IL-33 is a mediator rather than a trigger of the acute allergic response in humans.

BACKGROUND: IL-33 enhances FcεRI-induced mediator release in human basophils without inducing degranulation itself. In contrast, studies in mice suggested that in the presence of high IgE levels, IL-33 triggers degranulation and anaphylaxis of similar severity as specific allergen. Consistent with this view, sera of atopic patients contain elevated levels of IL-33 after anaphylaxis. In this study, we determined whether IL-33 is potentially anaphylactogenic in humans with high IgE levels by regulating exocytosis independent of FcεRI cross-linking. Furthermore, we investigated whether IL-33 is released upon allergen provocation in vivo. METHODS: In subjects with high serum IgE levels, we measured IL-33-induced histamine/LTC4 in vitro, CD63 translocation ex vivo, and responsiveness of mast cells in vivo by skin prick test (SPT). In asthma patients, release of IL-33 and its correlation with early (tryptase)- and late-phase markers (IL-13 levels, eosinophil numbers) of the allergic response were assessed in bronchoalveolar lavage fluids (BALFs) after allergen challenge. RESULTS: IL-33 itself does not trigger basophil degranulation in vitro and ex vivo, even in subjects with high serum IgE levels, and negative SPTs demonstrate that skin mast cells do not degranulate in response to IL-33. However, in response to allergen challenge, IL-33 is rapidly released into BALFs at levels that do not correlate with other immediate- and late-phase parameters. CONCLUSION: IL-33 is unlikely an independent trigger of anaphylaxis even in subjects with high IgE levels. However, the rapid release of IL-33 upon allergen provocation in vivo supports its role as a mediator of immediate allergic responses.

Cinnamon extract inhibits degranulation and de novo synthesis of inflammatory mediators in mast cells.

BACKGROUND: Mast cells (MC) are main effector cells of allergic and other inflammatory reactions; however, only a few anti-MC agents are available for therapy. It has been reported that cinnamon extract (CE) attenuates allergic symptoms by affecting immune cells; however, its influence on MC was not studied so far. Here, we analyzed the effects of CE on human and rodent MC in vitro and in vivo. METHODS: Expression of MC-specific proteases was examined in vivo in duodenum of mice following oral administration of CE. Release of mediators and phosphorylation of signaling molecules were analyzed in vitro in human MC isolated from intestinal tissue (hiMC) or RBL-2H3 cells challenged with CE prior to stimulation by FcεRI cross-linking. RESULTS: Following oral treatment with CE, expression of the mast cell proteases MCP6 and MC-CPA was significantly decreased in mice. In hiMC, CE also caused a reduced expression of tryptase. Moreover, in hiMC stimulated by IgE cross-linking, the release of ß-hexosaminidase was reduced to about 20% by CE. The de novo synthesis of cysteinyl leukotrienes, TNFα, CXCL8, CCL2, CCL3, and CCL4, was almost completely inhibited by CE. The attenuation of mast cell mediators by CE seems to be related to particular signaling pathways, because we found that activation of the MAP kinases ERK, JNK, and p38 as well as of Akt was strongly reduced by CE. CONCLUSION: CE decreases expression of mast cell-specific mediators in vitro and in vivo and thus is a new plant-originated candidate for anti-allergic therapy.

Colonoscopic allergen provocation test with rBet v 1 in patients with pollen-associated food allergy.

BACKGROUND: After consumption of fruits, nuts, and vegetables, several patients with pollen allergy experience gastrointestinal (GI) tract symptoms that are possibly caused by pollen-associated food allergy. The aim of this study was to evaluate the colonoscopic allergen provocation (COLAP) test using the recombinant birch pollen allergen Bet v 1 (rBet v 1) for in vivo diagnosis of pollen-associated food allergy manifesting in the GI tract. METHODS: Thirty-four patients with a history of adverse reactions to food, GI tract symptoms, and birch pollen pollinosis and five healthy controls underwent COLAP test. Twenty minutes after endoscopic challenge of the cecal mucosa with rBet v 1, the mucosal wheal and flare reaction was registered semiquantitatively, and tissue biopsy specimens were examined for eosinophil mucosal activation. RESULTS: The mucosal reaction to rBet v 1 was correlated with the presence of pollinosis (P = 0.004), history of adverse reaction to Bet v 1-associated food allergens (P = 0.001), and tissue eosinophils' activation (P < 0.001). A wheal and flare reaction in the COLAP test was observed in 13 of 16 patients (81%) with a history of GI tract symptoms associated with the ingestion of Bet v 1-related foods and in four of 18 (22%) patients with a negative history (P < 0.001). The control group did not develop visible mucosal reactions to rBet v 1. Systemic anaphylactic reactions did not occur. CONCLUSIONS: The mucosal administration of rBet v 1 by COLAP test provides a new diagnostic tool that might support the diagnosis of Bet v 1-associated food allergy manifesting in the GI tract.

Neurotrophin-3, but not nerve growth factor, promotes survival of human intestinal mast cells.

Neurotrophins are potent regulators of neuronal cell survival and function. Nerve growth factor (NGF) was shown to reduce apoptosis in cord blood-derived mast cells. Here, we examined the effect of the neurotrophins NGF and neurotrophin (NT)-3 on survival and mediator release of human intestinal mast cells. Mast cells isolated from normal intestinal tissue were cultured in the presence of NGF, NT-3, or stem cell factor (SCF) alone or in the presence of SCF together with each neurotrophin. NGF or NT-3 alone did not promote mast cell survival. In contrast, mast cell recovery was increased twofold when mast cells were cultured with NT-3 in addition to SCF for 14 days compared with control. Mast cell recovery was further increased following a combined addition of NT-3, SCF and IL-4. NT-3 mediated mast cell growth was dependent on the primary receptor for NT-3 TrkC. NGF in combination with SCF or with SCF and IL-4 showed no effect on mast cell survival. Histamine release and histamine content per mast cell remained unchanged, whereas leukotriene C4 release decreased if mast cells were cultured with NGF or NT-3 in addition to SCF. In summary, NT-3 affects mature human mast cells by promoting mast cell survival, whereas NGF does not.

Selective expression of histamine receptors H1R, H2R, and H4R, but not H3R, in the human intestinal tract.

BACKGROUND AND AIMS: Histamine is known as a regulator of gastrointestinal functions, such as gastric acid production, intestinal motility, and mucosal ion secretion. Most of this knowledge has been obtained from animal studies. In contrast, in humans, expression and distribution of histamine receptors (HR) within the human gastrointestinal tract are unclear. METHODS: We analysed HR expression in human gastrointestinal tissue specimens by quantitative reverse transcription-polymerase chain reaction and immunostaining. RESULTS: We found that H1R, H2R, and H4R mRNA were expressed throughout the gastrointestinal tract, while H3R mRNA was absent. No significant differences in the distribution of HR were found between different anatomical sites (duodenum, ileum, colon, sigma, and rectum). Immunostaining of neurones and nerve fibres revealed that H3R was absent in the human enteric nervous system; however, H1R and H2R were found on ganglion cells of the myenteric plexus. Epithelial cells also expressed H1R, H2R and, to some extent, H4R. Intestinal fibroblasts exclusively expressed H1R while the muscular layers of human intestine stained positive for both H1R and H2R. Immune cells expressed mRNA and protein for H1R, H2R, and low levels of H4R. Analysis of endoscopic biopsies from patients with food allergy and irritable bowel syndrome revealed significantly elevated H1R and H2R mRNA levels compared with controls. CONCLUSIONS: We have demonstrated that H1R, H2R and, to some extent, H4R, are expressed in the human gastrointestinal tract, while H3R is absent, and we found that HR expression was altered in patients with gastrointestinal diseases.

Growth, phenotype, and function of human intestinal mast cells are tightly regulated by transforming growth factor beta1.

BACKGROUND AND AIMS: Transforming growth factor beta1 (TGF-beta1) is expressed in the healthy human intestine and controls mucosal immune responses and inflammation by regulating the function of lymphocytes, macrophages, dendritic cells, and eosinophils. Here, we asked whether human intestinal mast cells (MC) might also be subject to immunoregulation by TGF-beta1. METHODS: MC were isolated from the intestinal mucosa, purified, and cultured in vitro in the presence of stem cell factor (SCF) with or without TGF-beta1. Growth characteristics, phenotype, and immunological mediator release of MC were analysed by reverse transcription-polymerase chain reaction, flow cytometry, immunocytochemistry, western blot, and different immunoassays, respectively. RESULTS: TGF-beta1 dose dependently (ED50 approximately = 0.1 ng/ml) inhibited SCF dependent growth of human intestinal MC by both enhancing apoptosis and decreasing proliferation. In parallel, TGF-beta1 increased the percentage of connective tissue-type MC expressing tryptase and chymase while downregulating expression of the receptors for IgE and SCF. Furthermore, TGF-beta1 dramatically changed the profile of mediators released by MC following IgE receptor crosslinking. Release of histamine, cysteinyl-leukotrienes, and tumour necrosis factor alpha was strongly reduced whereas prostaglandin D2 generation and cyclooxygenase 1 and 2 expression were upregulated by TGF-beta1. CONCLUSIONS: Our data indicate that TGF-beta1 acts as a novel potent inhibitor and modulator of human intestinal MC effector functions. The change in MC mediator release profile and protease expression induced by TGF-beta1 might be of relevance for the control of MC associated disorders of the intestine such as allergic reactions, Crohn's disease, irritable bowel syndrome, and parasitic infection.

Epsilon germ-line and IL-4 transcripts are expressed in human intestinal mucosa and enhanced in patients with food allergy.

BACKGROUND: The mechanisms of gastrointestinal (GI) food allergy (FA) are poorly understood. Immunoglobulin E (IgE) is increased in stools from patients with FA, as well as the number of cells carrying IgE in intestinal mucosa, but the origin of IgE production remains unknown. To investigate a local production of IgE in intestine, we analysed the levels of transcripts for epsilon germ-line (epsilonGT), and potential regulators of IgE production, IL-4, IL-13, IFN-gamma, IL-4Ralpha, STAT6 and FcepsilonRIalpha in intestinal mucosa of adult patients with FA. METHODS: Endoscopic biopsies were obtained from the caecum of 25 patients with FA and 14 control patients. The levels of epsilonGT, IL-4, IL-13, IFN-gamma, IL-4Ralpha, STAT6 and FcepsilonRIalpha mRNA were analysed by real-time RT-PCR and compared with unpaired nonparametric Mann-Whitney test. RESULTS: The mean epsilonGT transcript level in caecum was increased in FA patients compared with control patients (P < 0.05). IL-4 mRNA expression was also increased in FA patients (P < 0.05), whereas mRNA expression for IL-13, IFN-gamma, IL-4Ralpha, STAT6 and FcepsilonRIalpha mRNA expression was not altered. However, the ratio of IL-4 mRNA/IFN-gamma mRNA was significantly increased in FA patients (P < 0.05). No correlation was observed between epsilonGT transcripts expression in intestinal mucosa and total IgE levels in serum. CONCLUSIONS: This study shows that (i) epsilonGT transcripts are expressed in human intestinal mucosa; (ii) epsilonGT and IL-4 transcripts are increased in caecal mucosa from patients with FA. These results suggest local production of IgE in intestine that might be of importance for inflammatory reactions in the GI tract.

Substance P and other neuropeptides do not induce mediator release in isolated human intestinal mast cells.

Neuropeptides such as substance P (SP) and related peptides are supposed to act as mast cell agonists, and thus as mediators of neuroimmune interactions. The data supporting this hypothesis were obtained mostly from rodent experiments. Here, we studied for the first time the effect of SP and other peptides on mediator release in human intestinal mast cells, either unpurified or enriched to 85-99% purity. We found that SP at 0.1-100 micromol L(-1), or other peptides including neurokinin A and B, calcitonin gene-related peptide, vasoactive intestinal peptide and serotonin at 1 micromol L(-1) do not induce release of mediators such as histamine, sulphidoleukotrienes, and tumour necrosis factor alpha. The peptides also failed to cause mediator release in mast cells isolated from inflamed tissue derived from Crohn's disease. Using reverse transcriptase-polymerase chain reaction, flow cytometry and immunohistochemistry, we could show that human intestinal mast cells do not express the tachykinin receptors NK-1, NK-2, or NK-3 under basal conditions. However, upon stimulation by immunoglobulin E (IgE) receptor-crosslinking, which induces an extensive mediator release reaction, a subpopulation of mast cells clearly expressed NK-1, the SP receptor. In conclusion, our data show that SP and other neuropeptides do not act as secretagogues in human intestinal mast cells that have not been pre-activated by IgE receptor-crosslinking.

Cellular immune response to autologous blood transfusion in hip arthroplasty: whole blood versus buffy coat-poor packed red cells and fresh-frozen plasma.

BACKGROUND AND OBJECTIVES: Transfusion-induced immunomodulation by autologous blood is probably related to the buffy coat. Hence, in the present study, phagocytotic and oxidation activities of peripheral blood cells were investigated in hip arthroplasty patients exposed to autologous blood. MATERIALS AND METHODS: Blood from 60 autologous donors was allocated at random to storage as whole blood (WB) or as buffy coat-poor packed red cells and fresh-frozen plasma (RCP). Phagocytotic and oxidation activities of neutrophils and monocytes, incidence of infections and length of hospital stay were compared among the groups of transfused (WB and RCP) and non-transfused (NT) patients. RESULTS: Phagocytotic activities of neutrophils and monocytes were not significantly different among the WB, RCP and NT groups. CONCLUSION: In the perioperative setting, a specific cellular immune response to autologous transfusion is not detectable.

Humoral immune response to autologous blood transfusion in hip surgery: whole blood versus packed red cells and plasma.

BACKGROUND AND OBJECTIVES: The immune response to the transfused autologous buffy coat content in whole blood has, to date, not been studied in detail. SUBJECTS AND METHODS: Patients undergoing hip arthroplasty were studied according to whether they received autologous whole blood (WB) (n = 30), autologous fresh-frozen plasma and buffy coat-poor red cells (RC) (n = 40), or no transfusion (NT) (n = 27). Plasma levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and complement SC5b-9 were analysed by enzyme-linked immunosorbent assay (ELISA) 7 days after surgery. RESULTS: There were no significant between-group differences regarding the time course of TNF-alpha, IL-6 and complement SC5b-9 plasma level changes, the infection rate, or the length of hospital stay. CONCLUSION: In comparison to the impact of surgery on cytokine and complement levels, the transfusion of autologous buffy coat is not relevant.

Immune response to autologous transfusion in healthy volunteers: WB versus packed RBCs and FFP.

BACKGROUND: Storage of blood as packed RBCs and FFP is standard practice in allogeneic transfusion. Separation into components has been proposed for autologous transfusion, as well, but beneficial effects have not yet been shown. STUDY DESIGN AND METHODS: Twenty-four healthy male volunteers were randomly assigned to receive 1 unit of either autologous RBCs and FFP (RCP group) or WB (WB group) after 49 or 35 days of storage, respectively. The immune response was analyzed by ELISA for IL-6, C3a, terminal complement complex SC5b-9, TNF-alpha, and neopterin. Differential WBC counts and the phagocytosis of neutrophils and monocytes were measured by flow cytometry. RESULTS: Cell counts of monocytes (0.85 x 10(3) ng/microL) [corrected] and neutrophils (6.9 x 10(3) ng/microL) [corrected] increased 30 minutes after WB transfusion and then returned to close to the baseline values seen in the RCP group (0.47 and 2.9 x 10(3) ng/microL [corrected], respectively) throughout the monitored period (p<0.05). C3a (169 vs. 116 ng/microL) [corrected] and IL-6 (29 vs. 6 pg/mL) reached higher plasma concentrations in the WB group (n = 11) than in the RCP group (n = 10). Phagocytosis of opsonized Escherichia coli was increased in neutrophils and monocytes and lasted up to 7 days after the transfusion of whole blood. CONCLUSION: Autologous WB induces a modest immunomodulation, but this effect is not observed upon transfusion of autologous blood components.

Interleukin-4 induces a switch of human intestinal mast cells from proinflammatory cells to Th2-type cells.

BACKGROUND: During the last years, mast cells have been recognized as a potent cellular source of multiple cytokines. However, little is known about the regulation of cytokine production by mature human mast cells derived from mucosal sites. METHODS: Human mast cells were isolated from intestinal mucosa and cultured for 14 days in the presence of stem cell factor (SCF) alone or in combination with IL-4. Mast cells were then stimulated by IgE receptor cross-linking or bacterial infection and cytokine production was examined by RT-PCR and ELISA. RESULTS: We found that human intestinal mast cells produce proinflammatory cytokines such as TNF-alpha, IL-1beta and IL-6 without further stimulation. Stimulation of the cells with gram-negative bacteria (Escherichia coli and others) caused an upregulation of TNF-alpha expression. Following IgE receptor cross-linking, we found additional expression of the Th2 cytokines IL-3, IL-5 and IL-13. Interestingly, mRNA for IL-3, IL-5 and IL-13 was also expressed in unstimulated mast cells provided they were cultured in the presence of SCF and IL-4. Moreover, IL-4 rendered mast cells capable of releasing IL-5 in response to bacterial challenge. CONCLUSION: In the presence of the mast cell survival factor SCF, mature human mast cells produce predominantly proinflammatory cytokines, whereas in the presence of SCF and IL-4, mast cells produce not only proinflammatory but also Th2 cytokines.

Regulation of human intestinal mast cells by stem cell factor and IL-4.

Mature human mast cells are tissue-residing, key effector cells of immediate allergic reactions. Moreover, mast cells have been recognized as a potent cellular source of multiple cytokines, suggesting an important role in immunoregulation and host defense. Here, we report on the regulation of mature human mast cells isolated from intestinal tissues by stem cell factor (SCF) and interleukin (IL)-4. SCF is substantially necessary for mast cell survival and induces marginal mast cell proliferation in vitro, whereas IL-4 by itself has no effects on mast cell survival or proliferation. Most interestingly, in synergy with SCF, IL-4 strongly enhances mast cell proliferation. In the presence of SCF, mast cells predominantly produce pro-inflammatory cytokines including tumor necrosis factor (TNF)-alpha, IL-1beta, IL-6, IL-8, IL-16, and IL-18. Addition of IL-4 to the culture medium induces the expression of Th2-type cytokines (IL-3, IL-5 and IL-13), and a downregulation of pro-inflammatory cytokines, namely IL-6. Furthermore, SCF by itself supports the predominance of the tryptase/chymase double-positive mast cell subtype MCTC whereas the addition of IL-4 supports the chymase negative MCT subtype. In conclusion, SCF may primarily regulate resident mast cell survival, whereas IL-4 may promote local proliferation of mast cells and their expression of Th2-type cytokines.

[Mannheim concept of preoperative autologous blood donation and perioperative autologous blood transfusion in hip endoprosthesis surgery]. / Mannheimer Konzept der präoperativen Eigenblutspende und perioperativen Autotransfusion in der Hüftendoprothetik.

PURPOSE: To investigate the efficiency of preoperative autologous deposit and intra- and postoperative cell salvage (CS) to reduce homologous transfusion in hip arthroplasty and revision hip arthroplasty. METHODS: In a retrospective study, the data of 1402 patients scheduled for hip arthroplasty and for revision hip arthroplasty were analysed. RESULTS: 767 women and 635 men, age 62.9 +/- 9.8 years (x +/- s) were included in the study. 1270 were scheduled for hip arthroplasty, 132 for revision hip arthroplasty. Of the autologous donors, 51 patients predeposited four units, 1020 patients three, 204 patients two, 39 patients one unit. 88 patients who had not enrolled in the autologous donation program but received CS served as a control group. Blood loss in autologous donors amounted to 1620 (220-5620) ml in hip arthroplasty and 2830 (950-7910) ml in revision arthroplasty. CS was employed in part of the cases in arthroplasty and in all revision operations. 470 (0-2200) ml and 705 (0-2200) were retransfused. In hip arthroplasty 227 of 1182 patients (19.2%) received homologous blood. Homologous transfusion in patients with coxarthrosis due to acetabular protrusio, avascular necrosis of the femoral head and hip dysplasia showed a trend to higher values. Patients who had donated 3 units and received CS required homologous transfusion in 12.4% of the cases. CS reduced the homolgous transfusion rate significantly if the preoperative hemoglobin concentration was < or = 12 g/dl. A group of autologous donors receiving CS was matched with patients receiving CS only. 12 of 76 (15.8%) vs. 45 of 76 (59.2%) required homologous transfusion. In revision hip arthroplasty 58 of 132 patients (43.9%) required homologous blood. CONCLUSIONS: Preoperative deposit reduces homologous transfusion requirements considerably in hip surgery. Under the conditions studied CS should be employed in hip arthroplasty in addition to preoperative deposit if the preoperative hemoglobin concentration falls below 12 g/dl. In revision arthroplasty, 4 or more autologous units should be predeposited and CS should be used regularly.

[Complications in spinal anesthesia: steel introducer cannula breaks out of its handle]. / Komplikation bei einer Spinalanästhesie: Ausgebrochene Führungskanüle.

We report a complication in a spinal anesthesia caused by the steel introducer canula breaking out of its plastic handle.