Inflammation

Mannose binding lectin (MBL) and ficolins contribute to host defence through activation of the lectin pathway of complement. In this study, serum levels of ficolin-2 and MBL were determined in 276 patients with community-acquired pneumonia (CAP). MBL deficiency and ficolin-2 insufficiency were defined using previously validated cut-offs. No differences were observed in MBL or ficolin-2 between patients and controls. MBL-deficient patients (<500 ng/ml) were not at higher risk of 30-day mortality odds ratio (OR) 0.97 (0.38–2.48,p = 0.9) or a composite outcome of mortality, mechanical ventilation, vasopressor support (MV/VS) or complications OR 0.89 (0.44–1.77, p = 0.9). Although no significant relationship between ficolin-2 insufficiency and outcome was observed, very low ficolin-2 levels (<1,200 ng/ml) were associated with an OR 1.23 (0.15–10.1), p = 0.6 for 30-day mortality, 3.05 (0.61–15.2, p = 0.2) for MV/VS and OR 2.05 (0.52–8.1, p = 0.2) for the composite outcome. Low serum levels of MBL and ficolin-2 are not associated with CAP susceptibility. The high frequency of ficolin-2 insufficiency in patients with severe CAP would justify a larger investigation of ficolin-2 as a modifier of CAP severity.

The parasympathetic nervous system controls submandibular glands (SMG) functions in physiological and pathological conditions via muscarinic acetylcholine receptors (mAchR). We had previously demonstrated that IFNγ and carbachol stimulate amylase secretion in normal murine SMG by mAchR activation. While the cytokine action depended on nitric oxide synthase activation, the effect of the agonist was mediated by prostaglandin E2 (PGE2) production. Both IFNγ and carbachol triggered IFNγ secretion in SMG. We here show that during local acute inflammation (LAI) induced by intraglandular injection of bacterial endotoxin, lypopolisaccharide (LPS), amylase secretion is decreased in comparison to control glands. We also observed that the muscarinic agonist carbachol stimulates in a dose-dependent manner amylase activity by M2 and M3 mAchR activation. Moreover, cyclooxygenase-2 (COX-2) activation and subsequent PGE2 liberation, in a nitric oxide independent manner, seem to be involved in M3 and M2 receptor activation by carbachol. In contrast, the addition of exogenous IFNγ or carbachol inhibits the cytokine liberation in LAI glands.

Wishing to extrapolate in vitro observations of granulocyte function and pharmacology made with human cells to animal models of diseases in which we believe granulocyte stimulation to play a major role, we examined techniques for preparation of canine granulocytes and conducted a survey of the function and pharmacology of those cells. Isotonic density gradients of PercollTMproved a simple and highly satisfactory method of preparation. Canine granulocytes in most respects paralleled human cells in function and pharmacology, except that canine cells lacked receptors for formylated oligopeptides and resisted them as stimuli; canine plasma contained a heat-labile inhibitor of canine PMN aggregation, oxidative metabolism, and myeloperoxidase release; canine PMNs were not inhibited in aggregation by protease inhibitors such as aprotinin; canine response to ibuprofen and steroids was more variable than that of human cells, and synergy between those agents was less readily demonstrated; heterologous stimulation (canine cells by human C5a or vice versa) led to a different time course and maximum response from those observed in the homologous systems. Canine granulocytes were readily marked with indium-111, and functioned normally in vitro and survived well in vivo after marking. We conclude that the dog is a suitable animal for studying the role of stimulated PMNs in disease, as long as the observed differences are taken into account in experimental design and data interpretation.

The main causes of spinal cord ischemia are a variety of vascular pathologies causing acute arterial occlusions. We investigated neuroprotective effects of coumaric acid on spinal cord ischemia injury in rats. Rats were divided randomly into four groups of eight animals as follows: control, ischemia, ischemia + coumaric acid, and ischemia + methylprednisolone. In the control group, only a laparotomy was performed. In all other groups, the spinal cord ischemia was performed by the infrarenal aorta cross-clamping model. Levels of malondialdehyde and nuclear respiratory factor 1 were analyzed, as were the activity of superoxide dismutase. Histopathological and immunohistochemical evaluations were performed. Neurological evaluation was performed with the Tarlov scoring system. The ischemia + coumaric acid group was compared with the ischemia group, and a significant decrease in malondialdehyde and levels was observed. Nuclear respiratory factor 1 level and superoxide dismutase activity of the ischemia + coumaric acid group were significantly higher than in the ischemia group. In histopathological samples, the ischemia + coumaric acid group is compared with the ischemia group, and there was a significant increase in numbers of normal neurons. In immunohistochemical staining, hypoxia-inducible factor-1α and NF-kappa B immunopositive neurons were significantly decreased in the ischemia + coumaric acid group compared with that in the ischemia group. The neurological deficit scores of the ischemia + coumaric acid group were significantly higher than the ischemia group at 24 h. Our results revealed for the first time that coumaric acid exhibits meaningful neuroprotective activity following ischemia-reperfusion injury of the spinal cord.

KLF2 plays a protective role in antiinflammation and endothelial function, and can be regulated by promoter methylation alteration. Lipopolysaccharide (LPS) is a mediator of inflammatory responses, which causes epigenetic change of certain genes in host cells. We thus aimed to determine whether LPS could control the KLF2 expression by inducing methylation in promoter region. DNA methylation of 16 CpG sites within KLF2 promoter region was detected by bisulfite sequencing PCR. Results showed that methylation at 12 CpG sites were significantly increased in HUVECs after exposure to LPS among the total 16 sites, and the average level was increased by 57%. The KLF2 expressions assessed by reverse transcription quantitative real-time PCR and Western blot were significantly downregulated compared that without LPS simulation. Moreover, both messenger RNA and protein levels of KLF2 in HUVEC co-treated with LPS and DNA methyltransferase (DNMT) 1 small interfering RNA were dramatically higher than that treated with LPS only. Similar result was obtained when the cells were incubated in combination with LPS and 5-aza-2′-deoxycytidine (AZA), suggesting that the reduction of KLF2 expression induced by LPS can be reversed by DNMT1 inhibition. Finally, the presence of AZA changed the expression of genes that depends on KLF2 in LPS-stimulated HUVECs, which downregulated the E-selectin and VCAM and increased the eNOS and thrombomodulin expression. Our data demonstrated that LPS exposure resulted in hypermethylation in KLF2 promoter in HUVECs, which subsequently led to downregulation of the KLF2 expression. The study suggested that epigenetic alteration is involved in LPS-induced inflammatory response and provided a new insight into atherogenesis.

In gingival pockets of patients with periodontal disease, polymorphonuclear leukocytes (PMN) are in contact with a peculiar exudate, the gingival crevicular fluid (GCF). Because of the pivotal role played by PMN in periodontal disease, we evaluated the ability of GCF in modulating normal human PMN. GCF was obtained from two gingival sites with severe periodontitis (SP) and two gingival sites with only mild periodontitis (MP) in 12 patients. Purified PMN were exposed to GCF from SP and MP sites and, as a control, to sterile culture medium. GCF activity was evaluated by monitoring the modulation of membrane molecules relevant to cell function. Compared to control medium, GCF from SP and MP sites was able to induce an activation status in PMN evidenced by an increased CD11b (62 ± 9% and 28 ± 7%, respectively) and f-Met-Leu-Phe (56 ± 5% and 31 ±7%, respectively) receptor expression, with a concomitant reduction of CD62L expression (56 ± 8% and 23 ± 7%, respectively). Thus, reflecting the clinical status, GCF from SP sites was significantly more efficient in affecting PMN than GCF from MP sites. Cell size modifications, evaluated as an additional indicator of PMN activation, were consistent with membrane molecule modulation. The difference in PMN-activating capacity between SP and MP was abrogated by the successful completion of an appropriate periodontal therapy that dramatically improved clinical status. This is the first direct demonstration that GCF from periodontitis has the capacity to activate normal resting PMN and that this capacity reflects the magnitude of the inflammatory process that takes place in the gingiva.

Chronic subdural hematoma (CSDH) development involves inflammatory, angiogenetic, and fibrinolytic mechanisms, several components of which are now unraveled through intensive research. The urokinase plasminogen activator receptor (uPAR) is part of the plasminogen activator system and possesses inflammatory, angiogenetic, and fibrinolytic capabilities. As a first, this study aims to identify uPAR in the hematoma fluid, hematoma membrane, dura mater, and systemic blood from patients with CSDH and, if present, to investigate if the uPAR level at the time of surgery may be a predictor for later developing recurrent CSDH. uPAR expression in the hematoma membrane and dura mater was analyzed using immunohistochemistry and presented as the H-score of the positive immunostaining. The uPAR levels in the hematoma fluid and systemic blood were determined using a multiplex antibody bead kit (Luminex). Samples were collected at the time of the first CSDH surgery, and in the case of recurrent CSDH within 90 days, the samples were again collected at reoperation. A comparison of uPAR expression between the hematoma membrane and dura mater, as well as uPAR levels in systemic blood and hematoma fluid, was performed using the Wilcoxon rank sum test. We included 112 patients, 26 of whom had recurrent CSDH. The median hematoma uPAR level was 22,125 (14,845–33,237) and significantly higher than the median systemic blood level of 789 pg/L (465–2,088) (p < 0.001). Similarly, the uPAR level of the hematoma membrane was 14.3 (7.54–44.8) and significantly higher than the dural uPAR level of 0.81 (0.3–1.98) (p < 0.001). For the first time, we identified uPAR in the subdural fluid, hematoma membrane, dura mater, and systemic blood from patients with CSDH. The high expression of uPAR in the subdural fluid and hematoma membrane indicates that the mechanisms of CSDH are predominantly in the subdural fluid collection and surrounding hematoma membrane.

The effect of selected compounds with known immunoregulatory activity was examined in a 45-h sensitization period oxazolone contact-sensitivity reaction. Oxazolone sensitivity was induced by applying 0.1 ml of 5% oxazolone in absolute ethanol to the shaved abdomen of C57B1/6 mice on day 0. Challenge with oxazolone followed 45 h later and was accomplished by painting a 5% solution of oxazolone in absolute ethanol on the left hindpaw. The response at 24 h was determined plethysmographically. Histamine (0.062–1.0 mg/kg, subcutaneously, twice a day), concanavalin A (0.31–5.0 mg/kg, intravenously), penicillamine (6.25–25 mg/kg, subcutaneously), chloroquine (6.25– 25 mg/kg, subcutaneously), and thymosin fraction 5 (0.125–1.25 mg/kg subcutaneously) all stimulated the oxazolone reaction when administered on day 0. These data suggest that the low-grade oxazolone response may be a useful assay to detect immunostimulatory activity.

5-Lipoxygenase inhibitor zileuton has been demonstrated to attenuate ischemic brain damage in rats of permanent focal cerebral ischemia in previous work. To further investigate the mechanism underlying zileuton's neuroprotection, adult male Sprague-Dawley rats underwent permanent middle cerebral artery occlusion (MCAO), then received treatment with zileuton or vehicle after the onset of ischemia. Neurological deficit, cerebral infarction, and morphological characteristic were measured 6 and 24 h after MCAO. The enzymatic activity of myeloperoxidase (MPO) was assessed 6 and 24 h after MCAO and the lipid peroxidation levels were evaluated by malondialdehyde assay. Expression of nuclear factor-kappa B (NF-κB) p65 in rat brain was detected by immunohistochemistry and Western blot. Expression of inducible nitric oxide synthase (iNOS) in rat brain was determined by RT-PCR and Western blot. Nitric oxide production in rat brain was also measured 24 h after MCAO. The concentration of TNF-α and IL-1β in serum were detected by ELISA. Zileuton significantly reduced neurological deficit scores, cerebral infarct volume, MPO activity, and the lipid peroxidation levels. It also inhibited the expression of NF-κB and decreased the expression and activity of iNOS in rat brain. In addition, zileuton attenuated the release of TNF-α and IL-1β in serum. Our results suggest that zileuton reduces inflammatory reaction and brain damage in a rat model of permanent focal cerebral ischemia. The neuroprotective effect of zileuton in cerebral ischemia might be associated with the inhibition of inflammatory reaction.

Schisandrin B (Sch B), a dibenzocyclooctadiene lignan isolated from Schisandra chinensis (Turcz.) Baill, has been shown to have anti-inflammatory effect. The purpose of this study was to evaluate the effect of Sch B on LPS-induced inflammation in microglia and to investigate the molecular targets of Sch B. BV2 cells were stimulated by LPS in the presence or absence of Sch B. The results showed that the levels of TNF-α, IL-6, IL-1β, and PGE2 upregulated by LPS were significantly suppressed by Sch B. LPS-induced NF-κB activation was also inhibited by Sch B. Furthermore, Sch B was found to upregulate the expression of PPAR-γ in a concentration-dependent manner. In addition, the inhibition of Sch B on TNF-α, IL-6, IL-1β, and PGE2 production were reversed by PPAR-γ antagonist GW9662. In conclusion, these results suggested that Sch B inhibited LPS-induced inflammatory response by activating PPAR-γ.