Decreased infarct size after focal cerebral ischemia in mice chronically infected with Toxoplasma gondii

To determine whether Toxoplasma gondii infection could modify biological phenomena associated with brain ischemia, we investigated the effect of permanent middle cerebral artery occlusion (MCAO) on neuronal survival, inflammation and redox state in chronically infected mice. Infected animals showed a 40% to 50% decrease of infarct size compared with non-infected littermates 1, 4 and 14 days after MCAO. The resistance of infected mice may be associated with increased basal levels of anti-inflammatory cytokines and/or a marked reduction of the MCAO-related brain induction of two pro-inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma (IFNgamma). In addition, potential anti-inflammatory/neuroprotective factors such as nerve growth factor, suppressor of cytokine signaling-3, superoxide dismutase activity, uncoupling protein-2 and glutathione (GSH) were upregulated in the brain of infected mice. Consistent with a role of GSH in central cytokine regulation, GSH depletion by diethyl maleate inhibited Toxoplasma gondii lesion resistance by increasing the proinflammatory cytokine IFNgamma brain levels. Overall, these findings indicate that chronic toxoplasmosis decisively influences both the inflammatory molecular events and outcome of cerebral ischemia.

Effect of interferon-beta and atorvastatin on Th1/Th2 cytokines in multiple sclerosis

Beneficial effects by both interferon-beta and statin treatment in patients with multiple sclerosis (MS) may be linked to interference with the Th1/Th2 cytokine balance. We determined patterns of Th1/Th2 cytokines (interleukin (IL)-1beta, IL-2, IL-6, IL-12p70, tumor-necrosis factor (TNF)-alpha and interferon-gamma, and IL-4, IL-5 and IL-10, respectively) in the serum of patients with relapsing-remitting MS treated with 250microg interferon-beta 1b or with interferon-beta plus 40mg atorvastatin. In treatment naïve patients with MS, a trend for lower TNF-alpha serum levels compared to controls was detected (P=0.08). Interferon-beta treatment increased TNF-alpha levels, while a trend for lowering of IL-5 serum levels was found (P=0.07). Addition of atorvastatin raised IL-12p70 serum levels (P<0.05). Mean levels of two Th2 cytokines (IL-4, IL-10) showed a non-significant increase after addition of atorvastatin. We conclude that interferon-beta and atorvastatin exert divergent action on Th1/Th2 serum cytokines levels in MS. Supplemental atorvastatin might promote a Th1-type response by raising IL-12p70. Further studies are required to support a Th2 cytokine shift by atorvastatin in patients with MS.

The etiology of liver damage imparts cytokines transforming growth factor beta1 or interleukin-13 as driving forces in fibrogenesis

It is unknown whether transforming growth factor beta1 (TGF-beta1) signaling uniformly participates in fibrogenic chronic liver diseases, irrespective of the underlying origin, or if other cytokines such as interleukin (IL)-13 share in fibrogenesis (e.g., due to regulatory effects on type I pro-collagen expression). TGF-beta1 signaling events were scored in 396 liver tissue samples from patients with diverse chronic liver diseases, including hepatitis B virus (HBV), hepatitis C virus (HCV), Schistosoma japonicum infection, and steatosis/steatohepatitis. Phospho-Smad2 staining correlated significantly with fibrotic stage in patients with HBV infection (n = 112, P < 0.001) and steatosis/steatohepatitis (n = 120, P < 0.01), but not in patients with HCV infection (n = 77, P > 0.05). In tissue with HBx protein expression, phospho-Smad2 was detectable, suggesting a functional link between viral protein expression and TGF-beta1 signaling. For IL-13, immunostaining correlated with fibrotic stage in patients with HCV infection and steatosis/steatohepatitis. IL-13 protein was more abundant in liver tissue lysates from three HCV patients compared with controls, as were IL-13 serum levels in 68 patients with chronic HCV infection compared with 20 healthy volunteers (72.87 +/- 26.38 versus 45.41 +/- 3.73, P < 0.001). Immunohistochemistry results suggest that IL-13-mediated liver fibrogenesis may take place in the absence of phospho-signal transducer and activator of transcription protein 6 signaling. In a subgroup of patients with advanced liver fibrosis (stage > or =3), neither TGF-beta nor IL-13 signaling was detectable. Conclusion: Depending on the cause of liver damage, a predominance of TGF-beta or IL-13 signaling is found. TGF-beta1 predominance is detected in HBV-related liver fibrogenesis and IL-13 predominance in chronic HCV infection. In some instances, the underlying fibrogenic mediator remains enigmatic.

Induction of Bim limits cytokine-mediated prolonged survival of neutrophils

Under inflammatory conditions, neutrophil apoptosis is delayed due to survival-factor exposure, a mechanism that prevents the resolution of inflammation. One important proinflammatory cytokine involved in the regulation of neutrophil survival/activation is granulocyte-macrophage colony-stimulating factor (GM-CSF). Although GM-CSF mediates antiapoptotic effects in neutrophils, it does not prevent apoptosis, and the survival effect is both time dependent and limited. Here, we identified the proapoptotic Bcl-2 family member Bim as an important lifespan limiting molecule in neutrophils, particularly under conditions of survival factor exposure. Strikingly, GM-CSF induced Bim expression in both human and mouse neutrophils that was blocked by pharmacological inhibition of phosphatidylinositol-3 kinase (PI3K). Increased Bim expression was also seen in human immature bone marrow neutrophils as well as in blood neutrophils from septic shock patients; both cell populations are known to be exposed to GM-CSF under in vivo conditions. The functional role of Bim was investigated using Bim-deficient mouse neutrophils in the presence and absence of the survival cytokines interleukin (IL)-3 and GM-CSF. Lack of Bim expression resulted in a much higher efficacy of the survival cytokines to block neutrophil apoptosis. Taken together, these data demonstrate a functional role for Bim in the regulation of neutrophil apoptosis and suggest that GM-CSF and other neutrophil hematopoietins initiate a proapoptotic counterregulation that involves upregulation of Bim.

TH2 cytokines from malignant cells suppress TH1 responses and enforce a global TH2 bias in leukemic cutaneous T-cell lymphoma

PURPOSE In leukemic cutaneous T-cell lymphoma (L-CTCL), malignant T cells accumulate in the blood and give rise to widespread skin inflammation. Patients have intense pruritus, increased immunoglobulin E (IgE), and decreased T-helper (TH)-1 responses, and most die from infection. Depleting malignant T cells while preserving normal immunity is a clinical challenge. L-CTCL has been variably described as a malignancy of regulatory, TH2 and TH17 cells. EXPERIMENTAL DESIGN We analyzed phenotype and cytokine production in malignant and benign L-CTCL T cells, characterized the effects of malignant T cells on healthy T cells, and studied the immunomodulatory effects of treatment modalities in patients with L-CTCL. RESULTS Twelve out of 12 patients with L-CTCL overproduced TH2 cytokines. Remaining benign T cells were also strongly TH2 biased, suggesting a global TH2 skewing of the T-cell repertoire. Culture of benign T cells away from the malignant clone reduced TH2 and enhanced TH1 responses, but separate culture had no effect on malignant T cells. Coculture of healthy T cells with L-CTCL T cells reduced IFNγ production and neutralizing antibodies to interleukin (IL)-4 and IL-13 restored TH1 responses. In patients, enhanced TH1 responses were observed following a variety of treatment modalities that reduced malignant T-cell burden. CONCLUSIONS A global TH2 bias exists in both benign and malignant T cells in L-CTCL and may underlie the infectious susceptibility of patients. TH2 cytokines from malignant cells strongly inhibited TH1 responses. Our results suggest that therapies that inhibit TH2 cytokine activity, by virtue of their ability to improve TH1 responses, may have the potential to enhance both anticancer and antipathogen responses.

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory, GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.

Vaginal cytokines do not differ between postmenopausal women with and without symptoms of vulvovaginal irritation.

OBJECTIVE The aim of this exploratory pilot study was to determine if there are differences in vaginal cytokine levels between postmenopausal women with and without vulvovaginal irritative symptoms (itching, burning, or pain). METHODS Postmenopausal women (n = 34) not using hormone therapy and presenting with or without symptoms of vulvovaginal irritation were asked to volunteer for this study. Each participant underwent a vaginal examination and screening for vaginitis using Amsel criteria, pH, and light microscopy. A vaginal lavage with 5.0 mL of sterile saline was carried out, and a peripheral blood sample was obtained. The vaginal lavage and serum samples were assayed for interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α by specific enzyme-linked immunosorbent assays. Results were adjusted for total protein concentration and presented as the amount of cytokines per protein (pg/μg protein). Statistical analysis was performed using SAS version 9.3 (SAS Institute, Cary, NC). The means and SDs of all variables among women with and without vulvovaginal irritation were compared using independent-samples Student's t test. RESULTS A total of 26 postmenopausal women were enrolled into the study (symptomatic, n = 15; asymptomatic, n = 11). The mean (SD) vaginal pH for all participants was 5.9 (1.2). There were no significant differences (P > 0.05) in age, age at menopause, vaginal pH, and vaginal and serum cytokines and chemokines (IL-1β, IL-6, IL-8, and tumor necrosis factor-α) among symptomatic versus asymptomatic women. IL-8 was the most abundant vaginal cytokine, with mean (SD) vaginal IL-8 levels being 4.1 (3.4) and 3.1 (3.9) pg/μg protein in the symptomatic versus asymptomatic groups, respectively (P = 0.55). There were no significant linear correlations (P > 0.05) between serum and vaginal cytokine levels for all endpoints. CONCLUSIONS The presence or absence of postmenopausal vulvovaginal symptoms does not significantly differentiate vaginal inflammatory markers. Serum and vaginal cytokines are not significantly linearly correlated among postmenopausal women with and without symptoms commonly associated with vaginal atrophy, implying that this is a local reaction.

Correlation between altered central pain processing and concentration of peritoneal fluid inflammatory cytokines in endometriosis patients with chronic pelvic pain

Translational research has not yet elucidated whether alterations in central pain processes are related to peripheral inflammatory processes in chronic pain patients. We tested the hypothesis that the concentration of cytokines in the peritoneal fluid of endometriosis patients with chronic pain correlate with parameters of hyperexcitability of the nociceptive system. The concentrations of 15 peritoneal fluid cytokines were measured in 11 patients with chronic pelvic pain and a diagnosis of endometriosis. Six parameters assessing central pain processes were recorded. Positive correlations between concentration of some cytokines in the peritoneal fluid and amplification of central pain processing were found. The results suggest that inflammatory mechanisms may be important in the pathophysiology of altered central pain processes and that cytokines produced in the environment of endometriosis could act as mediators between the peripheral lesion and changes in central nociceptive processes.

Effects of stimulus with proinflammatory mediators on nitric oxide production and matrix metalloproteinase activity in explants of cranial cruciate ligaments obtained from dogs

OBJECTIVE To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators. SAMPLE POPULATION Tissue specimens obtained from 7 healthy adult Beagles that were (mean +/- SD) 4.5 +/- 0.5 years old and weighed 12.5 +/- 0.8 kg. PROCEDURE The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS). RESULTS All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and non-stimulated cultures. CONCLUSIONS AND CLINICAL RELEVANCE Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP.

Dominance of chemokine ligand 2 and matrix metalloproteinase-2 and -9 and suppression of pro-inflammatory cytokines in the epidural compartment after intervertebral disc extrusion in a canine model

BACKGROUND CONTEXT In canine intervertebral disc (IVD) disease, a useful animal model, only little is known about the inflammatory response in the epidural space. PURPOSE To determine messenger RNA (mRNA) expressions of selected cytokines, chemokines, and matrix metalloproteinases (MMPs) qualitatively and semiquantitatively over the course of the disease and to correlate results to neurologic status and outcome. STUDY DESIGN/SETTING Prospective study using extruded IVD material of dogs with thoracolumbar IVD extrusion. PATIENT SAMPLE Seventy affected and 13 control (24 samples) dogs. OUTCOME MEASURES Duration of neurologic signs, pretreatment, neurologic grade, severity of pain, and outcome were recorded. After diagnostic imaging, decompressive surgery was performed. METHODS Messenger RNA expressions of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF), interferon (IFN)γ, MMP-2, MMP-9, chemokine ligand (CCL)2, CCL3, and three housekeeping genes was determined in the collected epidural material by Panomics 2.0 QuantiGene Plex technology. Relative mRNA expression and fold changes were calculated. Relative mRNA expression was correlated statistically to clinical parameters. RESULTS Fold changes of TNF, IL-1β, IL-2, IL-4, IL-6, IL-10, IFNγ, and CCL3 were clearly downregulated in all stages of the disease. MMP-9 was downregulated in the acute stage and upregulated in the subacute and chronic phase. Interleukin-8 was upregulated in acute cases. MMP-2 showed mild and CCL2 strong upregulation over the whole course of the disease. In dogs with severe pain, CCL3 and IFNγ were significantly higher compared with dogs without pain (p=.017/.020). Dogs pretreated with nonsteroidal anti-inflammatory drugs revealed significantly lower mRNA expression of IL-8 (p=.017). CONCLUSIONS The high CCL2 levels and upregulated MMPs combined with downregulated T-cell cytokines and suppressed pro-inflammatory genes in extruded canine disc material indicate that the epidural reaction is dominated by infiltrating monocytes differentiating into macrophages with tissue remodeling functions. These results will help to understand the pathogenic processes representing the basis for novel therapeutic approaches. The canine IVD disease model will be rewarding in this process.