Axel Lorentz - Academia.edu (original) (raw)

Papers by Axel Lorentz

Methods in molecular biology (Clifton, N.J.), 2015

Mast cells are granulated immune cells typically located at barrier sites of the body, such as th... more Mast cells are granulated immune cells typically located at barrier sites of the body, such as the skin and the mucosa of the respiratory, urogenital, and gastrointestinal tract. They are well known for their capacity to participate in the orchestration of inflammatory and immune responses by releasing a broad array of mediators as a consequence of IgE-dependent and IgE-independent activation. Mast cells derive from myeloid progenitors, but in contrast to other myeloid cells, they leave the bone marrow in an immature state; therefore, mast cells are not visible in the blood under normal conditions. For full maturation, the tissue environment is necessary. Thus, mature mast cells can be only isolated from tissue such as skin or mucosal sites, which makes mast cell isolation complicated. This chapter describes methods to isolate, purify, and culture mast cells from the human intestinal mucosa. Human mucosal mast cells can be used to characterize their mediators and to study the mechan...

The Journal of allergy and clinical immunology, 2003

On activation by cross-linking the high-affinity IgE receptor (FcepsilonRI), expression of TNF-al... more On activation by cross-linking the high-affinity IgE receptor (FcepsilonRI), expression of TNF-alpha, IL-3, IL-5, and IL-13 is induced in human intestinal mast cells. We sought to examine, for the first time, FcepsilonRI signaling in mature human mast cells. Mast cells were isolated from intestinal tissue and cultured in the presence of stem cell factor. The cells were treated with specific inhibitors before stimulation by means of FcepsilonRI cross-linking. Cytokine mRNA expression was analyzed by means of RT-PCR, and activation of signaling molecules was determined by means of immunocytochemistry, RT-PCR, Western blotting, and protein kinase C (PKC) assay. We found that nuclear factor-kappaB (NF-kappaB), as well as c-Fos and c-Jun, the components of activator protein 1 (AP-1), are activated after FcepsilonRI cross-linking in human intestinal mast cells. Treatment of the cells with specific inhibitors revealed an involvement of NF-kappaB and nuclear factor of activated T cells, as ...

International archives of allergy and immunology

Recently we reported about a stem cell factor (SCF)-dependent culture system for human mast cells... more Recently we reported about a stem cell factor (SCF)-dependent culture system for human mast cells (MC), isolated from intestinal mucosa. Here we present a method to obtain highly purified human intestinal MC. MC were isolated from surgery specimens and purified by positive selection using the magnetic-activated cell sorting (MACStrade mark) system and subsequent culture of the MC in medium supplemented with SCF. In the presence of SCF, purified MC (50-85% purity after MACS) maintained in culture for up to 3 months. MC purity increased during culture and reached nearly 100%. During the first week of culture, MC numbers decreased, but after that time they started to proliferate. Cultured MC did not change their histamine content, phenotype or morphology. They were even more responsive towards IgE-dependent stimulation, which caused the release of high amounts of histamine, leukotrienes and cytokines such as TNF-alpha and IL-5. We show that mature human intestinal MC can be purified, m...

Frontiers in Immunology, 2012

Mast cells are known as inflammatory cells which exert their functions in allergic and anaphylact... more Mast cells are known as inflammatory cells which exert their functions in allergic and anaphylactic reactions by secretion of numerous inflammatory mediators. During an allergic response, the high-affinity IgE receptor, FcεRI, becomes cross-linked by receptor-bound IgE and antigen resulting in immediate release of pre-synthesized mediators -stored in granules -as well as in de novo synthesis of various mediators like cytokines and chemokines. Soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNARE) proteins were found to play a central role in regulating membrane fusion events during exocytosis. In addition, several accessory regulators like Munc13, Munc18, Rab GTPases, secretory carrier membrane proteins, complexins, or synaptotagmins were found to be involved in membrane fusion. In this review we summarize our current knowledge about the SNARE machinery and its mechanism of action in mast cell secretion.

Veterinary Immunology and Immunopathology, 2006

Several beneficial effects of probiotics have been described in studies using rodent disease mode... more Several beneficial effects of probiotics have been described in studies using rodent disease models and in human patients; however, the underlying mechanisms remained mostly unclear. Only a few studies focused on the effects of probiotics on the intestinal mucosal immune system. Here, we studied the effect of the probiotic strain E. coli Nissle 1917 (EcN) administered orally to young pigs at two concentrations (10(9) and 10(11)CFU/d for 21 days) on the gut-associated lymphatic tissue. This probiotic strain was shown recently to reduce recurrence of inflammation in ulcerative colitis patients. We quantified the number and distribution of intestinal immune cells (granulocytes, mast cells, CD4+, CD8+, CD25+, IgA+ lymphocytes) and the mucosal mRNA expression of cytokines (IFN-gamma, TNF-alpha, TGF-beta, IL-10) and antimicrobial peptides (PR-39, NK-lysin, prepro-defensin-beta 1, protegrins). The number and distribution of cells were highly different between small intestinal and colon segments in all groups, but were not influenced by EcN, except high dose EcN fed pigs (10(11) CFU/d) showing an increase in mucosal CD8+ cells in the ascending colon. The mRNA analysis revealed no changes associated with EcN feeding. In conclusion, according to our analyses EcN has only minor effects on the distribution of mucosal immune cells in the gut of healthy individuals. The well-established preventive effects of EcN might therefore be relate to other mechanisms than simple modulation of immune cell distribution.

Transfusion, 1991

The variations in plasma erythropoietin (EPO) concentration during preoperative deposit of autolo... more The variations in plasma erythropoietin (EPO) concentration during preoperative deposit of autologous blood were studied in 12 patients (8 men, 4 women). Four donations were scheduled at weekly intervals. A predonation hemoglobin concentration of 11 g per dL (1 10 /L) was required. Hemoglobin concentration decreased from 14.3 2 1.1 g per dL 7143 ? 11 g/L) (mean 2 SD) before the first donation to 11.7 f 0.7 g per dL (1 17 2 7 /L) on Day 22 (ps0.0001). Reticulocyte counts increased from a median of 31,800 ?range, 4900-95,000) per pL (median, 32 x 10 / [ 5-95 x 10e/L]) to 93,800 (16,800-194,900) per L (median, 94 x 10e/L ; zk$; y; :

Science, 1995

Centromeres attach chromosomes to the spindle during mitosis, thereby ensuring the equal distribu... more Centromeres attach chromosomes to the spindle during mitosis, thereby ensuring the equal distribution of chromosomes into daughter cells. Transcriptionally silent heterochromatin of unknown function is associated with centromeres in many organisms. In the fission yeast Schizosaccharomyces pombe, the silent mating-type loci, centromeres, and telomeres are assembled into silent heterochromatin-like domains. The Swi6 chromodomain protein affects this silencing, and now it is shown that Swi6p localizes with these three chromosomal regions. In cells lacking Swi6p, centromeres lag on the spindle during anaphase and chromosomes are lost at high rates. Thus, Swi6p is located at fission yeast centromeres and is required for their proper function.

Nucleic Acids Research, 1993

The gene rad22 of the fission yeast Schizosaccharomyces pombe has a function in DNA repair and ma... more The gene rad22 of the fission yeast Schizosaccharomyces pombe has a function in DNA repair and mating-type switching. We have cloned the rad22 gene from a genomic gene bank by functional complementation of the switching defect. An open reading frame coding for a putative protein of 469 amino acids was found by sequence analyses. The rad22 gene contains no intron. A region of 126 amino acids in the N-terminal half of the Rad22 protein has significant homologies (56% identity and 36% similarity) to the Rad52 protein of Saccharomyces cerevisiae. A rad22 disruption strain was constructed which seems to be inviable in a homothallic background. Southern blot analyses have shown that the rad22-67 mutant frequently gives rise to deletions in the mating-type region. These data indicate that the Rad22 protein has a function in the repair of DNA double-strand breaks.

Neurogastroenterology and Motility, 2004

Neuropeptides such as substance P (SP) and related peptides are supposed to act as mast cell agon... more 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 lmol L )1 , or other peptides including neurokinin A and B, calcitonin gene-related peptide, vasoactive intestinal peptide and serotonin at 1 lmol L )1 do not induce release of mediators such as histamine, sulphidoleukotrienes, and tumour necrosis factor a. 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.

Molecular Immunology, 2011

Challenge of human mast cells with both stem cell factor (SCF) and IL-4 enhances antigen-dependen... more Challenge of human mast cells with both stem cell factor (SCF) and IL-4 enhances antigen-dependent mediator release raising the assumption of intracellular crosstalk between the IL-4, SCF, and FcRI signaling pathways. Here, we analyzed the intracellular crosstalk of IL-4-, SCF-, and IgE-dependent activation pathways in mucosal mast cells isolated from human intestine. The release of ␤-hexosaminidase, leukotriene C 4 , and IL-8, but not IL-6, was strongly enhanced in response to sequential challenge of mast cells with IL-4, SCF and FcRI cross-linking compared to stimulation by FcRI cross-linking alone. Previous studies revealed that MAPK and other serine/threonine kinases are involved in mast cell activation processes. Here we found that activation of mast cells by FcRI cross-linking alone results in phosphorylation of ERK and p38, but not of Akt. Stimulation with SCF alone also induced phosphorylation of ERK and p38, and additionally of Akt. IL-4 priming enhanced activation of ERK, but blocked activation of p38. Activation of p38 was required for IL-6 production explaining the inhibitory effect of IL-4 on IL-6 expression in human mast cells. Moreover, IL-4 priming that anteceded FcRI cross-linking induced activation of Akt. The combined challenge of mast cells with IL-4, SCF and FcRI cross-linking substantially up-regulated activation of Akt, whereas blocking of Akt inhibited the pronounced production and release of IL-8 in response to the three mast cell agonists. In summary, our data demonstrate that ERK, p38, and especially Akt play an important role in cross-linking IL-4 priming, SCF signaling, and IgE-dependent activation of mature human mast cells.

MGG Molecular & General Genetics, 1992

The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in... more The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in homothallic (h 9°) strains of Schizosaccharomyces pombe. The MT region of h 9° comprises three cassette genes: the expression site mat1:1 and two silent loci, mat2.2 and mat3." 3. Besides reducing MT switching, the swi6 mutation leads to deletions in the MT region caused by intrachromosomal cross-overs between two paired cassettes. These deletions only arise if DNA double-strand breaks are present at mat1 : 1, which initiate MT switching. Furthermore, swi6 allows meiotic recombination in the K region, a region of 16 kb between mat2:2 and mat3: 3; in wild-type strains no recombination occurs in K. swi6 also allows the simultaneous expression of two different cassettes in the same haploid cell. Thus swi6 may have an influence on the general chromatin structure in the MT region.

The Journal of Immunology, 2000

Mast cells are recognized as a new type of immunoregulatory cells capable of producing different ... more Mast cells are recognized as a new type of immunoregulatory cells capable of producing different cytokines. So far, little is known about the cytokine profile of mature human mast cells isolated from intestinal tissue and cultured in the presence of stem cell factor (SCF). We observed that these cells express the proinflammatory cytokines TNF-alpha, IL-1 beta, IL-6, IL-8, IL-16, and IL-18 without further stimulation. Both IgE-dependent and IgE-independent agonists (e.g., Gram-negative bacteria) enhanced expression of TNF-alpha. Another set of cytokines consisting of IL-3, IL-5, IL-9, and IL-13 was expressed following activation by IgE receptor cross-linking. If mast cells were cultured in the presence of IL-4 and SCF, the production and release of IL-3, IL-5, and IL-13 was increased up to 4-fold compared with mast cells cultured with SCF alone. By contrast, IL-6 expression was completely blocked in response to culture with IL-4. In summary, our data show that mature human mast cells produce proinflammatory cytokines that may be up-regulated following triggering with IgE-independent agonists such as bacteria, whereas activation by IgE receptor cross-linking results in the expression of Th2-type cytokines. IL-4 enhances the expression of Th2-type cytokines but does not affect or even down-regulates proinflammatory cytokines.

Journal of Cellular and Molecular Medicine, 2011

The circadian clock in peripheral tissues can be entrained by restricted feeding (RF), a regimen ... more The circadian clock in peripheral tissues can be entrained by restricted feeding (RF), a regimen that restricts the duration of food availability with no calorie restriction (CR). However, it is not known whether RF can delay the occurrence of age-associated changes similar to CR. We measured circadian expression of clock genes, disease marker genes, metabolic factors and inflammatory and allergy markers in mouse serum, liver, jejunum and white adipose tissue (WAT) after long-term RF of 4 months. We found that circadian rhythmicity is more robust and is phase advanced in most of the genes and proteins tested under RF. In addition, average daily levels of some disease and inflammatory markers were reduced under

Journal of Allergy and Clinical Immunology, 2003

Objective: We sought to examine, for the first time, Fc RI signaling in mature human mast cells. ... more Objective: We sought to examine, for the first time, Fc RI signaling in mature human mast cells. Methods: Mast cells were isolated from intestinal tissue and cultured in the presence of stem cell factor. The cells were treated with specific inhibitors before stimulation by means of Fc RI cross-linking. Cytokine mRNA expression was analyzed by means of RT-PCR, and activation of signaling molecules was determined by means of immunocytochemistry, RT-PCR, Western blotting, and protein kinase C (PKC) assay.

Journal of Allergy and Clinical Immunology, 2002

Unidentified wheat presence in the food supply presents a serious health risk to both wheat-aller... more Unidentified wheat presence in the food supply presents a serious health risk to both wheat-allergic and celiac sprue patients. The Codex Alimentarius had previously set a level of less than 200 ppm for a food to be considered "gluten-free". Codex is considering changing this level to 20 ppm in the belief that this will better serve the celiac sprue patient. A commercially available wheat gluten detection kit that has a lower limit of detection of 10 parts per million wheat gluten [5 ppm gliadin] was used to test a variety of foods and ingredients for detectable gluten content. The test also exhibits some cross-reactivity to both barley and rye glutens. There is no commercial test available for total wheat protein, so this test is the only measure of a food's lack of wheat content. Foods tested included a variety of non-wheat foods at high risk for cross-contamination from the farm through food processing, foods labeled "wheat free" or "gluten free", and foods and food ingredients commonly avoided by celiac and wheat-allergic patients that originate from wheat or related cereals, but with questionable gluten content. Samples tested included at least two different lot numbers of each product. Results of this study indicate that some food products with questionable wheat content, such as alcoholic beverages, alcoholic carriers, alcohol-based flavorings, most types of vinegar, and most soy sauces, registered below 10 ppm in the assay. While seed oats did not show the presence of wheat gluten, oat-based cereal products had detectable wheat gluten content, probably due to contamination by wheat during commodity handling. Some food products labeled as wheat or gluten "free" exhibited detectable levels of gluten ranging from 22 to 71 parts per million. Based on the results of this study, the dietary choices for celiac sprue patients should not be as restricted as formerly assumed, and ingredients such as many vinegars and alcohol-based flavorings can likely be ingested without problem. However, wheat-allergic and celiac sprue patients must exercise care in choosing "gluten-free or wheat-free" -labeled foods, as some can contain appreciable amounts of wheat. Despite its worldwide consumption, beer has rarely been reported to elicit allergic reactions. We report a 59 year-old male patient who had suffered from angioedema and generalized urticaria 3 h after drinking 1.5 1 of WB and eating a pretzel. One month later, he had developed generalized pruritus, urticaria, and ultimately unconsciousness during a game of golf, 30 min after having drunk 1 1 of WB. Other alcoholic beverages, like wine or lager beers, were well tolerated. There was an immediate type skin prick test reaction to a commercial wheat flour extract, but not to a series of other commercial allergens. When using native material for skin prick testing, there were 3+ reactions to one brand of WB, 2+ reactions to wheat malt and another brand of WB, and a 1+ reaction to barley malt, which were all not observed in control subjects. There were specific IgE antibodies in the serum to wheat, barley and rye flour, to barley malt and to chicken meat. An oral challenge test with incremental doses of WB caused wheals occurring on the face after the ingestion of a cumulative dose of 1.6 1. Oral challenge with wheat flour also produced wheals on the face after the ingestion of a cumulative dose of 120 g. There have been sporadic reports of allergy to barley malt contained in lager beers. WB is brewed by using both barley and wheat malt, and our patient exhibited clinical reactions exclusively to wheat. This case is the first of a beer allergy specifically attributable to a sensitization to wheat.

Journal of Allergy and Clinical Immunology, 2003

RATIONALE: IL-3, tL-5 and GM-CSF exert overlapping biological functions in basophils and eosinoph... more RATIONALE: IL-3, tL-5 and GM-CSF exert overlapping biological functions in basophils and eosinophils, because their receptors have common signaling beta-chain. However, no comprehensive study has been performed to simultaneously compare the molar effects of IL-3, IL-5 and GM-CSF between different functions of these cells in the same experiment. METHODS: Survival enhancement, CDllb expression and CD69 expression were analyzed in both basophils and eosinophils. The enhancement of histamine release was studied in basophils. We also investigated the protein and mRNA expression levels of IL-3Ralpha, IL-5Ralpha and GM-CSFRalpha using flow cytometry and real-time PCR. RESULTS: The rank order of potency in basophils was IL-3 >> IL-5=GM-CSF for degranulation, survival and CDI lb expression. The ED50 of IL-3 was 0.5-1 pM, 0.03 pM and 7 pM, respectively, for these three functions. In eosinophils, the rank order was IL-5=GM-CSF > IL-3 for survival and CDI lb expression. The ED50 of IL-5 was 0.6 pM and 100pM for survival and CDI lb expression, respectively. However, in both cell types, CD69 expression was most potently induced by IL-3. Compared with eosinophils, basophils expressed a much higher level of IL-3Ralpha, similar or slightly lower level of IL-5Ralpha, and an apparently lower level of GM-CSFRaIpha. CONCLUSIONS: IL-3 is the most potent stimulator for basophils, whereas IL-5 and GM-CSF are most potent for eosinophils. In general, the rank order of potency of these cytokines corresponded exactly to their receptor expression levels. However, CD69 expression differed completely from this paradigm. Funding: Grant Monies t O1~ Cyclooxygenase (COX)Inhibition at the "time of Initial Antigen | U~J Presentation Results in Enhanced IL-13 and IgE

Journal of Allergy and Clinical Immunology, 2003

Journal of Allergy and Clinical Immunology, 2003

tor cross-linking. In this study, we examined the expression of cytokines by mast cells isolated ... more tor cross-linking. In this study, we examined the expression of cytokines by mast cells isolated from human intestinal mucosa following stimulation by stem cell factor (SCF). METHODS: Mast cells were purified by positive selection and cultured with SCF alone or with SCF and IL-4. Following pretreatment with specific inhibitors mast cells were stimulated with 1-100 ng/ml SCF for 90 min, Activation of mitogen-activated protein kinase (MAPK) was analyzed by Western blot and mRNA expression by RT-PCR. RESULTS: We did not detect an induction of cytokine expression by SCF in mast cells pre-cultured with SCF alone. In contrast, the mRNA for IL-5 and TNF-~ was strongly up-regulated in mast cells following stimulation with at least 10 ng/ml SCF if the cells were pre-cultured with SCF and IL-4. Cytokine expression was inhibited by apigenin but not by cyclosporin, wortmannin, and G0 6976. This indicates the necessity of mitogen-activated protein kinase (MAPK), but not of nuclear factor of activated T cells, phosphatidylinositol 3-kinase or protein kinase C. The activation of MAPK in response to SCF could be confirmed by Western blot. Consistently, we found activation of c-Fos, the downstream target of MAPK, a component of the transcription factor activator protein-1. CONCLUSIONS: In summary, our data show that SCF is capable of inducing cytokine expression in mature human mast cells pre-cultured with IL-4 through MAPK activation. RATIONALE: To characterize human cord blood derived mast cells in regard to chemokine oan chemokine receptor genes. METHODS: We have established/developed an in vitro method for generating very large quantities of mature an functionally intact human mast cells. Cells are derived from CD133+ progenitor cells isolated from cord blood and induced to differentiate into mast cells by cultivation in a serum free medium supplemented with stem cell factor and 1L-6 for 10 weeks. To further induce mast cell maturation, foetal calf serum wasadded during the subsequent 4 weeks. The availability of such large numbers of mast cells has for the first time made it possible to perform quantitative analyses at multiple cellular levels on a single cell sample: Flow cytometry, ELISA, Western blotting and RNAse protection assay. RESULTS: Following receptor-mediated or pharmacological activationof the mast cells for up to 24 hours the kinetics of induction of chemokine and chemokine receptor genes was monitored quantitatively at the level of transcription and translation. Moreover we demonstrate synthetic glucocorticoid and calcium signaling antagonist cyclosporin A quench different sets of chemokine genes in activated mast cells. CONCLUSIONS: We demonstrate the involvement of distinct signaling transduction pathways regulating various chemokine genes and their receptor genes upon activation of mast cells.

Methods in molecular biology (Clifton, N.J.), 2015

Mast cells are granulated immune cells typically located at barrier sites of the body, such as th... more Mast cells are granulated immune cells typically located at barrier sites of the body, such as the skin and the mucosa of the respiratory, urogenital, and gastrointestinal tract. They are well known for their capacity to participate in the orchestration of inflammatory and immune responses by releasing a broad array of mediators as a consequence of IgE-dependent and IgE-independent activation. Mast cells derive from myeloid progenitors, but in contrast to other myeloid cells, they leave the bone marrow in an immature state; therefore, mast cells are not visible in the blood under normal conditions. For full maturation, the tissue environment is necessary. Thus, mature mast cells can be only isolated from tissue such as skin or mucosal sites, which makes mast cell isolation complicated. This chapter describes methods to isolate, purify, and culture mast cells from the human intestinal mucosa. Human mucosal mast cells can be used to characterize their mediators and to study the mechan...

The Journal of allergy and clinical immunology, 2003

On activation by cross-linking the high-affinity IgE receptor (FcepsilonRI), expression of TNF-al... more On activation by cross-linking the high-affinity IgE receptor (FcepsilonRI), expression of TNF-alpha, IL-3, IL-5, and IL-13 is induced in human intestinal mast cells. We sought to examine, for the first time, FcepsilonRI signaling in mature human mast cells. Mast cells were isolated from intestinal tissue and cultured in the presence of stem cell factor. The cells were treated with specific inhibitors before stimulation by means of FcepsilonRI cross-linking. Cytokine mRNA expression was analyzed by means of RT-PCR, and activation of signaling molecules was determined by means of immunocytochemistry, RT-PCR, Western blotting, and protein kinase C (PKC) assay. We found that nuclear factor-kappaB (NF-kappaB), as well as c-Fos and c-Jun, the components of activator protein 1 (AP-1), are activated after FcepsilonRI cross-linking in human intestinal mast cells. Treatment of the cells with specific inhibitors revealed an involvement of NF-kappaB and nuclear factor of activated T cells, as ...

International archives of allergy and immunology

Recently we reported about a stem cell factor (SCF)-dependent culture system for human mast cells... more Recently we reported about a stem cell factor (SCF)-dependent culture system for human mast cells (MC), isolated from intestinal mucosa. Here we present a method to obtain highly purified human intestinal MC. MC were isolated from surgery specimens and purified by positive selection using the magnetic-activated cell sorting (MACStrade mark) system and subsequent culture of the MC in medium supplemented with SCF. In the presence of SCF, purified MC (50-85% purity after MACS) maintained in culture for up to 3 months. MC purity increased during culture and reached nearly 100%. During the first week of culture, MC numbers decreased, but after that time they started to proliferate. Cultured MC did not change their histamine content, phenotype or morphology. They were even more responsive towards IgE-dependent stimulation, which caused the release of high amounts of histamine, leukotrienes and cytokines such as TNF-alpha and IL-5. We show that mature human intestinal MC can be purified, m...

Frontiers in Immunology, 2012

Mast cells are known as inflammatory cells which exert their functions in allergic and anaphylact... more Mast cells are known as inflammatory cells which exert their functions in allergic and anaphylactic reactions by secretion of numerous inflammatory mediators. During an allergic response, the high-affinity IgE receptor, FcεRI, becomes cross-linked by receptor-bound IgE and antigen resulting in immediate release of pre-synthesized mediators -stored in granules -as well as in de novo synthesis of various mediators like cytokines and chemokines. Soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNARE) proteins were found to play a central role in regulating membrane fusion events during exocytosis. In addition, several accessory regulators like Munc13, Munc18, Rab GTPases, secretory carrier membrane proteins, complexins, or synaptotagmins were found to be involved in membrane fusion. In this review we summarize our current knowledge about the SNARE machinery and its mechanism of action in mast cell secretion.

Veterinary Immunology and Immunopathology, 2006

Several beneficial effects of probiotics have been described in studies using rodent disease mode... more Several beneficial effects of probiotics have been described in studies using rodent disease models and in human patients; however, the underlying mechanisms remained mostly unclear. Only a few studies focused on the effects of probiotics on the intestinal mucosal immune system. Here, we studied the effect of the probiotic strain E. coli Nissle 1917 (EcN) administered orally to young pigs at two concentrations (10(9) and 10(11)CFU/d for 21 days) on the gut-associated lymphatic tissue. This probiotic strain was shown recently to reduce recurrence of inflammation in ulcerative colitis patients. We quantified the number and distribution of intestinal immune cells (granulocytes, mast cells, CD4+, CD8+, CD25+, IgA+ lymphocytes) and the mucosal mRNA expression of cytokines (IFN-gamma, TNF-alpha, TGF-beta, IL-10) and antimicrobial peptides (PR-39, NK-lysin, prepro-defensin-beta 1, protegrins). The number and distribution of cells were highly different between small intestinal and colon segments in all groups, but were not influenced by EcN, except high dose EcN fed pigs (10(11) CFU/d) showing an increase in mucosal CD8+ cells in the ascending colon. The mRNA analysis revealed no changes associated with EcN feeding. In conclusion, according to our analyses EcN has only minor effects on the distribution of mucosal immune cells in the gut of healthy individuals. The well-established preventive effects of EcN might therefore be relate to other mechanisms than simple modulation of immune cell distribution.

Transfusion, 1991

The variations in plasma erythropoietin (EPO) concentration during preoperative deposit of autolo... more The variations in plasma erythropoietin (EPO) concentration during preoperative deposit of autologous blood were studied in 12 patients (8 men, 4 women). Four donations were scheduled at weekly intervals. A predonation hemoglobin concentration of 11 g per dL (1 10 /L) was required. Hemoglobin concentration decreased from 14.3 2 1.1 g per dL 7143 ? 11 g/L) (mean 2 SD) before the first donation to 11.7 f 0.7 g per dL (1 17 2 7 /L) on Day 22 (ps0.0001). Reticulocyte counts increased from a median of 31,800 ?range, 4900-95,000) per pL (median, 32 x 10 / [ 5-95 x 10e/L]) to 93,800 (16,800-194,900) per L (median, 94 x 10e/L ; zk$; y; :

Science, 1995

Centromeres attach chromosomes to the spindle during mitosis, thereby ensuring the equal distribu... more Centromeres attach chromosomes to the spindle during mitosis, thereby ensuring the equal distribution of chromosomes into daughter cells. Transcriptionally silent heterochromatin of unknown function is associated with centromeres in many organisms. In the fission yeast Schizosaccharomyces pombe, the silent mating-type loci, centromeres, and telomeres are assembled into silent heterochromatin-like domains. The Swi6 chromodomain protein affects this silencing, and now it is shown that Swi6p localizes with these three chromosomal regions. In cells lacking Swi6p, centromeres lag on the spindle during anaphase and chromosomes are lost at high rates. Thus, Swi6p is located at fission yeast centromeres and is required for their proper function.

Nucleic Acids Research, 1993

The gene rad22 of the fission yeast Schizosaccharomyces pombe has a function in DNA repair and ma... more The gene rad22 of the fission yeast Schizosaccharomyces pombe has a function in DNA repair and mating-type switching. We have cloned the rad22 gene from a genomic gene bank by functional complementation of the switching defect. An open reading frame coding for a putative protein of 469 amino acids was found by sequence analyses. The rad22 gene contains no intron. A region of 126 amino acids in the N-terminal half of the Rad22 protein has significant homologies (56% identity and 36% similarity) to the Rad52 protein of Saccharomyces cerevisiae. A rad22 disruption strain was constructed which seems to be inviable in a homothallic background. Southern blot analyses have shown that the rad22-67 mutant frequently gives rise to deletions in the mating-type region. These data indicate that the Rad22 protein has a function in the repair of DNA double-strand breaks.

Neurogastroenterology and Motility, 2004

Neuropeptides such as substance P (SP) and related peptides are supposed to act as mast cell agon... more 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 lmol L )1 , or other peptides including neurokinin A and B, calcitonin gene-related peptide, vasoactive intestinal peptide and serotonin at 1 lmol L )1 do not induce release of mediators such as histamine, sulphidoleukotrienes, and tumour necrosis factor a. 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.

Molecular Immunology, 2011

Challenge of human mast cells with both stem cell factor (SCF) and IL-4 enhances antigen-dependen... more Challenge of human mast cells with both stem cell factor (SCF) and IL-4 enhances antigen-dependent mediator release raising the assumption of intracellular crosstalk between the IL-4, SCF, and FcRI signaling pathways. Here, we analyzed the intracellular crosstalk of IL-4-, SCF-, and IgE-dependent activation pathways in mucosal mast cells isolated from human intestine. The release of ␤-hexosaminidase, leukotriene C 4 , and IL-8, but not IL-6, was strongly enhanced in response to sequential challenge of mast cells with IL-4, SCF and FcRI cross-linking compared to stimulation by FcRI cross-linking alone. Previous studies revealed that MAPK and other serine/threonine kinases are involved in mast cell activation processes. Here we found that activation of mast cells by FcRI cross-linking alone results in phosphorylation of ERK and p38, but not of Akt. Stimulation with SCF alone also induced phosphorylation of ERK and p38, and additionally of Akt. IL-4 priming enhanced activation of ERK, but blocked activation of p38. Activation of p38 was required for IL-6 production explaining the inhibitory effect of IL-4 on IL-6 expression in human mast cells. Moreover, IL-4 priming that anteceded FcRI cross-linking induced activation of Akt. The combined challenge of mast cells with IL-4, SCF and FcRI cross-linking substantially up-regulated activation of Akt, whereas blocking of Akt inhibited the pronounced production and release of IL-8 in response to the three mast cell agonists. In summary, our data demonstrate that ERK, p38, and especially Akt play an important role in cross-linking IL-4 priming, SCF signaling, and IgE-dependent activation of mature human mast cells.

MGG Molecular & General Genetics, 1992

The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in... more The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in homothallic (h 9°) strains of Schizosaccharomyces pombe. The MT region of h 9° comprises three cassette genes: the expression site mat1:1 and two silent loci, mat2.2 and mat3." 3. Besides reducing MT switching, the swi6 mutation leads to deletions in the MT region caused by intrachromosomal cross-overs between two paired cassettes. These deletions only arise if DNA double-strand breaks are present at mat1 : 1, which initiate MT switching. Furthermore, swi6 allows meiotic recombination in the K region, a region of 16 kb between mat2:2 and mat3: 3; in wild-type strains no recombination occurs in K. swi6 also allows the simultaneous expression of two different cassettes in the same haploid cell. Thus swi6 may have an influence on the general chromatin structure in the MT region.

The Journal of Immunology, 2000

Mast cells are recognized as a new type of immunoregulatory cells capable of producing different ... more Mast cells are recognized as a new type of immunoregulatory cells capable of producing different cytokines. So far, little is known about the cytokine profile of mature human mast cells isolated from intestinal tissue and cultured in the presence of stem cell factor (SCF). We observed that these cells express the proinflammatory cytokines TNF-alpha, IL-1 beta, IL-6, IL-8, IL-16, and IL-18 without further stimulation. Both IgE-dependent and IgE-independent agonists (e.g., Gram-negative bacteria) enhanced expression of TNF-alpha. Another set of cytokines consisting of IL-3, IL-5, IL-9, and IL-13 was expressed following activation by IgE receptor cross-linking. If mast cells were cultured in the presence of IL-4 and SCF, the production and release of IL-3, IL-5, and IL-13 was increased up to 4-fold compared with mast cells cultured with SCF alone. By contrast, IL-6 expression was completely blocked in response to culture with IL-4. In summary, our data show that mature human mast cells produce proinflammatory cytokines that may be up-regulated following triggering with IgE-independent agonists such as bacteria, whereas activation by IgE receptor cross-linking results in the expression of Th2-type cytokines. IL-4 enhances the expression of Th2-type cytokines but does not affect or even down-regulates proinflammatory cytokines.

Journal of Cellular and Molecular Medicine, 2011

The circadian clock in peripheral tissues can be entrained by restricted feeding (RF), a regimen ... more The circadian clock in peripheral tissues can be entrained by restricted feeding (RF), a regimen that restricts the duration of food availability with no calorie restriction (CR). However, it is not known whether RF can delay the occurrence of age-associated changes similar to CR. We measured circadian expression of clock genes, disease marker genes, metabolic factors and inflammatory and allergy markers in mouse serum, liver, jejunum and white adipose tissue (WAT) after long-term RF of 4 months. We found that circadian rhythmicity is more robust and is phase advanced in most of the genes and proteins tested under RF. In addition, average daily levels of some disease and inflammatory markers were reduced under

Journal of Allergy and Clinical Immunology, 2003

Objective: We sought to examine, for the first time, Fc RI signaling in mature human mast cells. ... more Objective: We sought to examine, for the first time, Fc RI signaling in mature human mast cells. Methods: Mast cells were isolated from intestinal tissue and cultured in the presence of stem cell factor. The cells were treated with specific inhibitors before stimulation by means of Fc RI cross-linking. Cytokine mRNA expression was analyzed by means of RT-PCR, and activation of signaling molecules was determined by means of immunocytochemistry, RT-PCR, Western blotting, and protein kinase C (PKC) assay.

Journal of Allergy and Clinical Immunology, 2002

Unidentified wheat presence in the food supply presents a serious health risk to both wheat-aller... more Unidentified wheat presence in the food supply presents a serious health risk to both wheat-allergic and celiac sprue patients. The Codex Alimentarius had previously set a level of less than 200 ppm for a food to be considered "gluten-free". Codex is considering changing this level to 20 ppm in the belief that this will better serve the celiac sprue patient. A commercially available wheat gluten detection kit that has a lower limit of detection of 10 parts per million wheat gluten [5 ppm gliadin] was used to test a variety of foods and ingredients for detectable gluten content. The test also exhibits some cross-reactivity to both barley and rye glutens. There is no commercial test available for total wheat protein, so this test is the only measure of a food's lack of wheat content. Foods tested included a variety of non-wheat foods at high risk for cross-contamination from the farm through food processing, foods labeled "wheat free" or "gluten free", and foods and food ingredients commonly avoided by celiac and wheat-allergic patients that originate from wheat or related cereals, but with questionable gluten content. Samples tested included at least two different lot numbers of each product. Results of this study indicate that some food products with questionable wheat content, such as alcoholic beverages, alcoholic carriers, alcohol-based flavorings, most types of vinegar, and most soy sauces, registered below 10 ppm in the assay. While seed oats did not show the presence of wheat gluten, oat-based cereal products had detectable wheat gluten content, probably due to contamination by wheat during commodity handling. Some food products labeled as wheat or gluten "free" exhibited detectable levels of gluten ranging from 22 to 71 parts per million. Based on the results of this study, the dietary choices for celiac sprue patients should not be as restricted as formerly assumed, and ingredients such as many vinegars and alcohol-based flavorings can likely be ingested without problem. However, wheat-allergic and celiac sprue patients must exercise care in choosing "gluten-free or wheat-free" -labeled foods, as some can contain appreciable amounts of wheat. Despite its worldwide consumption, beer has rarely been reported to elicit allergic reactions. We report a 59 year-old male patient who had suffered from angioedema and generalized urticaria 3 h after drinking 1.5 1 of WB and eating a pretzel. One month later, he had developed generalized pruritus, urticaria, and ultimately unconsciousness during a game of golf, 30 min after having drunk 1 1 of WB. Other alcoholic beverages, like wine or lager beers, were well tolerated. There was an immediate type skin prick test reaction to a commercial wheat flour extract, but not to a series of other commercial allergens. When using native material for skin prick testing, there were 3+ reactions to one brand of WB, 2+ reactions to wheat malt and another brand of WB, and a 1+ reaction to barley malt, which were all not observed in control subjects. There were specific IgE antibodies in the serum to wheat, barley and rye flour, to barley malt and to chicken meat. An oral challenge test with incremental doses of WB caused wheals occurring on the face after the ingestion of a cumulative dose of 1.6 1. Oral challenge with wheat flour also produced wheals on the face after the ingestion of a cumulative dose of 120 g. There have been sporadic reports of allergy to barley malt contained in lager beers. WB is brewed by using both barley and wheat malt, and our patient exhibited clinical reactions exclusively to wheat. This case is the first of a beer allergy specifically attributable to a sensitization to wheat.

Journal of Allergy and Clinical Immunology, 2003

RATIONALE: IL-3, tL-5 and GM-CSF exert overlapping biological functions in basophils and eosinoph... more RATIONALE: IL-3, tL-5 and GM-CSF exert overlapping biological functions in basophils and eosinophils, because their receptors have common signaling beta-chain. However, no comprehensive study has been performed to simultaneously compare the molar effects of IL-3, IL-5 and GM-CSF between different functions of these cells in the same experiment. METHODS: Survival enhancement, CDllb expression and CD69 expression were analyzed in both basophils and eosinophils. The enhancement of histamine release was studied in basophils. We also investigated the protein and mRNA expression levels of IL-3Ralpha, IL-5Ralpha and GM-CSFRalpha using flow cytometry and real-time PCR. RESULTS: The rank order of potency in basophils was IL-3 >> IL-5=GM-CSF for degranulation, survival and CDI lb expression. The ED50 of IL-3 was 0.5-1 pM, 0.03 pM and 7 pM, respectively, for these three functions. In eosinophils, the rank order was IL-5=GM-CSF > IL-3 for survival and CDI lb expression. The ED50 of IL-5 was 0.6 pM and 100pM for survival and CDI lb expression, respectively. However, in both cell types, CD69 expression was most potently induced by IL-3. Compared with eosinophils, basophils expressed a much higher level of IL-3Ralpha, similar or slightly lower level of IL-5Ralpha, and an apparently lower level of GM-CSFRaIpha. CONCLUSIONS: IL-3 is the most potent stimulator for basophils, whereas IL-5 and GM-CSF are most potent for eosinophils. In general, the rank order of potency of these cytokines corresponded exactly to their receptor expression levels. However, CD69 expression differed completely from this paradigm. Funding: Grant Monies t O1~ Cyclooxygenase (COX)Inhibition at the "time of Initial Antigen | U~J Presentation Results in Enhanced IL-13 and IgE

Journal of Allergy and Clinical Immunology, 2003

Journal of Allergy and Clinical Immunology, 2003

tor cross-linking. In this study, we examined the expression of cytokines by mast cells isolated ... more tor cross-linking. In this study, we examined the expression of cytokines by mast cells isolated from human intestinal mucosa following stimulation by stem cell factor (SCF). METHODS: Mast cells were purified by positive selection and cultured with SCF alone or with SCF and IL-4. Following pretreatment with specific inhibitors mast cells were stimulated with 1-100 ng/ml SCF for 90 min, Activation of mitogen-activated protein kinase (MAPK) was analyzed by Western blot and mRNA expression by RT-PCR. RESULTS: We did not detect an induction of cytokine expression by SCF in mast cells pre-cultured with SCF alone. In contrast, the mRNA for IL-5 and TNF-~ was strongly up-regulated in mast cells following stimulation with at least 10 ng/ml SCF if the cells were pre-cultured with SCF and IL-4. Cytokine expression was inhibited by apigenin but not by cyclosporin, wortmannin, and G0 6976. This indicates the necessity of mitogen-activated protein kinase (MAPK), but not of nuclear factor of activated T cells, phosphatidylinositol 3-kinase or protein kinase C. The activation of MAPK in response to SCF could be confirmed by Western blot. Consistently, we found activation of c-Fos, the downstream target of MAPK, a component of the transcription factor activator protein-1. CONCLUSIONS: In summary, our data show that SCF is capable of inducing cytokine expression in mature human mast cells pre-cultured with IL-4 through MAPK activation. RATIONALE: To characterize human cord blood derived mast cells in regard to chemokine oan chemokine receptor genes. METHODS: We have established/developed an in vitro method for generating very large quantities of mature an functionally intact human mast cells. Cells are derived from CD133+ progenitor cells isolated from cord blood and induced to differentiate into mast cells by cultivation in a serum free medium supplemented with stem cell factor and 1L-6 for 10 weeks. To further induce mast cell maturation, foetal calf serum wasadded during the subsequent 4 weeks. The availability of such large numbers of mast cells has for the first time made it possible to perform quantitative analyses at multiple cellular levels on a single cell sample: Flow cytometry, ELISA, Western blotting and RNAse protection assay. RESULTS: Following receptor-mediated or pharmacological activationof the mast cells for up to 24 hours the kinetics of induction of chemokine and chemokine receptor genes was monitored quantitatively at the level of transcription and translation. Moreover we demonstrate synthetic glucocorticoid and calcium signaling antagonist cyclosporin A quench different sets of chemokine genes in activated mast cells. CONCLUSIONS: We demonstrate the involvement of distinct signaling transduction pathways regulating various chemokine genes and their receptor genes upon activation of mast cells.