5 ng/ml compared to 35.7 ng/ml in the controls. Treatment of RST-stressed animals with the glucocorticoid receptor antagonist RU40555 resulted in healing rates comparable to those of control animals. Thus, the reduction in inflammation and delayed healing correlated with serum corticosterone levels and suggest that disruption of neuroendocrine homeostasis modulates wound healing.

An inhibitor of ornithine decarboxylase antagonizes superoxide generation by primed human polymorphonuclear leukocytes.

Tumor necrosis factor-alpha (TNF-alpha) induces a rapid increase in polymorphonuclear leukocyte (PMN) polyamine content which appears to be required for optimal priming of the respiratory burst. The objective of the present study was to determine whether inhibition of polyamine biosynthesis modifies PMN responses to lipopolysaccharide (LPS), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF). Treatment with alpha-difluoromethylornithine (DFMO), a selective inhibitor of the rate-limiting biosynthetic enzyme ornithine decarboxylase, produced dose-dependent inhibition of the respiratory burst in PMNs that were primed by these agents and subsequently activated by formyl-Met-Leu-Phe (fMLP). However, DFMO did not significantly inhibit fMLP-stimulated superoxide generation or alter the induction of PMN adhesion and interleukin-1 beta (IL-1 beta) mRNA expression by LPS or GM-CSF. Antagonism of priming by DFMO correlated with a dose-dependent attenuation of fMLP-induced intracellular Ca2+ mobilization (r > or = 0.96). Since Ca2+ plays an important role in modulating the respiratory burst in primed PMNs, this could, in part, account for the selective effects of DFMO.

Slowing of wound healing by psychological stress.

There is evidence that psychological stress adversely affects the immune system. We have investigated the effects of such stress, caused by caring for a relative with Alzheimer's disease, on wound healing. We studied 13 women caring for demented relatives (mean age 62.3 [SE 2.3] years) and 13 controls matched for age (60.4 [2.8] years) and family income. All subjects underwent a 3.5 mm punch biopsy wound. Healing was assessed by photography of the wound and the response to hydrogen peroxide (healing was defined as no foaming). Wound healing took significantly longer in caregivers than in controls (48.7 [2.9] vs 39.3 [3.0] days, p < 0.05). Peripheral-blood leucocytes from caregivers produced significantly less interleukin-1 beta mRNA in response to lipopolysaccharide stimulation than did controls' cells. Stress-related defects in wound repair could have important clinical implications, for instance for recovery from surgery.

Polyamines found in gingival fluid inhibit chemotaxis by human polymorphonuclear leukocytes in vitro.

Putrescine and spermidine occur at concentrations approaching 1 mM in gingival fluid at diseased periodontal sites. Previous work demonstrates that these polyamines potentiate Ca2+ signaling in polymorphonuclear leukocytes (PMNs), resulting in enhanced degranulation and superoxide generation. The present study extends this work by characterizing the effects of polyamines on PMN chemotaxis and phagocytosis, in which Ca2+ signaling plays a less defined regulatory role. Putrescine (1 mM) and spermidine (0.1 to 0.5 mM) significantly inhibited chemotaxis to fMet-Leu-Phe and C5a (P < 0.05). This inhibition was not strongly related to any effect polyamines have on PMN adhesion, actin polymerization, or formyl peptide receptor expression. Neither putrescine nor spermidine had a significant impact on phagocytosis of opsonized bacteria by PMNs. Thus, at concentrations similar to those found in gingival fluid, polyamines could potentially inhibit recruitment of PMNs to diseased pockets without impairing their ability to engulf invading bacteria.

An inhibitor of polyamine biosynthesis impairs human polymorphonuclear leukocyte priming by tumor necrosis factor alpha.

TNF primes polymorphonuclear leukocytes (PMNs) for enhanced oxidative and secretory activity and directly induces adhesion and IL-1 beta expression. Previous reports suggest that polyamine biosynthesis by ornithine decarboxylase (ODC) has an essential role in macrophage activation by TNF. In the current study, TNF induced rapid increases in the putrescine and spermine content of PMNs. Difluoromethylornithine (DFMO), a selective inhibitor of ODC, inhibited these increases and blunted the enhancement of superoxide generation and secondary granule release associated with priming by TNF. DFMO did not affect the expression of TNF receptors or block receptor-independent activation of the respiratory burst by phorbol esters. Moreover, DFMO did not antagonize induction of adhesion or IL-1 beta mRNA expression by TNF. Thus, polyamine biosynthesis plays an important role in priming by TNF, but is not involved in all PMN responses to this cytokine. This suggests that ODC is a potential target for selective chemotherapeutic modulation of the inflammatory response.

Cytokine-induced IL-1 beta gene expression in the human polymorphonuclear leukocyte: transcriptional and post-transcriptional regulation by tumor necrosis factor and IL-1.

We have previously demonstrated that Il-1 and TNF could rapidly, but transiently, induce gene expression of Il-1 beta in human polymorphonuclear leukocytes (PMN) at both the protein and mRNA level. Additionally, we demonstrated a cooperative effect of Il-1 and TNF on the kinetics of induction of Il-1 beta mRNA and protein. In order to better understand the molecular basis of Il-1 beta induction, we have further investigated the regulation of Il-1 and TNF-induced gene expression in the PMN. Using nuclear run-on transcription analysis, we found that within 1 h Il-1, TNF, and TNF plus Il-1 induced the transcription of the Il-1 beta gene by 33-, 61-, and 99-fold, respectively. By 2 h, the levels of transcription had been reduced to approximately 50% of peak levels for TNF- and TNF plus Il-1-treated PMN, and to near noninduced levels in Il-1-treated PMN. We also found that these cytokines induced stable mRNA, i.e., Il-1 beta mRNA t1/2 for Il-1-, TNF-, and TNF plus Il-1-induced PMN were 57, 94, and 86 min, respectively. By 2 h, when steady state levels of Il-1 beta mRNA were found to decrease, Il-1 beta mRNA t1/2 had fallen to approximately 18 min for all cytokine treatments. To determine if protein synthesis was required for induction of Il-1 beta gene expression, we treated PMN simultaneously with cytokines and cycloheximide, and found that cycloheximide enhanced the accumulation of Il-1-induced Il-1 beta mRNA, but abrogated the accumulation of Il-1 beta mRNA, by TNF- or TNF plus Il-1-treated PMN. This abrogation of Il-1 beta mRNA accumulation was not caused by inhibition of induction of Il-1 beta transcription because TNF induction of transcription of Il-1 beta was not affected by simultaneous treatment with cycloheximide. Thus, we report that Il-1 and TNF regulate IL-1 beta gene expression via both transcriptional and post-transcriptional mechanisms in vitro.

Characterization of cytokines present in middle ear effusions.

Retention of inflammatory mediators and cells in the middle ear cleft during chronic otitis media with effusion (COME), results in ongoing inflammation with the potential for pathologic changes and hearing loss. Cytokines are glycoproteins produced by macrophages and other cells. Activities of cytokines include fever production, osteoclast, fibroblast, phagocyte and cytotoxic cell activation, regulation of antibody formation, and inhibition of cartilage, bone and endothelial cell growth. Using enzyme-linked immunospecific assays we measured levels of six cytokines in middle ear effusions (MEE) from children with COME. Significant levels of four cytokines: interleukin-1-beta (greater than 50 pg/ml), interleukin-2 (greater than 300 pg/ml), tumor necrosis factor-alpha (greater than 40 pg/ml), and gamma-interferon (greater than 6.25 pg/ml) were found in 51%, 54%, 63%, and 19% of MEE, respectively. In contrast, levels of a fifth cytokine, granulocyte-macrophage colony-stimulating factor, and a sixth cytokine, interleukin-4, were undetectable. Age was observed to have a significant effect on the levels of specific cytokines. Interleukin-1 (IL-1) correlated inversely (P less than .02) with age such that the younger the child, the higher the level of IL-1 in MEE. Tumor necrosis factor-alpha (TNF) correlated directly (P less than .005) with age such that the older the child, the higher the level of TNF in MEE. Children undergoing tympanostomy on multiple occasions had average MEE TNF levels (234.2 +/- 109.1 pg/mg total protein) that were nearly 14 times higher (P less than .005) than those from children undergoing their first tympanostomy (16.9 +/- 3.0 pg/mg total protein). Thus IL-1 correlated with the early stages of COME, while TNF correlated with persistence of disease. The presence of these cytokines in MEE may be responsible for the mucosal damage, bone erosion, fibrosis, and resulting hearing loss seen in some cases of COME.

Cytokine regulation of IL-1 beta gene expression in the human polymorphonuclear leukocyte.

Although recently polymorphonuclear leukocytes (PMN) have been identified as producers of IL-1 beta in response to LPS and granulocyte/monocyte colony stimulating factor, little is known regarding the ability of other cytokines to induce the production of IL-1 beta in the PMN. Inasmuch as IL-1 and TNF have been shown to be important priming agents, as well as agents that induce migration of PMN, we investigated their effect on IL-1 beta gene expression in human peripheral blood PMN. In the present study, we demonstrate that human peripheral blood PMN produce IL-1 beta in response to IL-1 alpha, IL-1 beta, and TNF-alpha. Control (unstimulated) human PMN had virtually undetectable levels of IL-1 beta mRNA. Either IL-1 beta or TNF, induced PMN to transiently express IL-1 beta mRNA with peak expression at 1 h, returning to untreated levels by 2 h. A dose response indicated that as little as 0.05 ng/ml of IL-1 beta or TNF resulted in IL-1 beta induction, with maximal effects at 1 ng/ml of IL-1 beta and 5 ng/ml of TNF. IL-1 alpha or IL-1 beta exhibited similar dose responses in IL-1 beta mRNA induction. Inasmuch as cytokines have been shown to have synergistic effects in cell function studies, we induced PMN with a combination of maximally effective doses of TNF plus IL-1 beta. They demonstrated a cooperative effect on IL-1 beta gene expression, in that mRNA levels were sustained for three hours. IL-1 beta Ag expression, as measured by ELISA, paralleled IL-1 beta mRNA expression with cell associated peak levels at 2 to 4 h. IL-1 beta Ag levels in PMN lysates and supernatants correlated with IL-1 beta mRNA levels, i.e., TNF + IL-1 greater than TNF greater than IL-1. Thus, these studies represent the first demonstration of IL-1 and TNF induction of IL-1 beta gene expression in the PMN. Furthermore, the time course of induction is unique to the PMN, with peak induction of mRNA at 1 h, which is consistent with the short lived nature of these cells in inflammatory lesions.

An in vitro model of cell migration: evaluation of vascular endothelial cell migration.

In vivo vascular endothelial cell (VEC) migration is thought to play a central role in the development of new capillaries as well as the resurfacing of large vessels. Recently, we have developed an in vitro VEC migration assay system based on the ability of VEC to migrate off of tissue culture microcarrier beads. For these studies, bovine pulmonary artery VEC were grown to confluence on Cytodex 3 microcarrier beads (MCB). Next, the confluent VEC covered microcarrier beads were pipetted into 4-cm2 wells of a tissue culture plate and incubated at 37 degrees C/5% CO2. At various time intervals, the movement of the VEC off of the MCB onto the tissue culture surface was evaluated microscopically. Using this assay, we have studied the effect of endothelial cell growth supplement and various matrices (i.e., fibronectin, gelatin, and Matrigel) on VEC migration. These studies demonstrated that: (i) gelatin had no effect on normal or mitomycin C-pretreated VEC migration; (ii) fibronectin had no effect on normal VEC migration, but stimulated the relative migration of mitomycin pretreated VEC; and (iii) Matrigel significantly suppressed both normal and mitomycin C-pretreated VEC migration. Endothelial cell growth supplement (ECGS) stimulated both normal and mitomycin C-pretreated VEC migration on fibronectin at concentrations of 10 micrograms/ml ECGS. Pretreatment with ECGS had no effect of normal or mitomycin C VEC migration on gelatin. Finally, ECGS stimulated a statistically significant increase in the migration of normal and mitomycin C-pretreated VEC migration on Matrigel.(ABSTRACT TRUNCATED AT 250 WORDS)