Pattern Of Cytokine mRNA Expression Research Articles

Intrinsic Variability Present in Wharton's Jelly Mesenchymal Stem Cells and T Cell Responses May Impact Cell Therapy.

Wharton's jelly mesenchymal stem cells (WJ-MSC) exhibit immunomodulatory effects on T cell response. WJ-MSC are easy to collect, process, and proliferate rapidly in culture, but information on the variability of individual cell samples impacting upon in vitro expansion, immunomodulatory potential, and aging processes is still lacking. We propose to evaluate the immunomodulatory cytokine profile and capacity to inhibit T cell proliferation of WJ-MSC progressing to replicative senescence in order to analyze if expected responses are affected. Our results show that the gene expression of immunomodulatory molecules varied among samples with no specific pattern present. In coculture, all WJ-MSC were capable of inhibiting mitogen-activated CD3+ T cell proliferation, although to different degrees, and each PBMC responded with a different level of inhibition. Thus, we suggest that each WJ-MSC displays unique behavior, differing in patterns of cytokine mRNA expression and immunomodulatory capacity. We believe that variability between samples may play a role in the effectiveness of WJ-MSC employed therapeutically.

Distinct cytokine mRNA expression pattern in immunoglobulin G4-related kidney disease associated with renal cell carcinoma

We treated a 61-year-old man with immunoglobulin (Ig)G4-related kidney disease (IgG4-RKD). He had a history of allergic diseases and an allergic reaction and had received a diagnosis of autoimmune pancreatitis (AIP). He had also received a diagnosis of renal cell carcinoma (RCC) and had undergone segmental resection of the left kidney at 59 years of age. His serum amylase level and number of peripheral eosinophils increased after RCC development. We hypothesized that the RCC may have induced AIP and IgG4-RKD and we therefore examined the excised RCC tissue; typical findings of IgG4-RKD associated with RCC were recognized. We next evaluated the mRNA expression of cytokines in the excised tissues of this case and ten other ordinary RCC cases. In all cases, notable levels of IL-10 mRNA and high levels of TGF-β mRNA were seen. Although prominent differences were not observed in the mRNA expression of Th1, Th17 and Treg cytokines in all cases, the present case alone showed increased production of the Th2 cytokines IL-4 and IL-5, which were not detected in ordinary RCC cases. Although the mechanism underlying IgG4-RKD development has not yet been determined, Th2 and Treg cells are thought to play a prominent role in the pathogenesis. It is therefore likely that in this case, the association of these two diseases was not coincidental, and a distinct immune response against RCC may trigger IgG4-RKD development.

Sputum cytokine mapping reveals an ‘IL‐5, IL‐17A, IL‐25‐high’ pattern associated with poorly controlled asthma

Asthma is a heterogeneous disease with various clinical, inflammatory and molecular phenotypes. We studied sputum cytokine mRNA expression patterns in an unselected group of adult asthma patients to characterize the underlying inflammatory process. Differential cell counts and cytokine mRNA (quantified by real-time PCR) were analysed on sputum from 40 controls and 66 asthmatic adults. A 'cytokine-high' profile was defined if mRNA levels for that particular cytokine exceeded the 90th percentile value in the control population. Radar graphs were used to visualize cytokine profiles. Sputum mRNA analysis confirmed heterogeneity of cytokine patterns among patients. Thirty-six patients (55%) had a Th2 cytokine pattern: 'IL-5-high' (n = 13), 'IL-4-high' (n = 17) or 'IL-4- and IL-5-high' (n = 6). The 'IL-5-high' asthma profile (n = 13) coincided with the 'IL-25-high' (10/13) and surprisingly also with the 'IL-17A-high' (11/13) profile. The 'IL-5-/IL-25-/IL-17A-high profile was different from the 'IL-4-high' pattern. Patients with the 'IL-5, IL-17A, IL-25-high' pattern had significantly worse lung function parameters. Uncontrolled asthmatics [Asthma Control Test (ACT) < 20] had higher sputum IL-5, IL-17A and IL-25 mRNA levels compared to controlled asthmatics (P = 0.002; P = 0.002; P = 0.066) and uncontrolled asthma is more common among 'IL-5- and IL-17A-high' asthmatics compared to 'IL-5-, IL-17A-low' asthmatics (χ(2) = 3.7, P = 0.027; relative risk (RR): 1.8, 95% CI = 1.1-3.1). Patients with the 'IL-5, IL-17A, IL-25-high' airway inflammatory pattern are often uncontrolled asthmatics, despite daily treatment. It seems worthwhile to evaluate whether measuring sputum cytokine levels might be used to assess the response to increased doses of steroids in patients with asthma.

Dendritic cells are the major antigen presenting cells in inflammatory lesions of murine mycoplasma respiratory disease and contribute to disease severity (P3306)

Abstract Mycoplasmas cause chronic respiratory diseases in animals and humans, and vaccine development has been problematic. T cell responses were shown to confer protection as well as promote immunopathology in murine mycoplasma pneumonia. Because T cells play a critical role, the role of antigen presenting cells (APC) was examined as they likely influence either an increase in disease severity or promote protective immunity. The roles of APC, such as dendritic cells (DC) and macrophages, in mycoplasma disease are currently unknown. In this study, we examined the ability pulmonary APC populations and their contribution to T cell responses during disease pathogenesis. Both macrophages and DC increased in the lungs of mice after infection. These cell populations showed different patterns of cytokine mRNA expression, supporting the idea that these cells have different impacts on immunity in response to infection. In fact, DC from the lungs of infected mice were most capable of stimulating mycoplasma-specific T helper (Th) cell responses in vitro. In vivo, DC cells were co-localized with Th cells in inflammatory lesions in the lungs of mycoplasma-infected mice. Intratracheal inoculation of mycoplasma antigen-pulsed DC resulted in increased disease severity after subsequent infection. Thus, DC appear to be the major APC population responsible for pulmonary T cell stimulation in mycoplasma-infected mice, and these DC likely contribute to responses impacting disease pathogenesis.

Tu1683 Expression of Thymidine Phosphorylase in Ulcerative Colitis and Efficacy of Its Specific Inhibitor for the Treatment of Ulcerative Colitis

protein were collected. Cytokine levels in cell supernatants were determined using the Bioplex 3D suspension array system (Bio-Rad). Preadipocyte supernatants were also applied on cultures of human colonic NCM-460 cells for 24hs and RNA and protein were collected. Inflammatory molecule mRNA expression and activation of intracellular kinases were evaluated with Luminex inflammatory panels (FlexScript LDA), and phospho-protein multiplex panels, respectively. Results: Preadipocytes isolated from IBD patients demonstrated differential mediator secretion patterns compared to preadipocytes from control and obese patients. Of the 42 proinflammatory cytokines and growth factors tested, IL-7/IL-8/Gro/MCP3/VEGF/ PDGF-AA showed statistically significant differences between control, obese and IBD preadipocytes (p ,0.05). Notably, there were significant differences in mediator secretion between preadipocytes isolated from UC and CD patients, in particular VEGF and PDGF (p , 0.01 for both). NCM-460 cells had distinct GSK-3, AKT kinase activation (p , 0.05 for both) and cytokine mRNA expression patterns of TNF, IL-2, IL-3, IL-17, VEGF, MIP-1 β, MIP-3, RANTES (p,0.05) following exposure to preadipocyte supernatants from all three pathological conditions compared to controls. Conclusions: Our data suggest the existence of preadipocyte-disease-dependent mediators contributing to the generation of differential colonocyte responses. Together with reports indicating altered levels of several adipokines during IBD, our results provide the first direct evidence for the importance of adipose tissue homeostasis during obesity in determining the outcome of IBD. Supported by NIH P50 DK064539 (CP & IK), DK047343, DK060729 (CP), The Broad Foundation (BMRP) (IK, RA), and the Blinder Research Foundation for Crohn's Disease (AS).

Dendritic Cells Are the Major Antigen Presenting Cells in Inflammatory Lesions of Murine Mycoplasma Respiratory Disease

Mycoplasmas cause chronic respiratory diseases in animals and humans, and to date, development of vaccines have been problematic. Using a murine model of mycoplasma pneumonia, lymphocyte responses, specifically T cells, were shown to confer protection as well as promote immunopathology in mycoplasma disease. Because T cells play such a critical role, it is important to define the role of antigen presenting cells (APC) as these cells may influence either exacerbation of mycoplasma disease pathogenesis or enhancement of protective immunity. The roles of APC, such as dendritic cells and/or macrophages, and their ability to modulate adaptive immunity in mycoplasma disease are currently unknown. Therefore, the purpose of this study was to identify individual pulmonary APC populations that may contribute to the activation of T cell responses during mycoplasma disease pathogenesis. The present study indeed demonstrates increasing numbers of CD11c− F4/80+ cells, which contain macrophages, and more mature/activated CD11c+ F4/80− cells, containing DC, in the lungs after infection. CD11c− F4/80+ macrophage-enriched cells and CD11c+ F4/80− dendritic cell-enriched populations showed different patterns of cytokine mRNA expression, supporting the idea that these cells have different impacts on immunity in response to infection. In fact, DC containing CD11c+ F4/80− cell populations from the lungs of infected mice were most capable of stimulating mycoplasma-specific CD4+ Th cell responses in vitro. In vivo, these CD11c+F4/80− cells were co-localized with CD4+ Th cells in inflammatory infiltrates in the lungs of mycoplasma-infected mice. Thus, CD11c+F4/80− dendritic cells appear to be the major APC population responsible for pulmonary T cell stimulation in mycoplasma-infected mice, and these dendritic cells likely contribute to responses impacting disease pathogenesis.

P056 GED-0507–34 Levo, a novel modulator of PPARgamma as new therapeutic strategy in the treatment of intestinal fibrosis

protein were collected. Cytokine levels in cell supernatants were determined using the Bioplex 3D suspension array system (Bio-Rad). Preadipocyte supernatants were also applied on cultures of human colonic NCM-460 cells for 24hs and RNA and protein were collected. Inflammatory molecule mRNA expression and activation of intracellular kinases were evaluated with Luminex inflammatory panels (FlexScript LDA), and phospho-protein multiplex panels, respectively. Results: Preadipocytes isolated from IBD patients demonstrated differential mediator secretion patterns compared to preadipocytes from control and obese patients. Of the 42 proinflammatory cytokines and growth factors tested, IL-7/IL-8/Gro/MCP3/VEGF/ PDGF-AA showed statistically significant differences between control, obese and IBD preadipocytes (p ,0.05). Notably, there were significant differences in mediator secretion between preadipocytes isolated from UC and CD patients, in particular VEGF and PDGF (p , 0.01 for both). NCM-460 cells had distinct GSK-3, AKT kinase activation (p , 0.05 for both) and cytokine mRNA expression patterns of TNF, IL-2, IL-3, IL-17, VEGF, MIP-1 β, MIP-3, RANTES (p,0.05) following exposure to preadipocyte supernatants from all three pathological conditions compared to controls. Conclusions: Our data suggest the existence of preadipocyte-disease-dependent mediators contributing to the generation of differential colonocyte responses. Together with reports indicating altered levels of several adipokines during IBD, our results provide the first direct evidence for the importance of adipose tissue homeostasis during obesity in determining the outcome of IBD. Supported by NIH P50 DK064539 (CP & IK), DK047343, DK060729 (CP), The Broad Foundation (BMRP) (IK, RA), and the Blinder Research Foundation for Crohn's Disease (AS).

Alterations with age in peripheral blood lymphocyte subpopulations and cytokine synthesis in beagles.

The immune system is considered to be affected by aging, which is linked to various immune pathogeneses. The purpose of this study was to determine age-associated changes in immune function of healthy dogs (beagles), specifically those of naive and memory T lymphocytes, based on cytokine synthesis. Blood samples were obtained from 44 healthy beagles that were divided into three age-groups: young (<4 years), middle-aged (4-8 years), and older dogs (>8 years). Subpopulations of T lymphocytes were determined by flow cytometry. Transcriptional (mRNA) levels of cytokines were determined for primary-cultured leukocytes using quantitative real-time polymerase chain reaction. There were negative correlations between dogs' ages and the number of peripheral blood mononuclear cells, T cells, and B cells. In particular, the number of naive CD4+ CD45RA+ T cells and CD8+ CD45RA+ T cells significantly decreased with age. The mRNA levels for interleukin (IL)-2, IL-2Rα, and interferon-gamma were significantly higher in young or middle-aged dogs (P < 0.05), whereas IL-4 mRNA expression was not significantly different over the different age-groups. IL-2Rγ mRNA expression tended to decrease with age. Decreases of naive CD4+ and naive CD8+ T cells may be related to age-related immunosenescence in dogs. With regard to cytokine production, leukocyte IL-4 and IL-10 mRNA levels did not change with age, whereas IL-2, IL-2Rα, and IL-2Rγ mRNA levels decreased with age. These altered cytokine mRNA expression patterns may contribute to decreased naive T-cell function(s) with aging.

Inflammatory cytokine gene expression in THP‐1 cells exposed to Stachybotrys chartarum and Aspergillus versicolor

Very little is known about the mechanisms that occur in human cells upon exposure to fungi as well as their mycotoxins. A better understanding of toxin-regulated gene expression would be helpful to identify safe levels of exposure and could eventually be the basis for establishing guidelines for remediation scenarios following a water intrusion event. In this research, cytokine mRNA expression patterns were investigated in the human monocytic THP-1 cell line exposed to fungal extracts of various fragment sizes obtained from Stachybotrys chartarum RTI 5802 and/or Aspergillus versicolor RTI 3843, two common and well-studied mycotoxin producing fungi. Cytokine mRNA expression was generally upregulated 2-10 times following a 24 h exposure to fungal extracts. Expression of the proinflammatory interleukin-1β, interleukin-8, and tumor necrosis factor-α genes increased while the anti-inflammatory gene interleukin-10 also increased albeit at very low level, suggesting that negative feedback regulation mechanism of production of proinflammatory cytokines initiated upon 24 h of incubation. In addition, submicron size extracts of A. versicolor caused significant death of THP-1 cells, whereas extracts of S. chartarum caused no cell death while the mixture of the two fungi had an intermediate effect. There was no general correlation between gene expression and fragment sizes, which suggests that all submicron fragments may contribute to inflammatory response.

Cytokine mRNA profiling identifies B cells as a major source of RANKL in rheumatoid arthritis

Background and objectivesCytokines are important mediators involved in chronic inflammation and tissue destruction in rheumatoid arthritis (RA). Animal models of arthritis, in vitro studies, and observations made in patients treated...

IL-4 mRNA expression by peritoneal cells during episodes of peritonitis in patients on continuous ambulatory peritoneal dialysis.

Peritoneal cells were isolated from dialysates drained from nine patients on continuous ambulatory peritoneal dialysis (CAPD) during episodes of peritonitis. Levels of expression of mRNA for the regulatory cytokines, interferon-gamma (IFN-gamma) and IL-4, were investigated daily, where possible, during the first 5 days of peritonitis. Cytokine mRNA levels were compared with those of peripheral blood mononuclear cells (PBMC) stimulated in vitro with phorbol 12-myristate 13-acetate (PMA) plus phytohaemagglutinin (PHA). Peritoneal cells expressed low levels of IFN-gamma mRNA; for four of nine patients, IL-4 mRNA levels greater than those expressed by stimulated PBMC were detected. There was no pattern of cytokine mRNA expression associated with the types of organisms detected in dialysates at initiation of peritonitis. However, in contrast to those patients with a transient, resolving peritonitis, significant IL-4 mRNA expression was detected in cells isolated early in the episodes of peritonitis in patients who suffered recurrent peritonitis within 30 days of the initial peritonitis episode. These results suggest an association between early IL-4 mRNA expression and susceptibility to further infections. The known anti-inflammatory effects of IL-4 may explain this association.

Intrarenal gene expression of proinflammatory chemokines and cytokines in chronic proteinuric glomerulopathies

Proteinuria has been recently shown to be an independent risk factor for the progression of chronic nephropathies, but the actual mechanisms by which urinary protein load damages renal tissue in humans remain unsolved. Using real-time RT-PCR method we evaluated intrarenal mRNA expression of various cytokines and chemokines in patients with biopsy-proven IgA nephropathy (IgAN, n=11), membranous nephropathy (MN, n=6) and focal and segmental glomerulosclerosis (FSGS, n=6) who exhibited proteinuria over 0.5 g/day. There was a significant positive correlation between the proteinuria extent and the intrarenal RANTES (regulated upon activation normal T cell expressed and secreted) mRNA expression in patients with IgAN, a similar trend was also observed in patients with MN and FSGS. There were no clear relationships between the proteinuria and intrarenal mRNA expression of tumor necrosis factor alpha, transforming growth factor beta1 and monocyte chemoattractant peptide-1. There were no differences in the pattern of cytokine mRNA expression between different glomerulopathies. In conclusion, our results support the hypothesis that lymphocytes, macrophages and their products provoke tissue injury in response to proteinuria independently of the nature of renal diseases in man.

Influence of local and systemic CTLA4Ig gene transfer on corneal allograft survival

To analyse the effects of local (ex vivo) or systemic (in vivo) administration of adenovirus type 5 encoding CTLA4Ig (AdCTLA4Ig) on its influence to prolong corneal allograft survival and to study the underlying mechanisms. A MHC class I/II mismatched rat corneal transplant model was used. Recipients were randomly assigned to receive ex vivo gene-modified corneas expressing either CTLA4Ig, CTLA4Ig/IL-10 or a single intraperitoneal (i.p.) injection (1.0 x 10(9) or 1.0 x 10(10) infectious particles) of AdCTLA4Ig 1 day before transplantation and graft survival was analysed. The immunoregulatory effect of this treatment was examined by analysing intra-graft cytokine mRNA expression pattern at day 12 post-transplant. The anti-adenovirus immunity also was investigated. Ex vivo gene transfer resulted in a modest but significant prolongation of graft survival (p = 0.0036 compared to no treatment). In contrast, systemic gene therapy (1.0 x 10(9) or 1.0 x 10(10) infectious particles) significantly prolonged graft survival (p = 0.0007 and 0.0001, respectively, compared to no treatment). Systemic (1.0 x 10(10) infectious particles) therapy resulted in frequent indefinite survival of allogeneic grafts which was not observed in the other therapeutic regimens. Moreover, systemic therapy prevented the intra-graft accumulation and activation of T cells and resulted in a reduced mRNA expression of both TH1 and TH2 cytokines. The generation of anti-adenovirus antibodies was also efficiently inhibited. CTLA4Ig gene therapy is a successful strategy for the prevention of allogeneic graft rejection in corneal transplantation. Our work has further elucidated the mechanisms of corneal allograft rejection which may lead to novel therapeutic strategies.

Cytokine mRNA expression in unilateral ischemic-reperfused rat lung with salt solution supplemented with low-endotoxin or standard bovine serum albumin.

Our aim was to determine whether cytokine mRNA expression is induced by experimental manipulation including artificial perfusate or ischemia-reperfusion (I/R) in an isolated, perfused rat lung model. Constant pulmonary flow [Krebs-Henseleit solution supplemented with low-endotoxin (LE) or standard (ST) bovine serum albumin 4%, 0.04 ml/g body wt] and ventilation were maintained throughout. Right and left pulmonary arteries were isolated, and the left pulmonary artery was occluded for 60 min and then reperfused for 30 min. Analysis of tumor necrosis factor-alpha, IL-1 beta, IL-6, IL-10, and IFN-gamma mRNA expression by RT-PCR and evaluation of vascular permeability by bronchoalveolar lavage (BAL) fluid albumin content were conducted separately in right and left lung. Both LE and ST groups (each 12 rats) showed increases in vascular permeability by I/R (BAL fluid albumin content: 5.53 +/- 1.55 vs. 15.63 +/- 8.87 and 4.76 +/- 2.71 vs. 16.72 +/- 4.85 mg.ml BAL fluid-1.g lung dry wt-1, mean +/- SD; right vs. left lung in LE and ST groups, P < 0.05 between right and left). Cytokine mRNA expression was significantly higher in the I/R lung than in the control lung in the LE group, whereas it was higher in the control lung in the ST group (P < 0.05). mRNAs of not only proinflammatory but also anti-inflammatory cytokines were expressed in I/R lung, which are expected to aggravate I/R injury. The reversed pattern of cytokine mRNA expression in the ST group was possibly due to the longer perfusion of control lung with perfusate containing endotoxin, which caused no lung damage without I/R.

Kinetics of Cytokine Gene Expression in Human CD4+ and CD8+ T‐Lymphocyte Subsets Using Quantitative Real‐Time PCR

The time kinetics of five cytokines [interleukin-2 (IL-2), IL-5, interferon-gamma (IFN-gamma), granulocyte macrophage-colony stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha)] and one cytotoxic effector protein (granzyme B) was analysed by real-time quantitative polymerase chain reaction (PCR) following in vitro stimulation of human CD4 and CD8 T lymphocytes. Two stimuli were used, a mitogen [phytohemagglutinin (PHA)] and a recall antigen [purified protein derivative (PPD)]. The pattern of cytokine mRNA expression was found to be dependent on the T-cell subset and stimulus used. A wide interindividual variability in the cytokine gene expression pattern was demonstrated. Two expression patterns were observed. A bell-shaped expression profile was seen for most cytokines upon PHA activation in both subsets and PPD-activated CD4 T cells, whereas a biphasic/multiphasic expression pattern was noted in CD8 T cells upon PPD stimulation. For most cytokines, the time to induction was within 30 min of activation, and maximum accumulation seemed to be obtained after 4-8 h of activation. A sustained high level could, however, be noticed for up to 24 h. Granzyme B gene expression was also induced within 30 min of activation but showed a continuous gradual increase and late maximal accumulation (48-72 h). The findings of the present study are of importance when designing studies using the cytokine gene expression profile as a marker for antigen-specific T lymphocytes. It might be recommended that cytokine gene expression (IL-2, IL-5 and IFN-gamma) should be measured after 4-8 h of specific activation but also up to 24 h of stimulation is acceptable. Granzyme B should preferentially be measured after 48-72 h of activation.

Cytokine mRNA expression profiles in lymphoid tissues of pigs naturally affected by postweaning multisystemic wasting syndrome.

Fifteen 8-week-old conventional pigs were selected from a farm where pigs were suffering from postweaning multisystemic wasting syndrome (PMWS). Ten of the animals were diseased pigs showing typical signs of PMWS (wasting and respiratory disorders) and positive for infection with porcine circovirus type 2 (PCV2), and the other five animals selected as controls were pen-mate, apparently healthy pigs. Blood samples and lymphoid tissues were taken from each animal for haematological, serological and histopathological studies. Also, cytokine mRNA expression of IL-1beta, IL-2, IL-4, IL-8, IL-10, IL-12p40 and IFN-gamma from inguinal and bronchial lymph nodes, tonsils, spleen and thymus was determined by semi-quantitative RT-PCR. Pigs suffering from PMWS showed severe alterations of haematological parameters such as anaemia, lymphopenia with decrease of CD8(+) and IgM(+) cells, monocytosis and neutrophilia. Also, extensive lymphocyte depletion and altered cytokine mRNA expression patterns were seen in most of the examined lymphoid organs. Those cytokine mRNA alterations were characterized by an overexpression of IL-10 mRNA in thymus and IFN-gamma mRNA in tonsils, and by decreases in the mRNA expression of several cytokines as IL-2 and IL-12p40 in the spleen, IL-4 in tonsils, and IFN-gamma, IL-10, IL-12p40 and IL-4 in inguinal lymph nodes. Also, the IL-10 mRNA overexpression was histologically associated with the thymic depletion and atrophy observed in PMWS pigs. In conclusion, the cytokine mRNA imbalance, specially the increased mRNA levels of IL-10 in the thymus, jointly with the histopathological and haematological disorders, are highly indicative of a T-cell immunosuppression, enhancing the notion that the immune system of PMWS-affected pigs is severely impaired.

Patterns of CD4/CD8 T-cell ratio in dialysis effluents predict the long-term outcome of peritonitis in patients undergoing peritoneal dialysis.

The peritoneal immune compartment is a microenvironment with a particular T-cell repertoire and susceptible to local inflammation. To clarify the role of T lymphocytes in peritoneal immunity, the changes in T-cell subpopulations in peritoneal dialysis effluents (PDEs), and their influence on the response to the treatment of peritonitis and on its prognosis were studied in patients undergoing long-term, continuous ambulatory peritoneal dialysis (CAPD). A cohort of 36 patients treated with CAPD and who had histories of peritonitis were divided into a group with rapid and a group with delayed response to antibiotics, and were followed for 3 years. CD4/CD8 T-cell ratios, T-cell cytokine mRNA expression patterns and transforming growth factor-beta1 (TGF-beta1) concentrations were examined in PDE during bouts of peritonitis. The change in 4 h D/P creatinine during the peritoneal equilibration test (PET) between year 0 and year 3 was expressed as deltaD/P creatinine. The serial changes in T-cell subsets in PDE during peritonitis showed two patterns: (i) pattern 1, manifest as a progressive increase in the CD4/CD8 ratio, and associated with a rapid response to treatment; and (ii) pattern 2, manifest as a progressive decrease in the CD4/CD8 ratio, and associated with a delayed response to treatment. The major T-cell phenotypes in PDE during peritonitis were Th1-CD4(+) and Tc2-CD8(+), determined by cloning techniques, RT-PCR and double immunofluorescence staining. TGF-beta1 in the effluent was undetectable in pattern 1 after 7-8 days, but remained detectable at 2 weeks in pattern 2. Pattern 2 patients had a significantly greater decrease (deltaD/P creatinine: -0.198+/-0.086) in solute transport than pattern 1 patients (deltaD/P creatinine: -0.036+/-0.077, P<0.05). These results suggest that a progressive decrease of the CD4/CD8 ratio in PDE correlates with a persistent expression of TGF-beta1, and plays a pathogenetic role in the evolution of peritonitis, PET deterioration and peritoneal fibrosis. Therefore, patterns of CD4/CD8 T-cell ratio in PDE may predict clinical outcomes of peritonitis in CAPD patients.

Plasma cytokines do not reflect expression of pro- and anti-inflammatory cytokine mRNA at organ level after cardiopulmonary bypass in neonatal pigs.

Plasma concentrations of inflammatory markers are increased in response to the trauma of cardiac surgery and cardiopulmonary bypass (CPB). It is, however, unknown whether the plasma cytokine levels and cytokine mRNA expression at organ level reflect each other. Twenty-six piglets (17-19 days) were allocated to the sham-group (sternotomy only, n = 13) or to the CPB-group (sternotomy, 120 min CPB procedure with 60-min aortic cross-clamp, n = 13). The pigs were observed for 0.5 h or 4 h post-CPB. Plasma levels of IL-1beta, IL-6, IL-8 and IL-10 and mRNA expression of TNF-alpha, IL-1beta, IL-6, IL-8, IL-10 and iNOS in organs were registered with concomitant changes in oxygenation index (OI) and expiratory nitric oxide (NO). In pigs killed 0.5 h post-CPB there was a significant increase in IL-10 mRNA in the lungs and kidneys compared with the sham-group. IL-1beta mRNA was detectable in the kidneys and lungs of the CPB-pigs, while IL-6 mRNA was up regulated only in lungs. In pigs killed 4 h post-CPB a significantly higher IL-6 mRNA was found in heart tissue and a lower IL-10 mRNA was found in lungs of CPB pigs compared with the sham-group. There was a concomitant significant increase in OI and increased plasma IL-8 and IL-10 concentrations in the CPB-pigs compared with the sham-pigs. The cytokine mRNA expression pattern was very different for the pigs killed already 0.5 h after the CPB procedure compared with the pigs killed 4 h post-CPB. The plasma cytokine levels poorly reflected mRNA expression of the pro- and anti-inflammatory cytokines.

Cytokine mRNA expression patterns in the disease course of female adolescents with anorexia nervosa

Anorexia nervosa (AN) is a serious eating disorder characterized by extreme weight loss and abnormalities of the neuroendocrine and immune systems. Cytokines have been discussed to be involved in the pathomechanisms underlying cachexia. Therefore our study aimed at examining the mRNA expression pattern of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6) and interleukin-10 (IL-10) in whole blood of 11 female AN patients and 10 age and sex matched normal weight control subjects using a sensitive quantitative polymerase chain reaction (PCR) method. We found a significant increase in TNF-α and IL-6 mRNA expression in anorectic patients at admission (mean BMI 14.8 ± 1.3) when compared to controls. During follow-up, the expression of TNF-α mRNA remained significantly higher in formerly anorectic patients (mean BMI 18.7 ± 0.5) while IL-6 mRNA expression decreased. We interpret our results as suggesting that TNF-α may contribute to metabolic abnormalities in anorexia nervosa even after goal BMI is achieved.

Synovial MMP-3 and TIMP-1 levels and their correlation with cytokine expression in canine rheumatoid arthritis

The purpose of this study was to investigate whether synovial fluid levels of matrix metalloprotease-3 (MMP-3) and tissue inhibitor of metalloprotease-1 (TIMP-1) are specifically elevated in canine rheumatoid arthritis (CRA) compared to osteoarthritic joint disorders and if these markers are correlated with a specific pattern of cytokine mRNA expression. Synovial fluid samples of 17 dogs with CRA were analysed for MMP-3 and TIMP-1 by two canine sandwich ELISA (enzyme-linked immunosorbent assay) systems. The synovial mRNA content of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), transforming growth factor-β (TGF-β), and interferon-γ (IFN-γ) was determined by RT-PCR (reverse transcription-polymerase chain reaction). Dogs with osteoarthritis ( n=50) caused by anterior cruciate ligament rupture (ACLR) were used as controls. A significant rise of MMP-3 was found in the synovial fluid of joints with CRA that could not be balanced by sufficient amounts of TIMP-1. The 30-fold surplus of MMP-3 over TIMP-1 was strongly correlated with the synovial mRNA content of IL-1, IL-12 and TGF-β. Our results point to the potential use of the synovial levels of MMP-3 as a marker for RA in dogs.