[Magnetic resonance imaging manifestation of foot injury in amateur half marathon athletes].

Eighty-four amateur half marathon athletes (168 side feet) in Beijing from October 2018 to May 2021 were recruited, and their age, gender and whether they have foot pain were collected, including 44 males and 40 females, aged from 21 to 60 (40.7±9.3) years. All participants underwent bipedal magnetic resonance imaging (MRI) examinations, and the degree of foot pain was graded by foot ankle injury scale (FASS scale). The relationship between MRI features and the foot pain of amateur half marathon athletes were analyzed. The study found that the proportion of foot pain symptoms among amateur half marathon athletes in Beijing was high(122/168), and the MRI manifestations were mainly heel tendinitis and plantar fasciitis, which accounted for about 59.5% of all cases.

Genetic determinants of the defense response of resistant and susceptible pepper (Capsicum annuum) cultivars infected with Phytophthora capsici (Oomycetes; Pythiaceae).

Based on culture isolation and morphological observation blight-infected pepper plants in Shaanxi Province, China, we identified the pathogen causing pepper phytophthora blight as Phytophthora capsici. Varieties that differed in resistance (CM334, PBC602, and B27) were inoculated with this pathogen. The root activity of resistant CM334 variety was the highest while that of susceptible B27 variety was the lowest. Also, significant differences in the activity of POD, PAL, and ß-1,3-glucanase were found; there was a positive correlation between disease resistance and activity of these three enzymes. We inhibited mycelial growth and sporangia formation of P. capsici using crude ß-1,3-glucanase and PAL enzymes isolated from the resistant variety CM334 after it had been inoculated with P. capsici. These two enzymes had a synergistic effect on inhibition of P. capsici mycelial growth and sporangia formation. Expression of the defensive genes CaPO1, CaBGLU, CaBPR1, and CaRGA in the three varieties was higher in the leaves than in the roots. All three genes were upregulated in infected leaves and roots of the pepper plants, always expressing at higher levels in the resistant cultivar than in the susceptible cultivar, suggesting that the differences in resistance among the pepper genotypes involve differences in the timing and magnitude of the defense response.

Optimization of virus-induced gene silencing in pepper (Capsicum annuum L.).

Virus-induced gene silencing is currently a powerful tool for the study of gene function in plants. Here, we optimized the protocol for virus-induced gene silencing, and investigated factors that affect the efficiency of tobacco rattle virus-induced gene silencing in pepper plants. Consequently, an optimal protocol was obtained by the syringe-infiltration method in the leaves of pepper plants. The protocol involves 2-leaf stage plants, preparing the Agrobacterium inoculum at a final OD600 of 1.0 and then growing the inoculated plants at 22°C. Using this protocol, we achieved high efficiency in silencing CaPDS in different cultivars of pepper plants. We further used the CaPOD gene to illustrate the general reliability of this optimized protocol. Viral symptoms were observed on the leaves of inoculated plants of the Early Calwonder cultivar 25 days post-inoculation, indicating that this protocol can also be used to silence other genes in pepper plants. Real-time polymerase chain reaction analyses revealed that the expression levels of CaPDS and CaPOD were dramatically reduced in inoculated leaves compared to control plants. These results demonstrate that the optimized protocol can be applied to functional genomic studies in pepper to investigate genes involved in a wide range of biological processes.

Experimental study on angiogenesis in a rabbit VX2 early liver tumour by perfusion computed tomography.

Ten rabbits implanted with VX2 liver tumours were investigated by perfusion computed tomography (PCT) imaging 1 week (early) and 2 weeks (late) after tumour induction; 10 other rabbits were non-implanted controls. Time-density curves, perfusion parametric maps and perfusion parameters were obtained for tumour rim and normal tissue surrounding the tumour, and for liver tissue from the controls. In addition, microvessel density (MVD) and vascular endothelial growth factor (VEGF) were studied by immunohistochemistry 2 weeks after tumour implantation. A deconvolution mathematical model was used to calculate hepatic blood flow (HBF), hepatic blood volume (HBV), mean transit time (MTT), capillary vessel surface permeability (PS) and hepatic arterial index (HAI). At the tumour rim on the early PCT scan, MTT was significantly lower whereas HBF, HBV, HAI and PS were significantly higher than in surrounding normal tissue. There were no significant changes in perfusion parameters on the late PCT scan compared with the early scan. Significant linear correlations of MVD and VEGF were found with HBF, PS and HAI, but not with HBV or MTT. It is concluded that PCT imaging is useful for the evaluation of tumour angiogenesis and for the early detection of liver tumours.

The synthetic liver X receptor agonist GW3965 reduces tissue factor production and inflammatory responses in human islets in vitro.

AIMS/HYPOTHESIS: Optimising islet culture conditions may be one strategy for reducing islet loss prior to, and immediately after, islet transplantation. Liver X receptor (LXR) agonism has previously been shown to increase insulin release from pancreatic islets and reduce inflammation in leucocytes. Our aim was to investigate whether the synthetic LXR agonist GW3965 could modulate the inflammatory status of human pancreatic islets. METHODS: Levels of pro-inflammatory cytokines and tissue factor in isolated human islets were determined by TaqMan low density array and/or real-time quantitative RT-PCR (mRNA levels) and enzyme immunoassay (EIA) (protein levels). Islet viability was measured using intracellular ATP content, ADP/ATP ratio, mitochondrial dehydrogenase activity (XTT assay) and insulin secretion in a dynamic glucose-challenge model. Apoptosis was determined by EIA measurement of histone-DNA complexes present in cytoplasm and by assaying caspase-3/-7 activity. RESULTS: Treatment of LPS-stimulated human islets with the synthetic LXR agonist GW3965 (1 micromol/l) for 24 h reduced mRNA and protein levels of selected pro-inflammatory cytokines (IL-8, monocyte chemotactic protein-1 and tissue factor). Moreover, GW3965 had no adverse effect on insulin secretion, islet viability or apoptosis. No excess of lipid accumulation could be detected with the dosage and exposure time used. CONCLUSIONS/INTERPRETATION: LXR activation suppresses inflammation in human islets in vitro without adverse effects on islet viability. Short-term moderate activation of LXR prior to islet transplantation may represent a possible strategy for improving post-transplant islet survival.

Oral and systemic administration of beta-glucan protects against lipopolysaccharide-induced shock and organ injury in rats.

beta-Glucans are glucose polymers with a variety of stimulatory effects on the immune system. The objective of this study was to determine the effect of prophylactic oral administration of soluble Saccharomyces cerevisiae-derived beta-1,3/1,6-glucan (SBG) on the outcome of experimental endotoxaemia and shock-associated organ injury. Male Wistar rats were pretreated with SBG orally (SBGpo, 20 mg/kg/day) for 14 days, subcutaneously (SBGsc, 2 mg/kg/day) for 3 days, or vehicle (placebo). Rats were anaesthetized and subjected to endotoxaemia by intravenous infusion of Escherichia coli lipopolysaccharide (LPS) (6 mg/kg) or saline infusion (sham). We observed significant levels of plasma beta-glucan in the SBGpo group (P<0 x 5), although the SBGsc group had levels approximately 40-fold higher despite a 10-fold lower dose. SBG prophylaxis caused enhanced blood pressure recovery following LPS-induced blood pressure collapse. Oral treatment with SBG attenuated the LPS-induced rise in plasma creatinine levels (P<0 x 05), indicating protection against renal injury. SBG also attenuated the plasma levels of aspartate aminotransferase and alanine aminotransferase (SBGpo, P<0 x 01; SBGsc, P<0 x 01), indicating protection against LPS-induced hepatic injury. A moderate increase in baseline interleukin (IL)-1beta levels was observed in the SBGsc group (P< 0 x 05). In the LPS-challenged rats, plasma levels of proinflammatory cytokines was moderately reduced in both SBG-treated groups compared to placebo. SBG treatment, particularly oral administration, had a striking effect on the haemodynamics of LPS-treated rats, although only a minute fraction of the orally administered beta-glucan translocated to the circulation. Enhanced organ perfusion may thus be responsible for the attenuated levels of indicators of kidney and liver injury seen in SBG-treated rats.

Cytokine responses to CpG DNA in human leukocytes.

Previous studies have implicated a role of bacterial DNA, containing unmethylated cytosine-phosphate-guanosine (CpG) motifs, in the initiation of systemic inflammation. This is based on the ability of CpG-DNA to act in synergy with lipopolysaccharide (LPS) to trigger tumor necrosis factor alpha (TNFalpha) production in murine monocytes and to enhance LPS toxicity in rodents. In this study we investigated the capacity of CpG-DNA to trigger and modulate cytokine responses in human leukocytes. A human blood assay, as well as isolated cultures of monocytes and neutrophils, was exposed to the synthetic oligodeoxynucleotides (ODNs) CpG ODN (2006) and GpC ODN (2006-GC), alone or in combination with peptidoglycan or LPS. Plasma or supernatants were isolated and analyzed for TNFalpha, interleukin-1 beta (IL-1beta), IL-6 and IL-8 by ELISA. In the blood, 2006 (but not 2006-GC) induced the release of TNFalpha (P < 0.05) and possibly IL-1beta and IL-6. IL-8 was induced in a CpG-independent manner. When co-administered with peptidoglycan, both ODNs enhanced the release of cytokines, but not consistently CpG dependent. When co-administered with LPS, only IL-8 values were enhanced, whereas IL-6 was suppressed at early time points. In monocyte and neutrophil cultures, CpG dependent induction of cytokine release was not observed. However, both ODNs inhibited LPS-induced IL-6. In conclusion, the capacity of CpG DNA to trigger the release of TNFalpha and to enhance LPS-induced release of this cytokine is confirmed in human whole blood, but not in adherent human monocytes. Most effects of the ODNs on cytokine release in human leukocytes were CpG independent.

Anti-inflammatory properties of enamel matrix derivative in human blood.

BACKGROUND AND OBJECTIVE: Enamel matrix derivative (EMD), extracted from porcine tooth buds, has been shown to promote periodontal healing in patients with severe periodontitis. This involves modulation of the inflammatory response followed by the onset of periodontal regeneration. Based on these observations, we examined the ability of EMD to modulate the release of a pro-inflammatory cytokine [tumor necrosis factor (TNF)-alpha], an anti-inflammatory cytokine (interleukin-10) and a chemokine (interleukin- 8) in whole human blood challenged by bacterial cell wall components. MATERIAL AND METHODS: Whole blood from healthy donors was challenged by lipopolysaccharide or peptidoglycan and incubated with different concentrations of EMD or a cAMP analogue 8-(4-chlorophenyl)thio-cAMP (8-CPT-cAMP). TNF-alpha, interleukin-8 and interleukin-10 were analysed from plasma by enzyme-linked immunosorbent assay (ELISA) while cAMP levels of peripheral blood mononuclear cell lysates were analysed by enzyme immunoassay (EIA). RESULTS: We found that EMD attenuated the release of TNF-alpha and interleukin-8 in whole blood from healthy donors challenged by lipopolysaccharide or peptidoglycan, while the release of interleukin-10 was unchanged. Enamel matrix derivative also produced a four-fold increase in the cAMP levels of peripheral blood mononuclear cell lysates. Like EMD, 8-CPT-cAMP attenuated the formation of TNF-alpha, but not of interleukin-10, in blood challenged by lipopolysaccharide. CONCLUSION: Enamel matrix derivative limits the release of pro-inflammatory cytokines induced by lipopolysaccharide or peptidoglycan in human blood, suggesting that it has anti-inflammatory properties. We propose that this effect of EMD is, at least partly, secondary to an increase in the intracellular levels of cAMP in peripheral blood mononuclear cells.

Reduced capacity of whole blood leucocytes to express tumour necrosis factor-alpha and interleukin-10 following major orthopaedic surgery.

BACKGROUND: Severe trauma is a challenge to the immune response and may cause reduced immune capacity. As a marker of decreased cellular activity, studies with ex vivo lipopolysaccharide (LPS) stimulation of whole blood or isolated mononuclear cells from injured patients have revealed reduced production of inflammatory cytokines. To gain further insight into immune alterations in orthopaedic surgery, we studied LPS-induced tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 in whole blood of patients during peri- and postoperative phases of total hip replacement. METHODS: Four females and 3 males undergoing elective total hip replacement were included in the study. Ex vivo LPS-induced TNF-alpha and IL-10 were measured in a whole blood assay before, during and at 1 and 6 days after operation. In addition, the counts of white blood cells were determined. RESULTS: During the operation, there were significant reductions in the number of monocytes, but at day 1 and 6 after surgery, there were significant increases as compared to the levels before surgery. The capacity of whole blood to express TNF-alpha and IL-10 did not change significantly during the operation and the following postoperative day. At day 6, however, there were significant reductions in expression of both TNF-alpha and IL-10 as compared to the levels before the operation. In relation to the values of monocytes, there was a significant reduction in the expression of TNF-alpha also at day 1 after operation. CONCLUSION: Our data indicate that in the course of at least 6 days after a major orthopaedic trauma, there is suppression of the whole blood capacity to express the inflammatory cytokine TNF-alpha and the anti-inflammatory cytokine IL-10 when exposed to LPS. During this time, then, the patient is particular susceptible to septic complications.

Peptidoglycan of Staphylococcus aureus upregulates monocyte expression of CD14, Toll-like receptor 2 (TLR2), and TLR4 in human blood: possible implications for priming of lipopolysaccharide signaling.

Previous studies have indicated that peptidoglycan (PepG) from gram-positive bacteria can exert a priming effect on the innate immune response to lipopolysaccharide (LPS) from gram-negative bacteria. Here, we hypothesized that this priming effect may be preceded by enhanced expression of monocyte CD14, Toll-like receptor 2 (TLR2), and TLR4. In an ex vivo whole human blood model, we observed a substantial synergy between LPS and PepG in the release of tumor necrosis factor alpha and interleukin-1beta (IL-1beta) over the 24-h experimental period, whereas the effect on IL-8 and IL-10 release was more time dependent. The priming effect of PepG on cytokine release was preceded by a rapid upregulation of CD14, TLR2, and TLR4 expression on monocytes: at 3 hours there was a twofold increase in CD14 expression (P < 0.03), a fivefold increase in TLR2 expression (P < 0.03), and a twofold increase in TLR4 expression (P < 0.03). CD14 and TLR2 remained upregulated throughout the experimental period following exposure to PepG (P < 0.05). Only a transient upregulation of these monocyte receptors was observed following treatment with LPS or LPS plus PepG. In conclusion, the synergistic effect of LPS and PepG on cytokine release is preceded by a reciprocal upregulation of TLR2 and TLR4 by both bacterial cell wall components.

Cytokine responses to fungal pathogens in Kupffer Cells are Toll-like receptor 4 independent and mediated by tyrosine kinases.

Disseminated fungal infections are increasing. However, the interactions between the body's largest population of tissue macrophages, the Kupffer cells and the fungal pathogens are scarcely understood. The aim of this study was to examine the involvement of Toll-like receptor 4 (TLR4) signalling in cytokine production, using primary cultures of rat and murine Kupffer cells exposed to Aspergillus fumigatus and Candida albicans hyphae and conidia. All fungal components induced the release of tumour necrosis factor-alpha (TNF-alpha), but with delayed kinetics compared with lipopolysaccharide (LPS). Candida albicans was the most potent inducer of TNF-alpha protein and mRNA and the only inducer of interleukin-10 (IL-10) in rat Kupffer cells. All fungal components induced enhanced mRNA levels of macrophage inhibitory protein-2 (MIP-2) in the cells, similar to LPS. Inhibitors of Src tyrosine kinases added to cells prior to stimulation led to attenuation in the release of both TNF-alpha (60%, P < 0.05) and IL-10 (70%, P < 0.05) induced by C. albicans conidia but did not influence the LPS-mediated cytokine release. Murine Kupffer cells (C57BL/10J) also released TNF-alpha as well as the chemokines keratinocyte-derived chemokine (KC) and MIP-2 in response to fungal component. Surprisingly, Kupffer cells from TLR4-deficient C57BL/ScCr mice exhibited significantly enhanced production of KC and MIP-2 upon stimulation by fungal components compared with control littermates (P < 0.05). Our study demonstrates that Aspergillus and Candida components induce cytokine production in rat Kupffer cells and that the response to C. albicans conidia involves Src tyrosine kinases. The experiments with TLR4-deficient Kupffer cells suggest that the cytokine response in these cells to fungal component is not mediated by TLR4.

Peptidoglycan primes for LPS-induced release of proinflammatory cytokines in whole human blood.

The pathophysiological mechanisms involved in mixed bacterial infections caused by gram-positive and gram-negative bacteria are largely unknown. The present study examines the potential interaction between lipopolysaccharide (LPS) and peptidoglycan (PepG) in the induction of the sepsis-associated cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-10 in whole human blood. Plasma values of these cytokines were measured by enzyme immunoassays and a TNF bioassay. Co-administration of PepG (10 microg/mL) or muramyl dipeptide (MDP, 1 microg/mL) with LPS (10 ng/mL) caused significantly elevated values of TNF-alpha and IL-6 in the blood that could not be obtained by the sum of the values obtained by each stimulant alone, or by 3-fold higher doses of either bacterial component alone. This phenomenon was observed 1 h after stimulation, throughout the experimental period (24 h), and with different doses of LPS and PepG. In contrast, the release of IL-10 was not influenced by the co-administration of PepG or MDP with LPS. The TNF-alpha release induced by co-administration of LPS and PepG was abrogated after pretreatment with a monoclonal antibody against CD14 (18D11). Addition of PepG or MDP to whole blood caused a 2-fold increase in the surface expression of CD14 on monocytes, as measured by flow cytometry. In contrast, LPS caused decreased expression of this receptor. Our data suggest that PepG and MDP primes human whole blood leukocytes for LPS-induced release of proinflammatory cytokines. We speculate that synergy between PepG and LPS may contribute to the pathogenesis in sepsis caused by mixed bacterial infections.

Procalcitonin does not influence the surface expression of inflammatory receptors on whole blood leukocytes.

The plasma levels of procalcitonin (PCT) are increased in patients with severe bacterial infections. Its cellular origin and potential pathophysiological function in sepsis is, however, unclear. White blood cells have recently been described to express both PCT mRNA and protein. The aim of this study was to determine whether PCT has any influence on the surface expression of receptors, relevant in inflammation, on human whole blood leukocytes under normal and septic conditions. Venous blood from healthy donors was incubated with PCT (40 ng/ml or 1200 ng/ml) alone or in combination with lipopolysaccharide (LPS, 10 ng/ml) or peptidoglycan (PepG, 10 micrograms/ml) for 6 h. The surface expression of CD14, CD54, CD64, CD80, CD86 and HLA-DR was determined by flow cytometry. We could not detect any influence of PCT on the expression of these receptors. Further studies on potential effects on other cell types during infection seem warranted.

Peptidoglycan and lipoteichoic acid modify monocyte phenotype in human whole blood.

We examined the influence of the gram-positive cell wall products peptidoglycan (PepG) and lipoteichoic acid (LTA), compared to lipopolysaccharide (LPS), on the monocyte expression of receptors involved in antigen presentation (HLA-DR, B7.1, and B7.2), cell adhesion (intercellular adhesion molecule-1 [ICAM-1] and lymphocyte function associated antigen-3 [LFA-3]), phagocytosis (Fc gamma RI), and cell activation (CD14). We also evaluated possible influences of the immunosuppressive drugs cyclosporine A, tacrolimus, and sirolimus on the expression of these receptors. Pretreatment of whole blood for 4 h with the immunosuppressive drugs did not influence the expression of the surface receptors in normal or stimulated blood. Stimulation with both PepG and LTA caused significant up-regulation of the surface expression of ICAM-1 and HLA-DR on whole blood monocytes, similar to that obtained with LPS, whereas B7.1, B7.2, LFA-3, and Fc gamma RI were not modulated. PepG and LTA also caused increased expression of CD14, whereas LPS down-regulated this molecule. In contrast, we did not detect any significant influence of any of the bacterial products on the plasma concentration of soluble CD14. We hypothesized that the increased expression of surface CD14 in blood stimulated with PepG would prime for cellular activation by LPS. Indeed, we show that PepG and the partial PepG structure muramyl dipeptide acted in synergy with LPS to cause the release of tumor necrosis factor-alpha. The results suggest that PepG and LPS provoke partly different responses on monocyte phenotype and that CD14 may play different roles in the innate response to gram-positive and gram-negative bacteria.

Response of alkalinization or acidification by phytohemagglutinin is dependent on the activity of protein kinase C in human peripheral T Cells.

The increase of intracellular free calcium concentration ([Ca(2+)](i)) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human T cells. However, a rapid change in intracellular pH (pH(i)), acidification or alkalinization during the activation, is also associated after these two signals. The aim of this study was to define whether the change in pH(i) is affected by calcium and protein kinase C (PKC), in phytohemagglutinin (PHA)-stimulated T cells. T cells were isolated from human peripheral blood. The [Ca(2+)](i) and the pH(i) were measured using, respectively, the fluorescent dyes, Fura-2, and BCECF. In addition, down-regulation of PKC activity by PMA (1 microM, 18 h) was confirmed in these cells using a protein kinase assay. The results indicated that, (1) alkalinization was induced by PHA or PMA in T cells; the results of alkalinization was PKC-dependent and Ca(2+)-independent, (2) in PKC down-regulated T cells, PHA induced acidification; this effect was enhanced by pre-treating the cells with the Na(+)/H(+) exchange inhibitor, 5-(N,N-dimethyl)-amiloride, (DMA, 10 microM, 20 min), (3) the acidification was dependent on the Ca(2+) influx and blocked by removal of extracellular calcium or the addition of the inorganic channel blocker, Ni(2+), and (4) Thapsigargin (TG), a Ca(2+)-ATPase inhibitor, confirmed that acidification by the Ca(2+) influx occurred in T cells in which PKC was not down-regulated. These findings indicate two mechanisms, alkalinization by PKC and acidification by Ca(2+) influx, exist in regulating pH(i) in T cells. This is the first report that PHA stimulates the acidification by Ca(2+) influx but not alkalinization in T cells after down-regulation of PKC. In conclusion, the activity of PKC in T cells determines the response in alkalinization or acidification by PHA.

Involvement of CD14 and toll-like receptors in activation of human monocytes by Aspergillus fumigatus hyphae.

Invasive fungal infections represent an increasing problem associated with high mortality. The present study was undertaken to identify leukocyte subsets that are activated by hyphal fragments in a whole-human-blood model, as well as to examine the involvement of CD14 and Toll-like receptors (TLRs) in activation of monocytes by hyphae. Incubation of whole human blood with hyphal fragments from Aspergillus fumigatus and Scedosporium prolificans for 6 h caused induction of mRNAs for tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in T cells, B cells, and monocytes, but not in granulocytes, as analyzed by reverse transcription-PCR with mRNA isolated from very pure populations of these leukocyte subsets. In primary adherent human monocytes, induction of TNF-alpha by hyphal fragments was dependent on plasma. Heat treatment of plasma at 56 degrees C for 30 min strongly reduced the ability of plasma to prime for activation. Pretreatment of human monocytes with different concentrations (1, 3, and 10 microg/ml) of monoclonal antibody (MAb) HTA125 (anti-TLR4) or MAb 18D11 (anti-CD14) for 30 min inhibited the release of TNF-alpha induced by hyphal fragments in a dose-dependent manner. Maximal inhibitions of 35 and 70% were obtained with 10 microg of HTA125 and 18D11 per ml, respectively. In contrast, pretreatment with MAb TL2.1 (anti-TLR2) did not affect signaling induced by hyphae. Pretreatment with the lipid A antagonist B975 blocked lipopolysaccharide signaling but did not inhibit TNF-alpha production induced by hyphal fragments. Our results suggest that T cells, B cells, and monocytes are involved in the innate immune response to invasive fungal pathogens and that serum components are relevant for activation of monocytes by hyphae. CD14 and TLR4 may be involved in signaling of Aspergillus hyphae in monocytes, but further studies to elucidate this issue are warranted.

Sirolimus interferes with the innate response to bacterial products in human whole blood by attenuation of IL-10 production.

Current immunosuppressive strategies are aimed at abrogating the allospecific T-cell response against donor tissues or organs. However, little information is yet available on the potential influences of these drugs on innate immune responses. In order to address this, we have employed a whole blood model. Human whole blood was pretreated with sirolimus, cyclosporine A or tacrolimus in therapeutic as well as supra therapeutic doses, and subsequently stimulated with lipopolysaccharide (LPS), peptidoglycan (PepG) or lipoteichoic acid (LTA). Plasma cytokine analyses revealed a potent inhibitory effect of sirolimus on interleukin(IL)-10 production induced by all bacterial products tested. In contrast, cyclosporine A and tacrolimus inhibited the tumour necrosis factor (TNF)-alpha production in response to LPS, but not to PepG and LTA. Using a quantitative mRNA analyses, we also observed that sirolimus significantly decreased the IL-10 mRNA accumulation to sub-basal levels in peripheral blood mononuclear cells (PBMC). This suggests that the sirolimus inhibits IL-10 production by interfering with the IL-10 gene transcription. However, the molecular mechanism of this inhibition remains unclear. Based on the present study and observations by others, we postulate that the clinical use of the sirolimus may be associated with a dysregulated innate immune response to bacterial infection and thus an increased risk of hyperinflammation and sepsis.

Peptidoglycan and lipoteichoic acid from Staphylococcus aureus induce tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-10 production in both T cells and monocytes in a human whole blood model.

We have examined the ability of peptidoglycan (PepG) and lipoteichoic acid (LTA) isolated from Staphylococcus aureus to induce the release of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-10 in whole human blood and identified the cellular origins of these cytokines. Both PepG and LTA induced transient increases in TNF-alpha and IL-10 in plasma, with peak values at 6 and 12 h, respectively. IL-6 values increased throughout the experimental period (24 h). The TNF-alpha, IL-6, and IL-10 release induced by PepG and LTA was dose dependent. Only PepG was a potent inducer of TNF-alpha secretion. After stimulation of whole blood with PepG or LTA, very pure populations of monocytes (CD14 positive), T cells (CD2 positive), B cells (CD19 positive), and granulocytes (CD15 positive) were isolated by immunomagnetic separation and analyzed by reverse transcription-PCR for mRNA transcripts encoding TNF-alpha, IL-6, and IL-10. The TNF-alpha mRNA results were inconclusive. In contrast, PepG induced IL-6 and IL-10 mRNA accumulation in both T cells and monocytes. LTA, as well as lipopolysaccharide, induced IL-6 and IL-10 mRNA production in monocytes and possibly in T cells. Whether granulocytes and B cells produce cytokines in response to bacterial stimuli remains obscure. Blockade of the CD14 receptors with monoclonal antibodies (18D11) had no influence on the PepG-induced release of TNF-alpha but attenuated the LTA-induced release of the same cytokine. In conclusion, our data indicate that circulating T cells and monocytes contribute to cytokine production in sepsis caused by gram-positive bacteria.

Cytokine modulation in experimental endotoxemia: characterization of an ex vivo whole blood model.

A new model was developed to study cytokine regulation and modulation in whole blood ex vivo. The model is characterized by stable leukocyte counts and high leukocyte viability throughout the experimental period. Oxygen consumption per time decreased slowly, whereas carbon dioxide partial pressure increased accordingly throughout the experiment. In this model, the anti-inflammatory effects of recombinant human (rh) interleukin (IL)-4, rhIL-10 and rhIL-13 on lipopolysaccharide (LPS) stimulated (10 ng/ml) leukocytes were examined and compared by measuring their ability to inhibit the release and mRNA levels of tumor necrosis factor (TNF)alpha, IL-6 and IL-1beta. rhIL-10 potently inhibited the release of TNF-alpha, IL-6 and IL-1beta in a potent and dose-dependent manner, but did not influence the mRNA levels of these cytokines in CD14-positive cells. Also, rhIL-4 and rhIL-13 inhibited the release of IL-6 and IL-1beta in a potent and dose-dependent manner, however, stronger maximal inhibition of IL-1beta (85%) than of IL-6 (60%) was obtained. In contrast, rhIL-4 and rhIL-13 seemed to have both stimulatory and inhibitory effects on plasma values of TNF-alpha. The effects of 10 ng/ml LPS showed to be signalling through the CD14 receptor, since blood treated with a monoclonal anti-CD14 antibody did not produce any TNF-alpha. The whole blood model described in this study is in our opinion a useful tool for investigating immunomodulating effects on a mixed white blood cell population.

Regulation of tissue inhibitor of metalloproteinases-1 in rat Sertoli cells: induction by germ cell residual bodies, interleukin-1alpha, and second messengers.

In the testis, FSH has been shown to induce the expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) from Sertoli cells in vitro. This study was performed to elucidate further the cellular origin of testicular TIMP-1 and its expression by hormonal and paracrine factors. This is the first report on the expression of testicular TIMP-1 in vivo. TIMP-1 mRNA in whole testis was decreased after hypophysectomy and strongly increased by the injection of FSH-S17 to hypophysectomized rats. Primary cultures of both peritubular and Sertoli cells showed basal expression of TIMP-1 mRNA. In contrast, we were unable to detect TIMP-1 mRNA in Leydig cells, freshly isolated immature germ cells (primary spermatocytes and spermatids), or residual bodies. We further show that treatment of Sertoli cells with 8-(4-chlorophenyl)thio-cAMP (8-CPTcAMP) in combination with 12-O-tetradecanoylphorbol 13-acetate (TPA) or Ca(2+) inducers (calcium ionophore A23187 or thapsigargin) had additive (TPA) and synergistic effects (Ca(2+)) on the level of TIMP-1 mRNA and secreted protein. We also show that both the level of TIMP-1 mRNA and secreted protein from Sertoli cells were strongly increased by residual bodies, as well as by the cytokine interleukin-1alpha. TIMP-1 was not up-regulated by either 8-CPTcAMP or interleukin-1alpha in peritubular cells. In contrast to the regulated secretory fraction of TIMP-1, we also detected constitutively expressed immunoreactive TIMP-1 in the nucleus of Sertoli cells, suggesting a role of nuclear TIMP-1 in these cells. In conclusion, our data show that secretion of TIMP-1 from Sertoli cells is highly regulated by hormonal and local processes in the testis, indicating that TIMP-1 is of physiological importance during both testicular development and spermatogenesis.