Downregulation Of Proinflammatory Cytokines Research Articles

Anti‐inflammatory effects of atorvastatin on peripheral blood mononuclear cells and synovial fibroblasts in rheumatoid arthritis

Objective: Statins, such as atorvastatin (ATV), are 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitors known to exert lipid‐lowering but also anti‐inflammatory, effects. In this study, we analysed the in vitro effects of ATV on peripheral blood mononuclear cells (PBMCs) and fibroblast‐like synoviocytes (FLS) in rheumatoid arthritis (RA), a chronic inflammatory joint disease.Methods: PBMCs isolated from 25 RA patients and 20 healthy blood donors were stimulated in vitro with 0.1 µM ATV for 24 h. PBMC cultures were analysed for cell surface markers to characterize T‐cell subtypes (CD4, CD8, CD69, HLA‐DR) by flow cytometry and for T helper cell type 1 (Th1) and type 2 (Th2) cytokines [interferon‐γ (IFN‐γ), interleukin‐4 (IL‐4), IL‐10] in culture supernatants by enzyme‐linked immunosorbent assay (ELISA). Furthermore, RNA isolated from ATV‐stimulated RA‐FLS pre‐ and post‐ATV stimulation was analysed by microarray and quantitative reverse transcription polymerase chain reaction (RT‐PCR).Results: Flow cytometric analysis of T‐cell subsets revealed no significant differences for CD4, CD8, CD69, and HLA‐DR surface marker expression of PBMCs in RA patients and healthy controls after ATV stimulation. However, the proportion of IFN‐γ expressing CD4+ T cells and the IFN‐γ cytokine concentrations in culture supernatants were significantly reduced in T‐cell cultures from RA patients. In ATV‐stimulated FLS a significant downregulation of proinflammatory cytokine (IL‐6) and chemokine (IL‐8) expression was detected (p<0.001).Conclusions: Our study demonstrates a marked in vitro anti‐inflammatory activity of ATV in RA including a systemic effect on a pathogenic CD4+ T‐cell population (Th1) and a local effect on FLS. These findings may provide a scientific rationale for statins as add‐on therapy in RA.

Dissociation of Activated Protein C Functions by Elimination of Protein S Cofactor Enhancement

Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

Evidence that Cisplatin-induced Auditory Damage is Attenuated by Downregulation of Pro-inflammatory Cytokines Via Nrf2/HO-1

Recently, we demonstrated that pro-inflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 played a critical role in cisplatin-induced cochlear injury and that flunarizine, known as a T-type Ca(2+) channel antagonist, induced a cytoprotective effect against cisplatin cytotoxicity in HEI-OC1 cells by the activation of NF-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) cascade through PI3K-Akt signaling but calcium-independent pathway. We report here that flunarizine markedly attenuates cisplatin-induced pro-inflammatory cytokine secretion and their messenger RNA transcription as well as cisplatin cytotoxicity through the activation of Nrf2/HO-1 and downregulation of NF-kappaB. In HEI-OC1 cells, overexpression of Nrf2/HO-1 by gene transfer or pharmacological approaches attenuated cisplatin-induced cytotoxicity and pro-inflammatory cytokine production. On the contrary, inhibition of Nrf2/HO-1 signaling by pharmacological inhibitors or specific small interfering RNAs significantly abolished the beneficial effects of flunarizine. Flunarizine also attenuated cisplatin-mediated MAPK activation and pharmacological inhibition of MAPKs, especially MEK1/ERK, blocked cisplatin-induced NF-kappaB activation in HEI-OC1 cells. Furthermore, WT-Nrf2 overexpression effectively blocked MAPK activation after cisplatin exposure. Finally, orally administrated Sibelium, the trade name of flunarizine, suppressed the increase of pro-inflammatory cytokines by cisplatin in both serum and cochleas of mice, whereas it increased HO-1 expression in cochleas. These results indicate that flunarizine induces a protective effect against cisplatin ototoxicity through the downregulation of NF-kappaB by Nrf2/HO-1 activation and the resulting inhibition of pro-inflammatory cytokine production in vitro and in vivo.

Can Vitamin D Deficiency Impact Cardiovascular Health in the Elderly?

Life expectancy is increasing worldwide. Nevertheless, the burden of cardiovascular disease (CVD) morbidity and mortality is still high in developed countries and is rapidly increasing in developing countries [1]. During the last decade, it also became clear that insufficient and even deficient vitamin D status is a worldwide problem [2]. By far the most important reason for this phenomenon is an inadequate skin exposure to solar ultraviolet B radiation, since ultraviolet B-induced skin synthesis is the major source of vitamin D for humans. It has long been known that vitamin D plays a major role in bone mineral homeostasis by promoting the transport of calcium and phosphate to ensure adequate blood levels of these ions for normal bone mineralization. Bone diseases such as rickets and osteomalacia are well-known consequences of vitamin D deficiency in infants and adults, respectively. However, is there any rationale to assume that vitamin D deficiency might be a causal factor of CVD-related morbidity and mortality? In the last several decades, many studies have documented nontraditional roles of vitamin D, including immunomodulatory properties and the regulation of intracellular calcium concentrations. Receptors for the active form of vitamin D, 1,25-dihydroxyvitamin D, have been found in almost all human tissues and cells. Therefore, vitamin D is now regarded as an important protective factor for cellular health. Circulating levels and cellular availability of 1,25-dihydroxyvitamin D directly depend on blood concentrations of its precursor 25-hydroxyvitamin D (25[OH]D). Serum 25(OH)D is the hallmark for determining vitamin D status. Cut-offs are below 25 nmol/l for deficiency, 25–49.9 nmol/l for insufficiency, 50–74.9 nmol/l for borderline status and 75 nmol/l or greater for normal status. There is now increasing evidence that 1,25-dihydroxyvitamin D exerts important physiological actions in the vasculature. These mechanisms include the inhibition of vascular smooth muscle proliferation, the suppression of vascular calcification, the downregulation of proinflammatory cytokines, the upregulation of anti-inflammatory cytokines and the action of vitamin D as a negative endocrine regulator of the renin–angiotensin system. Studies in knockout mice have confirmed that the absence of vitamin D-receptor activation leads to hypertension, left ventricular hypertrophy and disturbed blood coagulation. Ecological studies have reported higher rates of coronary heart diseases and hypertension with increasing distance from the equator, a phenomenon that could be attributed to the higher prevalence of vitamin D deficiency in regions with less exposure to sunlight [3]. In addition, clinical studies have reported cross-sectional associations between lower vitamin D levels and prevalent cardiovascular disease. The most important results come from a recently published prospective longitudinal study demonstrating that individuals with 25(OH)D levels below 37.5 nmol/l had a multivariable-adjusted hazard ratio of 1.62 for incident cardiovascular events compared with those with 25(OH)D levels of 37.5 nmol/l or greater [4].

Taurine Reduces Testicular Ischemia/Reperfusion-Induced Neutrophil Recruitment to Testis Probably by Downregulation of Pro-Inflammatory Cytokines and E-Selectin

Testicular torsion is a urologic emergency. Testicular surgical detorsion is the current approach to therapy for testicular torsion. It seems that the main pathophysiology of testicular torsion-detorsion is ischemia-reperfusion injury of the testis. In the ischemia-reperfusion injury, overgeneration of reactive oxygen species (ROS) is thought to play a critical role in the loss of testicular spermatogenesis.1 ROS can cause tissue damage through cell membrane lipid peroxidation, protein denaturation, and DNA impairment.

Adrenomedullin and Adrenomedullin Binding Protein-1 Prevent Acute Lung Injury after Gut Ischemia-Reperfusion

Ischemic bowel remains a critical problem, resulting in up to 80% mortality. Acute lung injury, a common complication after intestinal ischemia/reperfusion (I/R), might be responsible for such a high mortality rate. Our previous studies have shown that administration of a novel vasoactive peptide adrenomedullin (AM) and its binding protein (AMBP-1) reduces the systemic inflammatory response in rat models of both hemorrhage and sepsis. It remains unknown whether administration of AM/AMBP-1 has any protective effects on intestinal I/R-induced acute lung injury. We hypothesized that administration of AM/AMBP-1 after intestinal I/R prevents acute lung injury through downregulation of proinflammatory cytokines. Intestinal I/R was induced by placing a microvascular clip across superior mesenteric artery (SMA) for 90 minutes in adult male Sprague-Dawley rats (275 to 325 g). On release of the SMA clamp, the animals were treated with either AM (12 mug/kg body weight) in combination with AMBP-1 (40 microg/kg body weight) or vehicle (1 mL normal saline) during a period of 30 minutes through a femoral vein catheter. Lung samples were collected at 4 hours after treatment or sham operation. Lung injury was assessed by examining lung water content, morphologic changes, and granulocyte myeloperoxidase activity. Tumor necrosis factor-alpha and interleukin-6 gene expression and their protein levels in the lungs were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. In additional groups of animals, AM/AMBP-1 or vehicle was administered at 1 hour after onset of reperfusion. Lung histology was examined at 3 hours after treatment. Intestinal I/R induced considerable lung injury, as characterized by lung edema, histopathologic changes, increased myeloperoxidase activity, and proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) levels in the lungs. Administration of AM/AMBP-1 after ischemia mitigated lung injury and dramatically downregulated proinflammatory cytokines. Lung injury was also ameliorated by delayed AM/AMBP-1 treatment as evidenced by improvement in lung histology. AM/AMBP-1 can be developed as a novel treatment to attenuate acute lung injury after an episode of gut ischemia. The protective effect of AM/AMBP-1 appears to be mediated through downregulation of proinflammatory cytokines.

In vitro down regulation of proinflammatory cytokines induced by LPS tolerance in pig CD14+ cells

LPS tolerance is characterized by a reduced sensitivity to subsequent challenge of LPS. In human and mouse models LPS tolerance is closely associated with marked unbalanced production of leukocyte-derived inflammatory mediators which, when overexpressed, led to septic syndrome and shock. Here we characterized the in vitro induction of LPS tolerance in porcine CD14+ spleen cells in order to give insights into LPS tolerance in pigs. Following LPS stimulation, TNF-α and, to a minor extent, IL-8 production showed a significant reduction in CD14+ spleen monocytes that were pretreated with LPS in comparison to naïve cells, while IL-1β production was slightly influenced by LPS stimulation and it was not affected by subsequent LPS challenge. Our findings showed that porcine CD14+ cells undergo a process, which resembles LPS tolerance, providing evidence that swine represent a valuable and useful model to perform experiments to study LPS tolerance and its biological significance.

Putting cardiovascular disease and vitamin D insufficiency into perspective

The aetiology of CVD is still not completely understood. The present review article summarises data supporting the hypothesis that an insufficient vitamin D status may contribute to the worldwide high prevalence of CVD. Human vitamin D status primarily depends on skin exposure to the UVB spectrum of the sunlight. Epidemiological data indicate that geographic latitude, altitude, season, and the place of residence (urban or rural) are associated with CVD mortality. Interestingly, all these factors also have an influence on human UVB exposure and thus on vitamin D status. Several mechanisms might be responsible for a protective role of vitamin D in CVD. These mechanisms include the inhibition of vascular smooth muscle proliferation, the suppression of vascular calcification, the down regulation of pro-inflammatory cytokines, the up regulation of anti-inflammatory cytokines, and the action of vitamin D as a negative endocrine regulator of the renin-angiotensin system. The first intervention trials indicate that vitamin D may suppress cardiovascular risk markers. However, more controlled clinical trials are needed to investigate whether optimal oral vitamin D supplementation is able to reduce CVD morbidity and mortality.

Transglutaminase Type II Is a Key Element in the Regulation of the Anti-Inflammatory Response Elicited by Apoptotic Cell Engulfment

A key feature of the macrophage-dependent clearance of apoptotic cells is the down-regulation of proinflammatory cytokines. Deficiency in the phagocytosis of apoptotic cells is often associated with the development of inflammatory reactions, resulting in chronic inflammatory and autoimmune diseases. The molecular mechanisms that regulate the engulfment process and particularly the immunomodulatory factors involved are still largely unknown in mammals. We have previously reported that the ablation of transglutaminase type II (TG2) in mice results in the defective clearance of apoptotic cells associated with the development of splenomegaly, autoantibodies, and glomerulonephritis. In this study we have investigated the mechanisms at the basis of the development of inflammation/autoimmunity associated with the defective clearance of apoptotic cells characterizing TG2 knockout mice. To this aim we compared the macrophage response to apoptotic cell exposure in wild-type vs TG2-null mice. We demonstrated that the lack of TG2 results in an impaired capacity of macrophages to engulf, but not to bind, apoptotic cells, which is paralleled by an abnormal inflammatory response both in vivo and in vitro. We have identified a differential response in the release of several cytokines in TG2(-/-) vs wild-type mice. Particularly relevant is the finding that both TGF-beta and IL-12 regulations were significantly altered in the absence of TG2. These results help explain the autoimmune phenotype developed by these mice and suggest that TG2 is a key regulatory element of the anti-inflammatory features of apoptosis.

ProbioticLactobacillus-induced improvement in murine chronic inflammatory bowel disease is associated with the down-regulation of pro-inflammatory cytokines in lamina propria mononuclear cells

IL-6/STAT-3 signals play key roles in inflammatory bowel disease (IBD). It is known that Lactobacillus casei strain Shirota (LcS) improves inflammatory disorders. This study aimed to elucidate the effect of LcS on murine chronic IBD and to clarify the mechanism. We focused the inhibitory effect of LcS on the production of IL-6 in lipopolysaccharide (LPS)-stimulated large intestinal lamina propria mononuclear cells (LI-LPMC) isolated from mice with chronic colitis and in RAW264.7 cells in vitro. We also determined in vivo the effect of LcS on murine chronic IBD models induced with dextran sodium sulphate and SAMP1/Yit mice. Finally, we examined the cellular determinants of LcS for the down-regulation of IL-6 secretion by LI-LPMC, RAW264.7 cells and peripheral blood mononuclear cells (PBMC) derived from patients with ulcerative colitis (UC). LcS, but not other strains of Lactobacillus, inhibited the production of IL-6 in LPS-stimulated LI-LPMC and RAW264.7 cells, down-regulating the nuclear translocation of NF-kappaB. The LcS-diet-improved murine chronic colitis is associated with the reduction of IL-6 synthesis by LI-LPMC. LcS also improved chronic ileitis in SAMP1/Yit mice. The release of IL-6 in vitro in LPS-stimulated LI-LPMC, RAW 264.7 cells and UC-PBMC was inhibited by a polysaccharide-peptidoglycan complex (PSPG) derived from LcS. This probiotic-induced improvement in murine chronic inflammatory bowel disease is associated with the down-regulation of pro-inflammatory cytokines such as IL-6 and IFN-gamma production in LPMC. Therefore, LcS may be a useful probiotic for the treatment of human inflammatory bowel disease.

Adrenomedullin and its binding protein attenuate the proinflammatory response after hemorrhage

The neuroendocrine response to hemorrhage is to maintain perfusion to the heart and brain, often at the expense of other organ systems. Systemic inflammation and tissue injury are important components of pathophysiologic consequences of hemorrhage. We have recently shown that administration of adrenomedullin (AM, a potent vasodilator peptide) and adrenomedullin binding protein-1 (AMBP-1) prevented the transition from the hyperdynamic to the hypodynamic stage in the progression of sepsis. However, the effect of AM/AMBP-1 on the inflammatory response after hemorrhage remains unknown. We therefore hypothesized that administration of AM/AMBP-1 during fluid resuscitation in hemorrhaged animals (i.e., posttreatment) attenuates tissue injury and the proinflammatory response. Prospective, controlled, and randomized animal study. A research institute laboratory. Male adult rats. Rats were bled, and then a mean arterial pressure was maintained at 40 mm Hg for 90 mins. They were then resuscitated by infusion of four times the volume of shed blood using Ringer's lactate solution for 60 mins. Fifteen minutes after the beginning of resuscitation, AM (12 microg/kg of body weight) in combination with AMBP-1 (40 microg/kg of body weight) was administered via a femoral venous catheter for 45 mins. Blood samples were collected 4 hrs postresuscitation and assayed for levels of liver enzymes (i.e., alanine aminotransferase and aspartate aminotransferase), lactate, creatinine, proinflammatory cytokines tumor necrosis factor and high mobility group box 1, and anti-inflammatory cytokine interleukin-10. The results indicate that levels of alanine aminotransferase, aspartate aminotransferase, creatinine, lactate, tumor necrosis factor, and high mobility group box 1 markedly elevated after hemorrhage and resuscitation, and AM/AMBP-1 treatment significantly attenuated these increases. In contrast, the serum concentration of anti-inflammatory cytokine interleukin-10 was increased by the treatment of AM/AMBP-1. Moreover, AM/AMBP-1 treatment significantly improved the survival rate from 35% in vehicle-treated animals to 73% in AM/AMBP-1-treated animals in a low-volume resuscitation model of hemorrhage. The combined administration of AM and AMBP-1 effectively suppresses hemorrhage-elicited organ injury and reduces hemorrhage-induced mortality, partly through down-regulation of proinflammatory cytokines (tumor necrosis factor and high mobility group box 1) and up-regulation of the anti-inflammatory cytokine interleukin-10.

Can worms defend our hearts? Chronic helminthic infections may attenuate the development of cardiovascular diseases

The established risk factors for atherosclerosis fail to fully explain the extent and severity of coronary artery diseases in 50% of the patients. Thus, the causative agents and processes, which may be involved in the pathogenesis of atherosclerosis, are being sought. Notoriously, atherosclerosis and cardiovascular event rates are much lower in developing countries. Clinically, severe infections by intracellular pathogens are widespread mostly in developing countries with poor sanitation, nutrition and massive worm infections. A link between atherosclerosis and helminth infections has never been examined. Based on the present knowledge of immune and infectious mechanisms related to atherosclerosis, it is proposed that chronic helminthic infections can have a significant bearing on the epidemiology of cardiovascular diseases. How can helminthic infections affect the cardiovascular risk? (1) Helminths evade or suppress host immune responses, by producing anti-inflammatory and other immunomodulatory molecules. (2) Helminths induce chronic Th2 activation, which can modify cytokine profiles and immunological responses to heat shock proteins, Chlamydia pneumoniae and cytomegalovirus. (3) The chronic Th2 profile may modulate monocyte activation and chemotaxis to inflammatory sites (atherosclerotic plaques). (4) Chronic Th2 activation may lead to a cytokine profile that could be beneficial for attenuation of atherosclerosis development (upregulation of IL-4, IL-10 and IL-13 and downregulation of proinflammatory cytokines). (5) Helminthic infections may reduce plasma LDL level not only by affecting the host nutrition, but also via modulation of naturally occurring antibodies to cholesterol. Studies are needed to clarify these suggestions. If the hypothesis that helminthic infections impact atherosclerosis is correct, it should be taken into consideration in atherosclerosis immunomodulation therapy and especially in the design of vaccines and vaccine trials.

Effects of Infliximab on Apoptosis and Reverse Signaling of Monocytes from Healthy Individuals and Patients with Crohn’s Disease

Infliximab, an anti-tumor necrosis factor (TNF) monoclonal antibody, might exert some of its long-term therapeutic effects in Crohn's disease (CD) by interacting directly with cells of the immune system such as monocytes and T lymphocytes via membrane TNF and by inducing apoptosis. Accordingly, the effects of inflix-imab on monocyte apoptosis and down-regulation of proinflammatory cytokines (reverse signaling) were assessed. To assess apoptosis, monocytes from healthy individuals (controls) and CD patients were incubated in the presence or absence of infliximab or the apoptotic agent gliotoxin for 24 hours. Annexin V staining and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-FITC nick end labeling assay were used to measure early and late apoptosis. To measure the effects of infliximab on reverse signaling, monocytes from healthy individuals pretreated in vitro with infliximab were stimulated with lipopolysaccharide or staphylococcal enterotoxin A, and the induction of the proinflammatory cytokines, TNF-alpha, interleukin (IL)-1beta, IL-6, and IL-8 was measured by reverse transcription polymerase chain reaction. The effect of in vivo infliximab treatment of monocytes was similarly determined by comparing the responses of monocytes from CD patients before and immediately after infliximab infusion. Infliximab did not induce apoptosis of monocytes from either healthy individuals or CD patients but rather stabilized them. However, monocytes from healthy individuals treated with infliximab in vitro, or from CD patients infused with infliximab, produced significantly less TNF and other proinflammatory cytokines when stimulated with the bacterial products lipopolysaccharide and staphylococcal enterotoxin A. Apoptosis of monocytes is not responsible for the therapeutic effects of infliximab. However, some of the therapeutic effects of infliximab may be caused by its ability to down-regulate proinflammatory cytokines production by monocytes exposed to bacterial antigens.

Hindlimb muscle inflammation caused by reperfusion injury persists despite the use of neutrophil-suppressive anesthesia

A significant role for innate immunity and resulting inflammation has been established in the pathogenesis of skeletal muscle ischemia and reperfusion injury. The degree to which neutrophils contribute to this local injury has been disputed and is difficult to study. In the past, our group has found few neutrophils in reperfused muscle, although data with inhibition of free oxygen radicals would argue otherwise. Specific depletion of neutrophils without also altering the complement system is not feasible. Ketamine anesthesia has anti-inflammatory properties, specifically in the down regulation of pro-inflammatory cytokines and neutrophil activation. We hypothesize that attenuation of neutrophil activity by ketamine will have little effect on complement-mediated ischemia/reperfusion injury. Two groups of mice ( n = 16 each), anesthetized with either intraperitoneal ketamine/xylazine or pentobarbital, underwent 2 h of tourniquet-induced hind limb ischemia and 18 h of reperfusion. The severity of injury was measured by the appearance of rhabdomyolysis as well as the mortality after 5 days. IgM and complement C3 deposition was indicative of the degree of inflammatory activation. Muscle and lung vascular permeability index (PI) was assessed by the extravasation of (125) I-radiolabeled albumin. Lung (Matrix Metalloproteinase) MMP-9 measured by gelatin zymography was used as a marker of local neutrophil activation. Ketamine anesthetized mice showed no decrease in overall muscle injury, immunohistochemical staining of IgM, C3, or 5-day mortality in comparison to pentobarbital anesthetized animals. There was, however, a decrease in neutrophil-induced MMP-9 activity in the lung as well as in the muscle and lung PI ( P < 0.05), suggesting neutrophils’ role only in remote downstream injury. From these data, we conclude that ketamine, while decreasing neutrophil activation as seen by attenuated MMP-9 production, affected neither complement activation, histological injury, nor survival after skeletal muscle ischemia/reperfusion injury.

Antibiotics improve survival and alter the inflammatory profile in a murine model of sepsis from Pseudomonas aeruginosa pneumonia.

Differing antibiotic regimens can influence both survival and the inflammatory state in sepsis. We investigated whether the addition and/or type of antimicrobial agent could effect mortality in a murine model of Pseudomonas aeruginosa pneumonia-induced sepsis and if antibiotics altered systemic levels of cytokines. FVB/N mice were subjected to intratracheal injection of pathogenic bacteria and were given gentamicin, imipenem, or 0.9% NaCl 2 h after surgery, which continued every 12 h for a total of six doses. Survival at 7 days (n = 24 in each group) was 100% for mice given gentamicin, 88% for mice given imipenem, and 8% for sham mice treated with 0.9% NaCl (P < 0.0001). Systemic interleukin (IL) 6 levels were assayed 6 h postoperatively on all mice to see if they were predictive of outcome. Plasma IL-6 levels above 3,600 pg/mL were associated with a 100% mortality, levels under 1,200 pg/mL were associated with a 100% survival, and levels between 1,200 and 3,600 pg/mL had no utility in predicting mortality. In a separate experiment, mice were sacrificed at 3, 6, 12 or 24 h after instillation of P. aeruginosa and were assayed for levels of TNF-alpha, IL-6, IL-10, and IL-12. Significant alterations in the proinflammatory cytokines TNF-alpha and IL-6 were present at all time points except 3 h between mice treated with antibiotics and sham controls. In contrast, statistically significant differences in the anti-inflammatory cytokine IL-10 were present between the groups only at 6 h, and levels of IL-12 were similar at all time points. These results indicate that both gentamicin and imipenem increase survival at least 10-fold in a model of pneumonia-induced monomicrobial sepsis, and this is predominantly associated with a down-regulation of proinflammatory cytokines.

Campylobacter surface-layers (S-layers) and immune evasion.

Many pathogenic bacteria have evolved mechanisms for evading host immune systems. One evasion mechanism is manifest by the surface layer (S-layer), a paracrystalline protein structure composed of S-layer proteins (SLPs). The S-layer, possessed by 2 Campylobacter species (C. fetus and C. rectus), is external to the bacterial outer membrane and can have multiple functions in immune avoidance. C. fetus is a pathogen of ungulates and immunocompromised humans, in whom it causes disseminated bloodstream disease. In C. fetus, the S-layer is required for dissemination and is involved in 2 mechanisms of evasion. First, the S-layer confers resistance to complement-mediated killing in non-immune serum by preventing the binding of complement factor C3b to the C. fetus cell surface. S-layer expressing C. fetus strains remain susceptible to complement-independent killing, utilizing opsonic antibodies directed against the S-layer. However, C. fetus has also evolved a mechanism for avoiding antibody-mediated killing by high-frequency antigenic variation of SLPs. Antigenic variation is accomplished by complex DNA inversion events involving a family of multiple SLP-encoding genes and a single SLP promoter. Inversion events result in the expression of antigenically variant S-layers, which require distinct antibody responses for killing. C. rectus is implicated in the pathogenesis of periodontal disease and also possesses an S-layer that appears to be involved in evading the human system. Although studied less extensively than its C. fetus counterpart, the C. rectus S-layer appears to confer resistance to complement-mediated killing and to cause the down-regulation of proinflammatory cytokines.

The cardiovascular response in sepsis: proposed mechanisms of the beneficial effect of adrenomedullin and its binding protein (Review)

Sepsis and its complications are leading causes of morbidity and mortality. A better understanding of the mechanisms responsible for the shift from the early, hyperdynamic phase of sepsis to the late hypodynamic phase could lead to novel therapies that might improve the outcome of the septic patient. Adrenomedullin is a vasodilatory peptide which shows sustained elevation starting early in sepsis and is important in initiating the hyperdynamic response. As sepsis progresses, however, the vascular response to adrenomedullin is blunted and this decreased sensitivity is important in producing the shift to the late, hypodynamic phase. The decline in the vascular response to adrenomedullin is related to a sepsis-induced decrease in the binding protein for adrenomedullin (i.e., adrenomedullin binding protein-1) rather than a change in gene expression of the components of adrenomedullin receptors. Treatment of septic animals with the combination of adrenomedullin and its binding protein prevents the transition to the late phase of sepsis, maintains cardiovascular stability, and reduces sepsis-induced mortality. We propose that the mechanisms responsible for the beneficial effect of adrenomedullin and adrenomedullin binding protein-1 in sepsis are associated with downregulation of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6), maintainence of endothelial constitutive nitric oxide synthase, and reduction of vascular endothelial cell apoptosis.

Toxicological consequences of altered peroxisome proliferator-activated receptor γ (PPARγ) expression in the liver: insights from models of obesity and type 2 diabetes

The pivotal role of peroxisome proliferator-activated receptor γ (PPARγ) in the liver, although important for the regulation of genes involved in glucose and lipid metabolism, has generally not been fully appreciated. This may be due to the fact that PPARγ, in contrast to PPARα or PPARδ, is not abundantly expressed in liver under normal conditions. However, recent findings have revealed that in several murine models of obesity and type 2 diabetes mellitus (T2DM), PPARγ mRNA and receptor protein are highly up-regulated in the liver, and that the receptor causes increased transcriptional activity as demonstrated by the activation of PPARγ-responsive genes in the liver. Prolonged treatment of obese and diabetic mice, but not of lean control mice, with the selective PPARγ ligands and activators, thiazolidinediones (TZDs), including troglitazone, rosiglitazone, or pioglitazone, has resulted in the development of severe hepatic centrilobular steatosis. In contrast to these effects in hepatocytes, TZD-mediated effects on Kupffer cells (down-regulation of proinflammatory cytokines) seem to be PPARγ-independent. In view of the findings that sustained hepatic steatosis can lead to steatohepatitis and/or fibrosis and that troglitazone (but not the other TZDs) has been associated with rare but serious hepatotoxicity in patients, further insight into PPARγ-mediated versus non-PPARγ-mediated effects of TZDs is desirable. It is concluded that liver-specific effects associated with TZD antidiabetics may become relevant under conditions of selective PPARγ up-regulation in the liver. Therefore, receptor expression in human liver tissue of obese and T2DM patients should deserve increased consideration in the future.

Regulation of human intestinal mast cells by stem cell factor and IL-4.

Mature human mast cells are tissue-residing, key effector cells of immediate allergic reactions. Moreover, mast cells have been recognized as a potent cellular source of multiple cytokines, suggesting an important role in immunoregulation and host defense. Here, we report on the regulation of mature human mast cells isolated from intestinal tissues by stem cell factor (SCF) and interleukin (IL)-4. SCF is substantially necessary for mast cell survival and induces marginal mast cell proliferation in vitro, whereas IL-4 by itself has no effects on mast cell survival or proliferation. Most interestingly, in synergy with SCF, IL-4 strongly enhances mast cell proliferation. In the presence of SCF, mast cells predominantly produce pro-inflammatory cytokines including tumor necrosis factor (TNF)-alpha, IL-1beta, IL-6, IL-8, IL-16, and IL-18. Addition of IL-4 to the culture medium induces the expression of Th2-type cytokines (IL-3, IL-5 and IL-13), and a downregulation of pro-inflammatory cytokines, namely IL-6. Furthermore, SCF by itself supports the predominance of the tryptase/chymase double-positive mast cell subtype MCTC whereas the addition of IL-4 supports the chymase negative MCT subtype. In conclusion, SCF may primarily regulate resident mast cell survival, whereas IL-4 may promote local proliferation of mast cells and their expression of Th2-type cytokines.

INCREASE OF ANTI-INFLAMMATORY CYTOKINES IN PATIENTS WITH ESOPHAGEAL CANCER AFTER PERIOPERATIVE TREATMENT WITH G-CSF

Granulocyte colony-stimulating factor (G-CSF) has been shown to effectively stimulate granulopoiesis, in both neutropenic and in non-neutropenic patients. Recently, other effects of G-CSF on the immune system have attracted interest in treating non-neutropenic patients with a high risk of severe infection. In this phase II trial, we measured the effects of G-CSF on the serum cytokine levels in patients with esophageal cancer undergoing esophagectomy. Twenty subsequent patients (study group, 19 evaluable) received G-CSF (rhG-CSF, Filgrastim) at standard doses (300μg or 480μg) subcutaneously 2 days before and up to 7 days after surgery. G-CSF was well tolerated. Leukocytes increased from 7600/μl at study entry (day −2) to a maximum of 45100/μl (day 6). In the study patients, we found a highly significant (P<0.001) postoperative increase of G-CSF, IL-1ra, sTNFRp55 and sTNFRp75 as compared with the baseline level. In contrast, IL-8 levels were decreased by a factor of 6.8; there were no changes in the very low TNF-α levels. The comparison of the study group with a control group of 21 cancer patients undergoing major surgery who were not treated with G-CSF showed significant differences in the serum levels of G-CSF, sTNFRp55, sTNFRp75, and IL-1ra, respectively. There was no infection in the study group up to 10 days after surgery as compared with 29.9% in a historical control group (P=0.008). Thus, the induction of anti-inflammatory cytokines and the downregulation of pro-inflammatory cytokines by G-CSF might be a promising adjuvant treatment of infectious complications in patients undergoing esophagectomy.