Haemorrhagic shock in mice--intracellular signalling and immunomodulation of peritoneal macrophages' LPS response

Haemorrhagic shock leads to decreased proinflammatory cytokine response which is associated with an increased susceptibility to bacterial infections. In the present study, the effect of GM-CSF on lipopolysaccharide (LPS)-induced TNF-alpha release and MAPkinase activation was analysed on the background of a possible immunostimulating activity of this substance. Male BALB/c mice were bled to a mean arterial blood pressure of 50 mmHg for 45 min followed by resuscitation. Peritoneal macrophages were isolated 20 h after haemorrhage and incubated with 10 ng/ml GM-CSF for 6h before LPS stimulation. TNF-alpha synthesis was studied in the culture supernatants using ELISA. Phosphorylation of ERK, p38MAPK and IkappaBalpha was detected by Western blotting. LPS-induced TNF-alpha production of peritoneal macrophages was significantly decreased 20 h after haemorrhage in comparison to the corresponding cells of sham-operated mice. In parallel the phosphorylation of IkappaBalpha was less in LPS-stimulated peritoneal macrophages from haemorrhagic mice. LPS-induced phosphorylation of ERK1/2 was also decreased in peritoneal macrophages isolated after haemorrhagic shock. In contrast, p38MAPK was phosphorylated more intensely after LPS-stimulation in macrophages collected from shocked mice. GM-CSF incubation elevated LPS-induced TNF-alpha response of macrophages from both sham-operated and shocked mice which was accompanied by an elevated IkappaB and ERK phosphorylation. In general, GM-CSF treatment in vitro enhanced peritoneal macrophages LPS-response both in terms of TNF-alpha synthesis and IkappaB and MAPK signalling, but the levels always stayed lower than those of GM-CSF-treated cells from sham-operated animals. In conclusion, GM-CSF preincubation could partly reactivate the depressed functions of peritoneal macrophages and may therefore exert immunostimulating properties after shock or trauma.     

Association Between Low-Grade Systemic Inflammation and Type 2 Diabetes Mellitus Among Men and Women from the ATTICA Study

AIM: To investigate the relationship between low-grade inflammation and several glycemic indices in a population-based sample of men and women. METHODS: The ATTICA study is a population-based cohort that randomly enrolled 1514 men and 1528 women (aged >18 years old), stratified by age and gender, from the Greater Athens area, during 2001-2002. Among several characteristics, inflammation markers (high sensitivity C-reactive protein, interleukin-6, tumor necrosis factor-alpha, homocysteine and amyloid A) and glycemic control indices (fasting blood glucose, insulin, HOMA) were measured in the participants. RESULTS: The prevalence of diabetes was 7.8% in men and 6.0% in women. The prevalence of impaired fasting glucose (IFG) was 21% in men and 12% in women. Diabetic subjects had 57% higher mean levels of C-reactive protein (p < 0.001), 22% higher mean levels of interleukin-6 (p < 0.001) and 60% higher levels of tumor necrosis factor-alpha (p < 0.001) compared to non-diabetic subjects. Homocysteine and serum amyloid A levels did not show significant differences among groups. CONCLUSION: Our study supports a positive association between low-grade inflammation and diabetes in a population-based sample of men and women without any evidence of cardiovascular disease, which is independent of demographic, clinical and lifestyle characteristics, including physical activity and dietary factors.     

Interleukin 10 Inhibits TNF-Alpha Production in Human Monocytes Independently of Interleukin 12 and Interleukin 1 Beta

Previously we demonstrated that endogenously produced Interleukin (IL-)IO suppressed the production of tumor necrosis factor-alpha (TNF-alpha) in CD3 activated T-cells via downregulation of paracfine IL-12 secretion from APC. Here we investigated the effect of endogenous IL-10 on TNF-alpha production in purified lipopolysaccharide (LPS) stimulated monocytes and its mechanism. Similarly to its effects on T-cells, EL-10 inhibited monocyte TNF-alpha production by about half Unlike in T-cells, however, this effect was not mediated via IL-12. While blockade of endogenous IL-10 binding to the IL-10 receptor enhanced the autocrine production of TNF-alpha, IL-12 and IL-1 beta, the neutralization of IL-12 or IL-1 beta did not affect the EL-10 effects on TNF-alpha production. This suggests that despite its inhibitory effects on IL-12 and IL-1 beta, which is quite similarly observed in T-cells, in purified monocytes IL-10 does not effect its TNF-alpha suppression by this mechanism. These findings indicate that IL-10 regulates production of pro-inflammatory cytokines by distinct mechanisms in different cells and tissues. Our study thus adds to the appreciation of the complex cytokine regulation of the immune system.     

Risk of adverse post-transplant events after kidney allograft transplantation as predicted by CTLA-4 +49 and TNF-alpha -308 single nucleotide polymorphisms: a preliminary study

Genetic differences between donor and recipient HLA haplotypes are of major importance for transplant rejection. Other genetic variations occurring in genes encoding cytokines and costimulatory molecules also appear to exert an influence on the manner the host immune system recognizes the allograft. The aims of this work were: 1) to study selected single nucleotide polymorphisms (SNPs) at the loci encoding the T-cell regulatory molecule CTLA-4 (CD152), and the cytokines interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-10, and transforming growth factor (TGF)-beta1 in a sample of healthy volunteers and a group of kidney-transplanted patients; and 2) to investigate whether an association exists between any of the SNPs studied and acute or chronic rejection, or non-responsiveness to steroid treatment during episodes of acute rejection (AR) after kidney allograft transplantation. When healthy volunteers were compared with transplanted patients, no significant differences were found in the distribution of genetic frequencies for any of the SNPs analyzed. However, in transplanted patients who received a kidney from a living related donor (KdTxL), a statistically significant association was found between carrying the CTLA-4 +49 A/A genotype and protection from experiencing acute rejection. No such association was found in the group of transplanted patients who received a kidney from a cadaveric non-related donor (KdTxCad). In both, KdTxL and KdTxCad patients, responsiveness to steroid treatment during acute rejection was also in association with the CTLA-4 (+49A/G) SNP. The CTLA-4 +49G allele was found at a very low frequency among steroid-resistant compared with steroid-sensitive patients. Finally, a statistically significant association was found between the presence of the TNF-alpha -308A allele and protection to suffer from chronic rejection. The genetic differences found may serve as risk predictors of adverse post-transplant events.     

Pharmacokinetic and Tolerability Assessment of a Pediatric Oral Formulation of Pentoxifylline in Kawasaki Disease

Background: In infants and children, treatment of Kawasaki disease (KD) with high-dose intravenous immunoglobulin (IVIG) and acetylsalicylic acid ([ASA] aspirin) diminishes inflammatory response and reduces the risk for coronary artery abnormalities. However, patients with high serum concentrations of tumor necrosis factor (TNF)-alpha, which is associated with vascular damage, may develop coronary artery lesions even with treatment. The hemorheologic agent pentoxifylline blocks the production of TNF-alpha and may be an appropriate adjunctive therapy to IVIG and ASA.Objective: The objective of this study was to assess the pharmacokinetic characteristics and tolerability of a new oral syrup formulation of pentoxifylline as an adjunct to IVIG and ASA in the treatment of KD in children.Methods: Hospitalized boys and girls aged 6 months to 5 years and who were diagnosed with KD within the first 10 days of illness were eligible. Patients were assigned to 1 of 4 pentoxifylline treatment groups, by dose level (dose levels 1, 2, 3, and 4: 10, 15, 20, and 25 mg/kg daily, respectively, divided into 3 doses). Six plasma samples collected at the time the first dose was administered, and 4 samples collected after administration of the last dose on study day 6, were assessed by high-performance liquid chromatography using noncompartmental and 1-compartment pharmacokinetic analyses for pentoxifylline and its active metabolite (M-1). TNF-alpha levels on days 1 and 6 were assessed using electroimmunoassay.Results: Fourteen boys and 10 girls were enrolled. The mean age, body weight, and illness day at study entry were 34.5 months, 13.8 kg, and 6, respectively. Pentoxifylline exhibited nonlinear kinetic characteristics, with median area under the plasma concentration-time curve from time 0 to infinity(AUC_0-∞) values of 622, 3428, 8416, and 10,347 ng/mL · h for dose levels 1 to 4, respectively, on study day 1. Pentoxifylline noncompartmental oral clearance and volume of distribution were significantly lower, and dose-normalized AUC_0-∞ was significantly higher, for dose level 3 than dose level 1. M-1 parameters were not significantly different between dose levels. No accumulation of pentoxifylline or M-1 was noted. Fifteen of 24 patients (63%) reported mild to moderate adverse events that may or may not have been treatment related. Frequency and severity did not differ significantly between dose levels.Conclusions: In the children in this study, pentoxifylline was well tolerated at the doses studied. No notable differences in clinical outcomes were observed between dose levels, and dose levels 3 and 4 (20 and 25 mg/kg daily, respectively) resulted in similar exposure to both pentoxifylline and M-1. Future efficacy and tolerability studies should use a daily dose of 20 mg/kg of pentoxifylline in acute KD.     

Up-regulation of A(2A) adenosine receptors by proinflammatory cytokines in rat PC12 cells

The purpose of this study was to examine the regulation of A(2A) adenosine receptor (A(2A) AR) gene expression induced by proinflammatory cytokines in PC12 cells. The A(2A) AR mRNA levels were substantially increased following 3-48 hr PC12 cell treatment with interleukin 1 beta (500 unit/mL) or tumor necrosis factor alpha (1000 unit/mL), as revealed by RT-PCR analysis. In parallel, cell cytokine treatment induced an up-regulation of A(2A) receptor protein. Equilibrium radioligand binding studies on treated-cells showed a significant increase in maximum density of [3H] 2-(carboxyethylphenylethylamino) adenosine-5'-carboxamide binding sites, with no significant changes in the affinity constant value. The increase in A(2A) receptor density was also demonstrated by Western blot analysis. Interleukin 1 beta and tumor necrosis factor alpha effects on A(2A) AR mRNA and protein levels were detectable after 3 hr cytokine treatment and reached a maximum within 24 and 48 hr, respectively. These results demonstrated the existence of heterologous regulation of A(2A) ARs by proinflammatory cytokines. The biological significance of this regulation might be associated with modulating cellular activity in response to tissue damage associated with inflammatory mediator production.     

Estrogen protects the liver and intestines against sepsis-induced injury in rats

BACKGROUND AND AIM: Sepsis is commonly associated with enhanced generation of reactive oxygen metabolites, leading to multiple organ dysfunctions. The aim of this study was to examine the putative protective role of estradiol against sepsis-induced oxidative organ damage. MATERIALS AND METHODS: Sepsis was induced by cecal ligation and puncture method in Wistar albino rats. Sham-operated (control) and sepsis groups received saline or estradiol propionate (10 mg/kg) intraperitoneally immediately after the operation and at 12 h. Twenty-four hours after the surgery, rats were decapitated and malondialdehyde, glutathione levels, and myeloperoxidase activity were determined in the liver and ileum, while oxidant-induced tissue fibrosis was determined by collagen contents. Tissues were also examined microscopically. Serum aspartate aminotransferase, alanine aminotransferase levels, and lactate dehydrogenase were measured for the evaluation of liver functions and tissue damage, respectively. Tumor necrosis factor-alpha was also assayed in serum samples. RESULTS: In the saline-treated sepsis group, glutathione levels were decreased significantly, while the malondialdehyde levels, myeloperoxidase activity, and collagen content were increased in the tissues (P < 0.01 to P < 0.001), suggesting oxidative organ damage, which was also verified histologically. In the estradiol-treated sepsis group, all of these oxidant responses were reversed significantly (P < 0.05 to P < 0.01). Liver function tests and tumor necrosis factor-alpha levels, which were increased significantly (P < 0.001) following sepsis, were decreased (P < 0.05 to P < 0.001) with estradiol treatment. CONCLUSION: The results demonstrate the role of oxidative mechanisms in sepsis-induced tissue damage, and estradiol, by its antioxidant properties, ameliorates oxidative organ injury, implicating that treatment with estrogens might be applicable in clinical situations to ameliorate multiple organ damage induced by sepsis.     

Ocular toxicity of intravitreous adalimumab (Humira) in the rabbit

Purpose To evaluate the ocular toxicity of escalating doses of intravitreous adalimumab (Humira) in the rabbit eye. Methods Twelve New Zealand albino rabbits received unilateral intravitreous injections of 0.1 ml of adalimumab 0.25 mg (three eyes), 0.50 mg (three eyes), 1.0 mg (three eyes) or 0.1 ml balanced salt solution (BSS, threeeyes). Slit-lamp biomicroscopy and fundoscopy were carried out at baseline, day 1, 7 and 14 following intravitreous injection, while electroretinography (ERG) was carried out at baseline and day 14. Animals were euthanized on day 14, and histopathological examination of the eyes was performed. Results Slit-lamp biomicroscopy and fundoscopy were normal in eyes having received BSS, 0.25 mg or 0.50 mg adalimumab; however, inflammation was present in two of three eyes having received 1.0 mg adalimumab. Similarly, comparison of scotopic and photopic ERG light at baseline and day 14 demonstrated no changes in eyes receiving BSS, 0.25 mg or 0.50 mg adalimumab, but two of three eyes having received 1.0 mg adalimumab showed a greater than 30% reduction in a and b wave. Finally, histopathology demonstrated no differences between eyes receiving BSS, 0.25 mg or 0.50 mg of adalimumab, but two of three eyes injected with 1.0 mg demonstrated inflammatory cell infiltration of the vitreous and anterior chamber, with one of these eyes demonstrating retinal necrosis. Conclusions Escalating doses of intravitreous adalimumab in rabbit eyes caused no detectable functional or structural ocular toxicity up to a dose of 0.50 mg. Administration of 1.0 mg in 0.1 ml was associated with an inflammatory reaction and retinal necrosis. None of the authors has any proprietary interest in any technique or product described herein. The authors have full control of all primary data and they agree to allow Graefes Archive for Clinical and Experimental Ophthalmology to review their data.     

The skin tissue is adversely affected by TNF-alpha blockers in patients with chronic inflammatory arthritis: a 5-year prospective analysis

OBJECTIVE:

We evaluated the incidence of and the main risk factors associated with cutaneous adverse events in patients with chronic inflammatory arthritis following anti-TNF-α therapy.

METHODS:

A total of 257 patients with active arthritis who were taking TNF-α blockers, including 158 patients with rheumatoid arthritis, 87 with ankylosing spondylitis and 12 with psoriatic arthritis, were enrolled in a 5-year prospective analysis. Patients with overlapping or other rheumatic diseases were excluded. Anthropometric, socioeconomic, demographic and clinical data were evaluated, including the Disease Activity Score-28, Bath Ankylosing Spondylitis Disease Activity Index and Psoriasis Area Severity Index. Skin conditions were evaluated by two dermatology experts, and in doubtful cases, skin lesion biopsies were performed. Associations between adverse cutaneous events and clinical, demographic and epidemiological variables were determined using the chi-square test, and logistic regression analyses were performed to identify risk factors. The significance level was set at p<0.05.

RESULTS:

After 60 months of follow-up, 71 adverse events (73.85/1000 patient-years) were observed, of which allergic and immune-mediated phenomena were the most frequent events, followed by infectious conditions involving bacterial (47.1%), parasitic (23.5%), fungal (20.6%) and viral (8.8%) agents.

CONCLUSION:

The skin is significantly affected by adverse reactions resulting from the use of TNF-α blockers, and the main risk factors for cutaneous events were advanced age, female sex, a diagnosis of rheumatoid arthritis, disease activity and the use of infliximab.     

Estrogen Protects against Oxidative Multiorgan Damage in Rats with Chronic Renal Failure

The impact of sex dimorphism on chronic renal failure (CRF)-induced oxidative multiorgan damage and the effects of estradiol (E₂) loss and E₂ supplementation on the progress of CRF were studied. Sprague-Dawley rats underwent 5/6 nephrectomy (CRF), and a group of female rats had bilateral ovariectomy (OVX), while the sham-operated rats had no nephrectomy or OVX. Rats received either estradiol propionate (50 μg/kg/day) or vehicle for six weeks. Serum BUN levels were elevated in both male and female CRF groups treated with vehicle, while creatinine level was not significantly changed in the female CRF group. CRF-induced elevation in serum TNF-α of male rats was abolished when the animals were treated with E₂, while OVX exaggerated TNF-α response. In OVX and male rats with CRF, E₂ treatment reversed the malondialdehyde elevations in all the studied tissues (kidney, heart, lung, ileum, brain, liver, and gastrocnemius muscle), while depletion of glutathione in these tissues was prevented by E₂ treatment. Similarly, increased levels of myeloperoxidase activity, lucigenin chemiluminescence, and collagen in most of the tissues were reversed by E₂ treatment. The findings show that the extent of tissue injuries was relatively less in females, while ovariectomy exacerbated all the indices of oxidative injury. Moreover, the administration of E₂, with its potent anti-oxidant and anti-inflammatory effects, markedly improved CRF-induced systemic inflammatory outcomes in both male and female rats by depressing tissue neutrophil infiltration and modulating the release of inflammatory cytokines.