Higher Cytokine Concentrations Research Articles

Peritonitis in Childhood: Clinical Relevance of Cytokines in the Peritoneal Exudate

The aim of the study was to assess pro-inflammatory cytokine (tumor necrosis factor alpha [TNF-alpha], interleukin 6 [IL-6]) and anti-inflammatory cytokine (interleukin 10 [IL-10]) responses in children with peritonitis secondary to hollow viscus perforation, and to evaluate the influence of peritoneal microbial status on cytokine expression and morbidity. The peritoneal fluid of 13 children with perforated appendicitis was examined with qualitative analysis of bacteria, and measurement of cytokine levels, which were compared to cytokine plasma levels, over a five-day period following operation. All fluid specimens showed permanently elevated levels of TNF-alpha and IL-10. IL-6 tended to decrease to normal levels by the 5th postoperative day. Peritoneal exudate levels of TNF-alpha and IL-10 were more than 100 - 1000 times greater than those in plasma. The most common bacterial species isolated in the peritoneal fluid was Escherichia coli. Despite persisting high cytokine concentrations and bacterial load of the peritoneal cavity for 5 days postoperatively, the children recovered uneventfully and the systemic signs of infection disappeared rapidly. Neither the bacterial nor the pro-inflammatory cytokine load of the peritoneal cavity proved to be associated with the clinical course. We hypothesize that in peritonitis in childhood a significant and clinically relevant cytokine-mediated inflammatory response is compartmentalized in the peritoneal cavity. Therefore adjuvant surgical measures in addition to appendectomy and intraoperative debridement seem not to be necessary, at least for peritonitis due to perforated appendicitis in children.

Lung epithelial cells downmodulate neutrophil‐endothelial interactions in response to inflammatory stimuli

Endothelial cells (EC) are in close proximity to epithelial cells (EpC) in the lung. To mimic this in vitro, we established a bilayer culture model on Transwell filters to examine the interactions between EC and lung EpC during leukocyte adhesion/transendothelial migration. Human umbilical vein EC (HUVEC) were seeded on one surface of a filter with 0.4 μm pores, and alveolar epithelial-derived A549 cells were seeded on the other side of the same filter. Using various stimuli, including interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and killed E. coli, human neutrophil adhesion and transendothelial migration was diminished when HUVEC were grown adjacent to lung EpC compared with when grown alone. This was observed regardless of whether the endothelium was activated apically or basolaterally. Similarly, E-selectin upregulation on endothelium grown in bilayers was significantly less than that of endothelium grown alone with the same stimuli as above. However, these differences were more pronounced when the endothelium was activated basolaterally, suggesting that the epithelium mediates its protective effects both through its function as a physical barrier and via yet to be defined mechanisms. At high concentrations of cytokines, these differences were abrogated, suggesting that the epithelium loses its protective effect under conditions of intense inflammation. These findings indicate that lung EpC may be protective or may downmodulate endothelial activation for neutrophil recruitment during pulmonary inflammation and this protection is mediated by barrier-dependent and barrier-independent mechanisms. Supported by NSHRF, IWK Health Centre and CIHR

Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch

Pleiotropism is a hallmark of cytokines and growth factors; yet, the underlying mechanisms are not clearly understood. We have identified a motif in the granulocyte macrophage-colony-stimulating factor receptor composed of a tyrosine and a serine residue that functions as a binary switch for the independent regulation of multiple biological activities. Signalling occurs either through Ser585 at lower cytokine concentrations, leading to cell survival only, or through Tyr577 at higher cytokine concentrations, leading to cell survival as well as proliferation, differentiation or functional activation. The phosphorylation of Ser585 and Tyr577 is mutually exclusive and occurs via a unidirectional mechanism that involves protein kinase A and tyrosine kinases, respectively, and is deregulated in at least some leukemias. We have identified similar Tyr/Ser motifs in other cell surface receptors, suggesting that such signalling switches may play important roles in generating specificity and pleiotropy in other biological systems.

Heat-inducible amplifier vector for high-level expression of granulocyte-macrophage colony-stimulating factor

Purpose: In cytokine immunotherapy of cancer it is critical to deliver sufficiently high local cytokine concentrations in order to reach the therapeutic threshold needed for clinical efficacy. Simultaneously, for optimal clinical safety adverse effects caused by high systemic cytokine levels must be minimized. One of the most promising anti-cancer therapeutic cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), has elicited anti-tumour immune responses in animal studies and clinical trials. However, the clinical efficacy has been limited, with local GM-CSF levels being therapeutically insufficient and systemic toxicity being a limiting factor.Methods: To address these problems we have developed a novel GM-CSF expression vector, pAD-HotAmp-GM-CSF, which can provide high levels of GM-CSF expression, and induction of cytokine expression to limited tissue areas. This expression system combines inducible and amplifying elements in a single multi-genic construct. The first transcriptional unit contains the inducible element, the heat shock protein 70B (HSP70B) promoter that regulates expression of the transcription-activating factor tat.Results: Upon the binding of tat to the second promoter, the HIV2 long terminal repeat amplifies downstream gene expression of the therapeutic cytokine GM-CSF. Moderate hyperthermia at 42°C for 30 min induced GM-CSF expression in pAD-HotAmp-GM-CSF that was over 2.5- and 2.8-fold higher than levels reached with HSP70B promoter alone and the prototypical human cytomegalovirus promoter.Conclusions: Thus, the inducible amplifier vector, pAD-HotAmp-GM-CSF, represents a novel system for regulated and enhanced GM-CSF expression, which enables both greater efficacy and safety in cytokine immunotherapy of cancer.

Neutrophil Recruitment by Fetal Porcine Endothelial Cells: Implications in Scarless Fetal Wound Healing

Fetal dermal wounds heal scarlessly and with a minimal inflammatory response. When a robust inflammatory response is induced at the site of fetal dermal wounds by the application of cytokines, healing results in fibrosis. To test the hypothesis that the reduced inflammatory response in fetal wounds is due to impaired fetal leukocyte-endothelial interactions, the contributions of fetal endothelial cells to the inflammatory response in the fetus were investigated. Endothelial cells isolated from blood vessels of adult and mid-gestational fetal pigs were cultured in media until confluent monolayers were established. Adult porcine neutrophils were isolated and resuspended at a concentration of 1 million cells/mL. Interactions between neutrophils and endothelial cells were observed under static and flow conditions. Endothelial monolayers were exposed to neutrophils with and without prior stimulation of the endothelial cells with tumor necrosis factor alpha (TNF-alpha) for 4 h. The neutrophil-endothelial interactions were observed and analyzed for neutrophil adhesion, rolling velocity, and transmigration. Endothelial P-selectin mRNA expression was determined by real-time polymerase chain reaction (PCR). A novel in vitro model of fetal inflammation is described. Both adult and fetal endothelial cells demonstrated a dose-dependent increase in neutrophil adhesion and transmigration with increasing doses of TNF-alpha. The fetal response was significantly lower than the adult. As expected, rolling velocity was lower at higher cytokine concentrations and had an inverse correlation with P-selectin mRNA expression. Fetal endothelial cells are less permissive to adhesion and transmigration of neutrophils than adult endothelial cells. This may contribute to the paucity of inflammation seen in the fetal response to dermal injury.

Differential effect of imipenem treatment on wild-type and NK cell-deficient CD8 knockout mice during acute intra-abdominal injury

CD8 knockout mice depleted of natural killer (NK) cells by treatment with anti-asialoGM1 (CD8KO/alphaAsGM1 mice) are resistant to injury caused by cecal ligation and puncture (CLP). However, CLP-induced injury is complex. Potential sources of injury include bacterial dissemination, cecal ischemia, and translocation of bacterial toxins. We treated wild-type and CD8KO/alphaAsGM1 mice with imipenem after CLP to decrease bacterial dissemination. Additional mice were subjected to cecal ligation without puncture of the cecal wall or cecal ligation and removal of cecal contents. Imipenem treatment decreased bacterial counts by at least two orders of magnitude. However, all wild-type mice, whether treated with saline or imipenem, died by 42 h after CLP and exhibited significant hypothermia, metabolic acidosis, and high plasma cytokine concentrations. Wild-type mice subjected to cecal ligation without puncture also died, despite very low bacterial counts in blood, but wild-type mice subjected to cecal ligation and washout of cecal contents survived. In CD8KO/alphaAsGM1 mice subjected to CLP, imipenem treatment increased survival from 50% to 100%. After cecal ligation without puncture, long-term survival was 80-90% in CD8KO/alphaAsGM1 mice. Hypothermia, metabolic acidosis, and cytokine production were attenuated in CD8KO/alphaAsGM1 mice compared with wild-type controls. These results indicate that bacterial dissemination is not a major source of injury in wild-type mice after CLP, but the presence of gut flora in the cecal lumen is required for induction of systemic inflammation after cecal injury. CD8KO/alphaAsGM1 mice are resistant to the systemic manifestations of cecal injury.

Omega-3 fatty acids and anorexia

To review the mechanisms of action of omega-3 fatty acids and their role in the brain, as well as their therapeutic implications in anorexia. Recent studies have demonstrated that omega-3 fatty acids modulate changes in the concentrations and actions of several orexigenic and anorexigenic neuropeptides in the brain, including neuropeptide Y, alpha-melanocyte stimulating hormone and the neurotransmitters serotonin and dopamine. In patients with acute and chronic inflammatory conditions, low tissue concentrations of omega-3 fatty acids and high concentrations of proinflammatory cytokines are found, in association with anorexia and decreased food intake. The data suggest that omega-3 fatty acid supplementation suppresses proinflammatory cytokine production and improves food intake by normalizing hypothalamic orexigenic peptides and neurotransmitters. Based on current data, omega-3 fatty acid supplementation has a role in the treatment of anorexia by stimulating the production and release of orexigenic neurotransmitters in food intake regulatory nuclei in the hypothalamus.

Association of Osteoprotegerin With Human Abdominal Aortic Aneurysm Progression

Abdominal aortic aneurysm (AAA) is characterized by destruction of the arterial media associated with loss of vascular smooth muscle cells, infiltration of mononuclear cells, and high concentration of metalloproteinases (MMPs) and cytokines. Osteoprotegerin (OPG) has recently been identified in atherosclerosis. The presence and functional importance of OPG in human AAA was investigated. In 146 men with small AAA followed up by ultrasound for 3 years, serum OPG was weakly correlated with aneurysm growth rate. Western analysis showed 3-, 8-, and 12-fold-greater OPG concentrations in human AAA biopsies compared with biopsies of atherosclerotic narrowed aorta (1.4+/-0.1 versus 0.5+/-0.1 ng/mg tissue; P=0.002), postmortem nondiseased abdominal aorta (1.4+/-0.1 versus 0.2+/-0.1 ng/mg tissue; P<0.001), and nondiseased thoracic aorta (1.4+/-0.1 versus 0.1+/-0.06 ng/mg tissue; P<0.001). Healthy human aortic vascular smooth muscle cells incubated with recombinant human (rh)OPG (0 to 20 ng rhOPG/10(5) cells per 1 mL per 24 hours) developed an aneurysmal phenotype defined by impaired cell proliferation (P<0.001), increased apoptosis (P<0.01), and increased MMP-9 (92 kDa) expression (P<0.001). Incubation of monocytic THP-1 cells with 1 ng rhOPG/10(5) cells per 1 mL per 24 hours induced a 2-fold increase in MMP-9 expression (P<0.001), a 1.5-fold increase in MMP-2 activity (P=0.005), and a 2-fold stimulation of IL-6 production in these cells (P=0.02). Finally, secretion of OPG from human AAA explant was abrogated by treatment with the angiotensin II blocker irbesartan, with the reduction in secreted levels averaging 63.0+/-0.9 ng/mg tissue per 48-hour period. These findings support a role for OPG in the growth of human AAA and suggest a potential benefit for angiotensin II blockade in slowing aneurysm expansion.

368. Differential Sensitivity of Normal and CML-Derived CD34+Cells to Inhibition of SHP2, Gab2 and Stat5 Gene Expression by RNA Interference (RNAi)

RNA interference has rapidly become an efficient tool for functional genomics in a variety of organisms. Stable expression of shRNA driven by pol III promoters upon lentiviral gene transfer can induce long-term gene silencing in mammalian cells. We recently demonstrated that lentivirus-mediated anti bcr-abl RNAi can specifically silence bcr-abl gene expression, inhibit oncogene driven cell proliferation, and eradicate leukemic cells depending on the dose of lentivirus-mediated shRNA expression. However, since effective depletion requires a threshold of lentiviral integrations into target cell genomes, the risk of insertional mutagenesis may limit the therapeutic value of this approach. To search for new potential therapeutic targets in CML, we studied the function of several signaling molecules in purified normal and CML CD34+cells from chronic phase CML patients harvested at initial diagnosis. We selected SHP2, Gab2, and Stat5 based on their constitutive tyrosine phosphorylation in bcr-abl+ cell lines. Several shRNAs for each target gene were evaluated using a bicistronic EGFP-encoding reporter plasmid system as described earlier. Effective shRNA expression cassettes were cloned into lentiviral plasmids encoding RFP to track lentiviral transduction. Lentivirus-mediated RNAi targeting SHP2, Gab2, or Stat5 results in a reduction of mRNA and protein by more than 90 % and induces a rapid depletion of transduced bcr-abl+ and negative cell lines. In addition, RNAi against all three targets enhances imatinib induced depletion of bcr-abl+K562 cells. We next transduced normal and CML-derived primary CD34+cells with control and anti-SHP2, Gab2, and Stat5 lentiviruses, and analysed colony-formation of transduced, i.e. RFP+progenitor cells in methylcellulose cultures. To eliminate effects of different transduction rates we plated CD34+cells for each transfection in the presence of high (GM-CSF: 20 ng/ml; IL-3: 10 ng/ml) or low (GM-CSF: 0.2 ng/ml; IL-3: 0.1 ng/ml) cytokine concentrations indicating the functional relevance of the respective RNAi-target for CFU-colony formation. Whereas anti-SHP2, Gab2, and Stat5 RNAi did not reduce the proliferation of normal transduced CFU (n=5), proliferation of transduced CFU from CML patients was specifically reduced between 50 to 85 % under low cytokine concentration (n=9-13). Furthermore, co-expression of both anti-bcr-abl and anti-SHP2 shRNA from a single lentiviral vector strongly cooperates to inhibit colony formation of CML-progenitors. This study establishes lentivirus-mediated RNAi as a valuable tool for functional genomics in primary hematopoietic cells and demonstrates that normal CFUs are more resistant to inhibition of SHP2, Gab2, and Stat5 gene expression than CML-progenitors. Furthermore, the cooperative effects of RNAi targeting different genes simultaneously and the combined effects of protein and gene expression antagonists suggest that co-treatment with a leukemia-specific and a non-specific inhibitor may harbor significant therapeutic potential in CML.

Role of cytokines in cardiovascular diseases: a focus on endothelial responses to inflammation

Complex cellular and inflammatory interactions are involved in the progress of vascular diseases. Endothelial cells, upon exposure to cytokines, undergo profound alterations of function that involve gene expression and de novo protein synthesis. The functional reprogramming of endothelial cells by cytokines is of importance especially in patients with chronic vascular inflammation. The intercellular network of dendritic cells, T-lymphocytes, macrophages and smooth muscle cells generates a variety of stimulatory cytokines [e.g. TNF-alpha (tumour necrosis factor-alpha), IL (interleukin)-1, IL-6 and IFN-gamma (interferon-gamma)] and growth factors that promote the development of functional and structural vascular changes. High concentrations of proinflammatory cytokines increase oxidative stress, down-regulate eNOS (endothelial nitric oxide synthase) bioactivity and induce endothelial cell apoptosis. Chemoattractant cytokines [e.g. VEGF (vascular endothelial growth factor), TGF-beta1 (transforming growth factor-beta1) and IL-8] are important regulators of inflammation-induced angiogenesis and are directly modulated by nitric oxide. This review will focus on the vascular mechanisms orchestrated by cytokines and summarizes the current knowledge concerning the contribution of cytokines to cardiovascular diseases.

Activation of Virus-Specific CD8+ T Cells by Lipopolysaccharide-Induced IL-12 and IL-18

Virus-specific T cells represent a hallmark of Ag-specific, adaptive immunity. However, some T cells also demonstrate innate functions, including non-Ag-specific IFN-gamma production in response to microbial products such as LPS or exposure to IL-12 and/or IL-18. In these studies we examined LPS-induced cytokine responses of CD8(+) T cells directly ex vivo. Following acute viral infection, 70-80% of virus-specific T cells will produce IFN-gamma after exposure to LPS-induced cytokines, and neutralization experiments indicate that this is mediated almost entirely through production of IL-12 and IL-18. Different combinations of these cytokines revealed that IL-12 decreases the threshold of T cell activation by IL-18, presenting a new perspective on IL-12/IL-18 synergy. Moreover, memory T cells demonstrate high IL-18R expression and respond effectively to the combination of IL-12 and IL-18, but cannot respond to IL-18 alone, even at high cytokine concentrations. This demonstrates that the synergy between IL-12 and IL-18 in triggering IFN-gamma production by memory T cells is not simply due to up-regulation of the surface receptor for IL-18, as shown previously with naive T cells. Together, these studies indicate how virus-specific T cells are able to bridge the gap between innate and adaptive immunity during unrelated microbial infections, while attempting to protect the host from cytokine-induced immunopathology and endotoxic shock.

Differential Sensitivity of Normal and CML-Derived CD34+ Cells to Inhibition of SHP-2 Gene Expression by RNA Interference (RNAi).

RNA interference (RNAi) has rapidly evolved into an efficient tool for functional genomics in a variety of organisms. Stable expression of shRNA (short hairpin RNA) driven by pol III promoters upon retro- or lentiviral gene transfer can induce long-term gene silencing in mammalian cells, including human hematopoietic cells. We recently demonstrated that lentivirus mediated anti bcr-abl RNAi can specifically silence bcr-abl gene expression, inhibit oncogene driven cell proliferation, and eradicate leukemic cells depending on the dose of lentivirus-mediated shRNA expression (Scherr et al. Gene Therapy 2004). Since effective depletion requires a threshold of lentiviral integrations into target cell genomes, the risk of insertional mutagenesis may limit the therapeutic value of this approach. We therefore applied lentivirus-mediated RNAi for functional genomics in purified primary normal and CD34+ cells from chronic phase CML patients harvested at initial diagnosis. Several SHP-2 shRNAs were generated according to established rules and were functionally evaluated using a bicistronic reporter system as described earlier. Effective shRNA expression cassettes were subsequently cloned into lentiviral plasmids encoding RFP to track lentiviral transduction. Transduction of K562, U937, NB-4 and TF-1 cells with lentiviral supernatants results in a reduction of SHP-2 mRNA and protein by more than 90 %. Interestingly, anti-SHP-2 shRNA induced almost complete depletion of RFP+ cells in all four cell lines, demonstrating that SHP-2 expression is essential for proliferation and survival in these cells. We next transduced normal and CML-derived CD34+ cells with a puritiy of > 95% with control and anti-SHP-2 lentiviruses, and stimulated methylcellulose cultures of the cells with high (GM-CSF: 20 ng/ml; IL-3: 10 ng/ml) or low (GM-CSF: 0.2 ng/ml; IL-3: 0.1 ng/ml) cytokine concentrations. This assay relies on the fact that colony formation of CML-CFU is mediated by both cytokine receptor and bcr-abl signaling. Therefore differential numbers of transduced, i.e. RFP+ colonies under different cytokine stimulations reflect the role of the RNAi-target in normal or malignant CFU. Whereas anti-SHP-2 RNAi did not reduce the proliferation of normal transduced CFU (n=5), proliferation of CFU from CML patients was specifically reduced between 50 to 85 % under low cytokine concentration (n= 9). These data suggest that primary normal cells are more resistant to inhibition of SHP-2 gene expression than leukemic cell lines and CD34+ cells from CML patients and identify SHP-2 as a potential target for anti bcr-abl therapy.

Prestorage leucocyte filtration of blood: effects on cytokine generation and complement activation

Each unit of whole blood normally is separated into several components before storage and transfusion. Blood contains several complex cascade systems and biologically active substances that can be activated during the processing of blood components and subsequent storage. With a transfusion, cellular components of blood and coagulation factors are transferred as desired and required. However, inflammatory mediators that may be responsible for adverse transfusion reactions are also transferred. High concentrations of cytokines, released from residual leucocytes, appear to be the cause of most febrile non-haemolytic transfusion reactions to platelet concentrates. To reduce adverse transfusion reactions, an increasing number of blood banks are introducing reduction of leucocytes in blood components before storage. Prestorage leucocyte filtration of platelet concentrates and red cells diminishes the accumulation of leucocyte-derived cytokines during storage but transfusion reactions are not eliminated. The prestorage leucoreduction of plasma has been shown to activate the complement cascade from the very beginning of storage, and complement activation and accumulation may explain some of the remaining transfusion reactions. Many severe conditions require transfusion of blood components. The clinical significance of inflammatory mediators in transfused blood products remains unclear, but it is speculated that they may have an additive and detrimental effect on the complex activated immune network in severe conditions, especially following massive transfusion.

IL-10 plasma and bronchoalveolar lavage levels used as markers of candida bronchopneumonia during fungal sepsis

Nosocomial bloodstream fungal infections by Candida species in ICU has risen dramatically in the past two decades. In fact, fungal infections in the critically ill patient are difficult to diagnose and are associated with a high mortality rate. The high concentration of some proinflammatory cytokines registered in the serum and in bronchoalveolar lavage (BAL) of patients with severe bronchopneumonia and the different kind of host response during fungal infections are well defined. Our aim is to correlate the IL-10 serum and BAL levels in critically ill patients with confirmed bronchopneumonia associated with candidiasis or bacterial sepsis and to research whether these levels consent to prove the fungal origin of severe pneumonia associated with severe sepsis.

Second-Trimester Maternal Serum Placental Growth Factor and Vascular Endothelial Growth Factor for Predicting Severe, Early-Onset Preeclampsia

In Brief OBJECTIVE To determine whether alterations in second-trimester maternal serum cytokine concentrations can identify women at risk for developing severe, early-onset preeclampsia. METHODS Patients with severe preeclampsia requiring delivery prior to 34 weeks (n = 20) were each matched by gestational age, gravidity, parity, and sample freezing time with three healthy controls who delivered at term (n = 60). By using second-trimester maternal sera originally collected for fetal aneuploidy screening, the concentrations of placental growth factor, vascular endothelial growth factor, granulocyte colony-stimulating factor, endothelin-1, and human chorionic gonadotropin were compared between patients and controls. Logistic regression analysis was used to estimate odds ratios for high versus low (median split) cytokine concentrations with respect to the development of severe, early-onset preeclampsia. Receiver operating characteristic (ROC) curves based on a second logistic regression, using actual cytokine values, were plotted to illustrate reciprocal impact on sensitivity and specificity. RESULTS Placental growth factor and vascular endothelial growth factor levels were significantly lower in patients than in controls. No significant differences were observed for the other cytokines. The odds ratios (with 95% confidence intervals) were 15.54 (3.29, 73.40) for vascular endothelial growth factor and 4.20 (1.35, 13.06) for placental growth factor. Receiver operating characteristic analysis of placental growth factor and vascular endothelial growth factor confirmed that both were useful in discriminating between patients and controls. Models combining both vascular endothelial growth factor and placental growth factor provided the best performance for identifying patients at risk for developing severe, early-onset preeclampsia, according to both odds ratios and ROC analyses. CONCLUSION Combined analysis of placental growth factor and vascular endothelial growth factor is potentially useful as a tool for early identification of patients at risk for developing severe, early-onset preeclampsia. Combined analysis of second-trimester maternal serum placental growth factor and vascular endothelial growth factor levels can identify patients at risk for developing severe, early-onset preeclampsia.

Second-trimester maternal serum placental growth factor and vascular endothelial growth factor for predicting severe, early-onset preeclampsia.

To determine whether alterations in second-trimester maternal serum cytokine concentrations can identify women at risk for developing severe, early-onset preeclampsia. Patients with severe preeclampsia requiring delivery prior to 34 weeks (n = 20) were each matched by gestational age, gravidity, parity, and sample freezing time with three healthy controls who delivered at term (n = 60). By using second-trimester maternal sera originally collected for fetal aneuploidy screening, the concentrations of placental growth factor, vascular endothelial growth factor, granulocyte colony-stimulating factor, endothelin-1, and human chorionic gonadotropin were compared between patients and controls. Logistic regression analysis was used to estimate odds ratios for high versus low (median split) cytokine concentrations with respect to the development of severe, early-onset preeclampsia. Receiver operating characteristic (ROC) curves based on a second logistic regression, using actual cytokine values, were plotted to illustrate reciprocal impact on sensitivity and specificity. Placental growth factor and vascular endothelial growth factor levels were significantly lower in patients than in controls. No significant differences were observed for the other cytokines. The odds ratios (with 95% confidence intervals) were 15.54 (3.29, 73.40) for vascular endothelial growth factor and 4.20 (1.35, 13.06) for placental growth factor. Receiver operating characteristic analysis of placental growth factor and vascular endothelial growth factor confirmed that both were useful in discriminating between patients and controls. Models combining both vascular endothelial growth factor and placental growth factor provided the best performance for identifying patients at risk for developing severe, early-onset preeclampsia, according to both odds ratios and ROC analyses. Combined analysis of placental growth factor and vascular endothelial growth factor is potentially useful as a tool for early identification of patients at risk for developing severe, early-onset preeclampsia.

High-level expression of interleukin-4 following electroporation-mediated gene transfer accelerates Type 1 diabetes in NOD mice

Nonobese diabetic (NOD) mice develop T cell-dependent autoimmune disease. Administration of interleukin-4 (IL-4), one of the T helper 2 (Th2) cytokines, is reported to prevent either insulitis or diabetes or both in NOD mice. We examined the effect of transferring an IL-4-expressing plasmid vector into muscle by in vivo electroporation on the progression of diabetes in NOD mice. Plasmid DNA expressing murine IL-4 (pCAGGS-IL-4) was introduced into the muscles of 4- and 6-week-old female NOD mice using an in vivo electroporation technique we developed previously. The serum IL-4 levels reached 2000–8000 pg/ml 3 days after the delivery of pCAGGS-IL-4 and remained detectable (>5 pg/ml) for over 4 weeks. In contrast to the previous reports, 88% of the mice treated with pCAGGS-IL-4 developed overt diabetes by 30 weeks of age, while only 25% of nontreated mice and 19% of the mice treated with control pCAGGS developed overt diabetes by then ( p<0.01). Therefore, highly expressed IL-4 introduced by in vivo electroporation may have caused a Th1 shift, resulting in the promotion of diabetes in NOD mice. The high serum concentration of cytokines attained by our method is likely to unveil previously unknown cytokine functions.

Antenatal inflammation and infection in chronic lung disease of prematurity.

In spite of improved neonatal care, chronic lung disease of prematurity (CLD) remains a major cause of morbidity and mortality in extremely preterm infants. Our current understanding is that antenatal infection can trigger intra-uterine inflammation which then promotes preterm labour. Recent studies suggest that antenatal infection and inflammation can also increase the preterm infant's susceptibility to develop CLD. It may be that exposure of the fetal lung to high concentrations of pro-inflammatory cytokines is the cause of this increased susceptibility. One candidate for initiating intra-uterine inflammation is ascending infection by the vaginal commensal Ureaplasma urealyticum (Uu). Antibiotics administered to mothers prior to delivery appear to improve the neonatal outcome in cases of preterm prolonged rupture of membranes, but not in cases of preterm labour with intact membranes. Uu can be transmitted vertically to the airways of the preterm infant, but the role of Uu in causing CLD remains uncertain. Small trials of antibiotics given to preterm infants after delivery have not shown any consistent benefit in reducing CLD. Although CLD remains a significant problem for the extremely preterm infant, it is likely that molecular biology techniques, such as the polymerase chain reaction, will enhance the detection of antenatal infection and further our understanding of the pathogenesis of CLD.

Prolonged inflammatory response to acute Pseudomonas challenge in interleukin-10 knockout mice.

Cystic fibrosis (CF) lung disease is characterized by a neutrophilic infiltrate that is excessive relative to the burden of infection. Decreased interleukin-10 in CF airways may impair proper termination of inflammation, leading to persistence of neutrophils after acute infections have been cleared. This could explain reports of lung inflammation in the absence of bacteria in infants with CF. We evaluated the kinetics of inflammation after transient Pseudomonas aeruginosa challenge in IL-10 knockout (KO) and wild-type (WT) mice. Both types of mice cleared the infection by Day 6 (p > or = 0.29). However, IL-10 KO mice had more neutrophils in bronchoalveolar lavage fluid than did WT mice on Days 4 (p < 0.0001), 6 (p < 0.0001), and 8 (p = 0.042). IL-10 KO mice had high concentrations of proinflammatory cytokines in BAL on Days 2 and 4, with some cytokines detectable on Days 6 and 8, whereas cytokines in BAL from WT mice were greatest on Day 2 and undetectable by Day 4. Moreover, IL-10 KO mice failed to regenerate IkappaBalpha once degraded and subsequently had prolonged activation of NF-kappaB. These data suggest that IL-10 deficiency contributes to prolonged inflammatory responses early in CF, when infection may be transient.

Embryotoxicity of peritoneal fluid in women with endometriosis. Its relation with cytokines and lymphocyte populations.

The goals of the present work were to study the embryotoxic effects of peritoneal fluid (PF) in women with or without endometriosis, and to relate any embryotoxicity to the severity of endometriosis, infertility or achievement of pregnancy, cytokine concentrations and lymphocyte populations. Sixty-six consecutive women of reproductive age, 54 with endometriosis (21 infertile) and 12 infertile without endometriosis, and another 12 fertile women as control group, were included in this study. They all underwent laparoscopy or laparotomy in the second half of the cycle, and PF was collected from the pouch of Douglas. The embryotoxicity of the PF was assessed by means of a mouse embryo assay, and expressed as the number of embryos that did not reach blastocyst stage. Cytokines and lymphocyte populations present in PF were also studied and correlated with embryotoxicity. PF embryotoxicity was increased in women with endometriosis, but there was little correlation with the severity of the disease. However, although a clear relationship to the presence of infertility was not found, embryotoxicity appeared to be lower in those infertile patients with endometriosis who later became pregnant. We found a significant increase in embryotoxicity in the presence of high cytokine concentrations, especially with interleukin-6, and less so with interleukin-8 (P < 0.05). No good correlation was observed with lymphocyte populations, but CD56 (NK) cells were significantly increased in the PF of women with endometriosis. In general, the correlations for embryotoxicity were better when PF was diluted at 20% (91.4 +/- 17 versus 68.1 +/- 31, P < 0.01). These results suggest that alteration in the production of cytokines in the PF, especially IL-6, besides contributing to the endometriosis and its evolution, probably increases embryotoxicity. However, no correlation was found between the latter and associated infertility.