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Cholesterol is a determinant of the permeability barrier to NH3 in colonic crypts
colitis (weight loss, diarrhea, prolapse etc.) beginning at 2~3 weeks. The onset of dise~tse was delayed in the mice co-transferred with B cells. Surprisingly, disease was attenuated by mesenteric CD19+IgM~ B cells, but not splenic MZ B cells. Flow cytometry demonstrated colonization of CD19+IgMh~but not MZ B cells in the large intestine and mesenteric nodes of Rag2-/- recipients. The results suggest that CD19+IgMhk B cells localized in the gutassociated lymphoid tissue promote mucosal immunologic homeostasis and play the protective function against IBD susceptibility. Supported by NIH DK46763 and the Crohn's and Colitis Foundation of America.
M1144 IFN'y and Mast Ceils Are Responsible for Delayed Changes in Colonic Paracellular Permeability Induced by Acute Stress Demaude Julien, Laurent Femer, Nicolas Cenac, Rafael Garcia-Villar, Jean Fioramonti, Lionel Bueno Background: Stressful events are known to produce various effects on the gastro-intestmal tract, but pathways involved are not clearly defined. Mast cells (MC) contain chemotactic factors for T ceils, released upon activation, and IFN3, produced by stress-induced T calls is involved in the increase of gut epithelial cell permeability. This study aimed to characterize long-term (four days) changes in permeability triggered by stress and the possible implication of mast cells and IFN3t. Methods: Twelve groups of Swiss mice and two groups, B6 iafg~(IFN'y-deficient) and their control C57BL6/J mice were used in this study. Swiss mice (two groups) pretreated by the mast cell membrane stabilizer, doxantrazole (10 mg/kg) or its vehicle, were submitted during two hours to an acute stress session consisting of contention plus acoustic stimuli. Five of the groups of Swiss mice and the B6 infg4 mice and their control C57BL6/J received one injection of the mast cell degranulator, Brx-537A (bromofasalocid ; 2 mg/kg IF). Colonic paracellular permeability was assessed by intracolonic infusion of 500 ~il ~Cr-EDTA (0.7 ~tCi) for two hours daily (14:00 to 16:00) from day one to day four, after a single stress session or MC stimulation. Colons were collected and permeability expressed as the ratio between body and total (body plus colon) radioactivities. Results: In Swiss mice, the two-hour permeability increased significantly only at day 4 after a single stress session (3.9% -+ 0.4 vs 1.7% _+ 0.3), while no significant change was observed at days 1, 2 and 3 (2.3% _+ 0.3, 0.8% -+ 0.3 and 1.7% _-4"0.6 respectively). The same effect was obtained at day 4 after mast cell degranulatiun with Brx-537A (3.9% -+ 0.8 vs 0.9% + 0.1). Previous treatment with doxantrazole prevented the effects of stress on permeability seen at day 4 (1.5% -+ 0.3 vs 3.9% + 0.4). In B6 infg~, we did not observe the increased permeability seen at day 4 in C57BL6/J mice (0.9% -+ 0.2 vs 4.1% _+ 0.3). Conclusion: An increase of colonic paraceUular permeability is observed at day 4 after acute stress or mast cell degranulation, and IFN7 is required in the genesis of this delayed effect. These data support that mast cells are implicated in the increase of paracellufar permeability observed after an acute stress.
Ml147 Alteration of enteric biota by T cell-induced chronic colitis Yingzi Cong, Wayne Duck, Fengxia Qi, Stephame Stahlbunk, Charles O. Elson We have shown that transfer of cecal bacteria-specific C3H/HeJBir Thl cells into scid mice induces focal colitis predominately in cecum. Transfer of CD4 + CD45RBhi T cells also induces colitis in scid recipients, however with a diffuse pancolitis. The aim of present study was to test whether different colitis patterns might be explained by differences in gut bacterial species. Methods. Control C3H-scids, scid recipients of CD45RBhi T cells, and scid recipients of enteric hacteria-sprcific Bir-Thl ceils were housed in same environment and cecal bacteria taken at 8 wks post transfer. Colitic C3HIL-2-/- and normal C3HIL-2+/- mice were also included. 16s rDNA of bacteria was amplified by PCR with bacterial-specific universal primers and separated by denaturing gradient gel electrophoresis. The bands of interest were excised, cloned, sequenced, and categorized against known sequences in GenBank database. Results. Within each group the pattern of 16s rDNA bands were similar, however there were notable differences between normal control and colitic scid mice. The pattern of bands, and predominant members of microbiota, was altered in colitic recipients of CD45RBhi T ceils as compared to control scid mice. The pattern of bands was also altered in colitic scid recipients of Bir Thl cells, but different from CD45RBhi T cell recipients. A predominam band was present in all normal scid mice and colitic Bir-Thl recipients but not in colitic CD45RBhi recipients. This band was identical to a mouse Helicobacter ganmani. Two bands were present in all colitic Bir-Thl and CD45RBhi recipients but not in control scid mice. One of them was identified as Helicobacter hepaticus plus others, probably Porphyromonas sp. and Dysgonomonas sp. The other band inchided Clostridinm indolis plus others, probably Porphyromonas sp. and Bacteroides sp. The pattern of bands present in colitic 11.-2 4- mice was different from either of adoptive scid recipients, with many fewer bands present compared to control 11-2 +/- mice. A dominant band present in IL-2-/- mice but not in control 11-2 +/- mice was identified as mouse Helicubacter rodemium sp. Conclusion. Chronic T cell-mediated colitis altered the composition of the enteric microbiota. Even though colitis was induced by C3H/HeJBir CD4 + T cells in each model, the alterations of the enteric microbiota appeared distinct in each instance. The differences in the pattern of colitis after adoptive transfer of CD45RBhi T cells vs. pathogenic Bir Thl cells might be related to the different shifts in the enteric microbiota in these two models.
M1145 Cholesterol is a Determinant of the Permeability Barrier to NH3 in Colonic Crypts Satish K. Singh, Selvi Krishnan BACKGROUND & AIM: The weak base, ammonia, is a fermentation product found at high levels (-20 raM) in the colon. NH3 freely crosses most membranes by diffusing via a lipid path, resulting in rapid intracelhifar alkalinization. However, we previously described a unique apical barrier to NH3 diffusion in rabbit proximal colonic crypts; this barrier appears to shield cells of the mid-crypt region from the acid-base extremes of the luminal compartment. As the apical lipid domain of colonic epithelial cells is highly cholesterol.enriched, we sought to determine if depletmg its cholesterol content would increase apical permeability to NH3in intact crypts. METHODS: We perfused single crypts dissected from rabbit proximal colon while monitoring intracelhilar pH (pHi) by digitally imaging the fluorescent dye BCECF. All crypts were himinaUy pre-perfused with the mucolytic dithiothreitol (1 raM) for 10 min followed by a 45 min perfusion with either a HEPES Ringer alone or one supplemented with 2% J3--cyclodextrin, an extracellular cholesterol acceptor known to deplete membrane cholesterol. RESULTS: In control experiments we again observed that exposing the crypt lumen to 20 mM NI-I4C[,pH 7.4 for up to 15 rains caused no change from baseline pHi. (7.04-+0.03 to 7.05-+0.02, mean-+ SEM, N = 5 8 cells from 7 cwpts) while a 20 ram NH,CI pulse applied from the basolateral side of the same crypt caused an expected acute alkalinization of -0.2 pH units; increasing lumen NI-hCI fivefuld (100 raM, pH 7.4) did not unmask an alkalinizatinn. However, in crypts pretreated with luminal I~-cyclodextrin, a rapid alkalinization was observed when 100 mM NI-hC1 was introduced into the lumen (7.22-+0.01 to 7.28-+0.02, N = 6 0 cells from 6 crypts), consistent with apical NH3 entry. Observed alkalinization was not blocked by bath or lumen amiloride (1 mM) suggesting that Na-H exchange was not homenstatically stimulated to alkalinize phi. Subsequent removal of luminal NH4C1 resulted in a prompt return to baseline pHi. No extravasation of the cdlimpermeant tracer, fluorescein-dextran (40,000 MW) was observed at the conclusion of experiments suggesting that crypt integrity and intercellular junctions remained intact. CONCLUSION: The apical borders of rabbit proximal colonic crypts possess art apical diffusion barrier to NH3 that is, at least in part, determined by apical membrane cholesterol content; thus, dietary, genetic and/or environmental factors that affect crypt cell cholesterol content may, in fact, profoundly affect colonic epithelial cell health.
Ml148 Lymphocytes are Required for the Development of Chronic Enterocolitis in Mice with a Myeloid Cell Specific Star3 Deficiency Wolfgang Reindl, Hans A. Lehr, Hermann Wagner, lrmgard Forster Stat3 is the key signal transducer downstream of cytokine receptors signalling through gpl30. Using the Cre~~ recombmase system, mice with a cell type specific disruption of the Stat3 gene in macrophages and neutrophils (lysozyme M (LysM)credStat3 ~~ were generated (Takeda et al. Immunity 10, 39-49). Analysis of these mice demonstrated that Stat3 is indispensable for ILIO signalling in macrophages and neutrophils. This signalling defect causes a spontaneous, chronic enterocolitis and impaired weight gain observed in LysMcre/Stat3 a~ mice. The hyperreactivity of myeloid cells is also the reason for the high susceptibility to endotoxin shock seen in these animals. To elucidate the significance of lymphocytes in the pathogenesis of colitis and endotoxin shock we crossed the conditional knockout mice to a RAG deficient background (LysMcre/Stat3 n~ RAG'). Peritoneal macrophages isolated from LysMcre/Stat3~~ and LysMcre/Stat3 "~ RAGr showed identical levels of IFN~/, IL-12 and NO production after activation with LPS. However, histology displayed no signs of colitis in LysMcre/Stat3 ~ RAG~ mice and their increase in body weight was similar to littermate controls. Despite the similar potential of myeloid cells to produce proinflammatory mediators, RAG~- LysMcre/Stat3 ~ - mice showed enhanced resistance to LPS challenge compared to LysMcre/Stat3 ~ mice. Nevertheless LysMcre/Stat3 r'~ RAG/- mice were far more susceptible to endotoxin than wildtype controls. These results demonstrate, that despite the fact that myeloid cells predominate in fully developed lesions of LysMcre/Stat3 n~ mice, T and/or B-lymphocytes play a key role in the initiation and perpetuation of colitis.
Ml146 M1149 Mesenteric C d l 9 § , Igm TM B Cells Play a Protective Function in Gcti2 T Cell Transferred Colitis Mouse Model Bo Wei, Peter Velazquez, Matthew Schrage, Tiffany Huang, Jonathan Braun
Nod2 is expressed by Myeloid Blood Dendritic Cells, mature Nod2 mutant and wild-type Blood Dendritic Cells induce similar T-cell phenotypes Henri Braat, Jan Van Den Bmnde, Esther De Jong, A. Van Bodegraven, Martien Kapsenberg, Daan Hommes, Sander Van Deventer
Commensal enteric microflora trigger abnormal mucosal CIM T cell reactivity in susceptible hosts leading to the development of inflammatory bowel disease. Genetic and adoptive transfer experiments, notably by Bhan, Mizoguchi and colleagues, have suggested a rote of B cells in mucosal immunologic homeostasis and IBD resistance. Our previous work demonstrated that enteric bacteria and GPCIVGai2 signaling is required for marginal zone B cell development. Impaired formation of these B cell subsets in restricted flora or Gai2-/mice is associated with increased susceptibility to immune colitis. Since MZ B cells are the predominant B ceil producer of ID 10, and a major component of resistance to Gram-positive bacteria, we predicted that MZ-like B cells might play protective role in IBD development. In current study, we evaluated the immunoregulatory action of splenic MZ B cells and the related mucosal B cell subset in mesenteric nodes (CD19+IgM h') on Gai2-/- colitis. Purified Gcti2-/- CD3 + T cells were transferred i.p. into Rag2-/- mice either alone, or with co-transfer of wildtype splenic MZ or mesenteric CD19+IgMh*B cells. Gai2-/- T cells alone induced
AGA
Abstracts
Introduction: Nod2 is an important pattern recognition receptor of the innate immune system which is constitutively expressed in dendritic cells. The Nod2 3020insC mutation is present within a subpopulation of patients with Crohn's Disease (CD) and might lead to dendritic cell dysfunction due to a shortening of the pattern recognition domain of the Nod2 protein. Mature dendritic ceils are potent APC's able to initiate T-cell specific immune responses indispensable of CD. Two peripheral subtypes of dendritic cells have been described: the myeloid- and plasmacytoid Blood Dendritic Cells (BDC) are characterized by BDCA-1 and BDCA-4 expression respectively. Aim: To assess functional differences in BDC from patients with and without the Nod?. mutation. Methods: Peripheral blood from three patients with and three patients without the homozygous Nod?. mutation was used to isolate BDCA-1 + and BDCA-4 + BDC by a magnetic separation procedure. The expression of Nod2
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M1144 IFN'y and Mast Ceils Are Responsible for Delayed Changes in Colonic Paracellular Permeability Induced by Acute Stress Demaude Julien, Laurent Femer, Nicolas Cenac, Rafael Garcia-Villar, Jean Fioramonti, Lionel Bueno Background: Stressful events are known to produce various effects on the gastro-intestmal tract, but pathways involved are not clearly defined. Mast cells (MC) contain chemotactic factors for T ceils, released upon activation, and IFN3, produced by stress-induced T calls is involved in the increase of gut epithelial cell permeability. This study aimed to characterize long-term (four days) changes in permeability triggered by stress and the possible implication of mast cells and IFN3t. Methods: Twelve groups of Swiss mice and two groups, B6 iafg~(IFN'y-deficient) and their control C57BL6/J mice were used in this study. Swiss mice (two groups) pretreated by the mast cell membrane stabilizer, doxantrazole (10 mg/kg) or its vehicle, were submitted during two hours to an acute stress session consisting of contention plus acoustic stimuli. Five of the groups of Swiss mice and the B6 infg4 mice and their control C57BL6/J received one injection of the mast cell degranulator, Brx-537A (bromofasalocid ; 2 mg/kg IF). Colonic paracellular permeability was assessed by intracolonic infusion of 500 ~il ~Cr-EDTA (0.7 ~tCi) for two hours daily (14:00 to 16:00) from day one to day four, after a single stress session or MC stimulation. Colons were collected and permeability expressed as the ratio between body and total (body plus colon) radioactivities. Results: In Swiss mice, the two-hour permeability increased significantly only at day 4 after a single stress session (3.9% -+ 0.4 vs 1.7% _+ 0.3), while no significant change was observed at days 1, 2 and 3 (2.3% _+ 0.3, 0.8% -+ 0.3 and 1.7% _-4"0.6 respectively). The same effect was obtained at day 4 after mast cell degranulatiun with Brx-537A (3.9% -+ 0.8 vs 0.9% + 0.1). Previous treatment with doxantrazole prevented the effects of stress on permeability seen at day 4 (1.5% -+ 0.3 vs 3.9% + 0.4). In B6 infg~, we did not observe the increased permeability seen at day 4 in C57BL6/J mice (0.9% -+ 0.2 vs 4.1% _+ 0.3). Conclusion: An increase of colonic paraceUular permeability is observed at day 4 after acute stress or mast cell degranulation, and IFN7 is required in the genesis of this delayed effect. These data support that mast cells are implicated in the increase of paracellufar permeability observed after an acute stress.
Ml147 Alteration of enteric biota by T cell-induced chronic colitis Yingzi Cong, Wayne Duck, Fengxia Qi, Stephame Stahlbunk, Charles O. Elson We have shown that transfer of cecal bacteria-specific C3H/HeJBir Thl cells into scid mice induces focal colitis predominately in cecum. Transfer of CD4 + CD45RBhi T cells also induces colitis in scid recipients, however with a diffuse pancolitis. The aim of present study was to test whether different colitis patterns might be explained by differences in gut bacterial species. Methods. Control C3H-scids, scid recipients of CD45RBhi T cells, and scid recipients of enteric hacteria-sprcific Bir-Thl ceils were housed in same environment and cecal bacteria taken at 8 wks post transfer. Colitic C3HIL-2-/- and normal C3HIL-2+/- mice were also included. 16s rDNA of bacteria was amplified by PCR with bacterial-specific universal primers and separated by denaturing gradient gel electrophoresis. The bands of interest were excised, cloned, sequenced, and categorized against known sequences in GenBank database. Results. Within each group the pattern of 16s rDNA bands were similar, however there were notable differences between normal control and colitic scid mice. The pattern of bands, and predominant members of microbiota, was altered in colitic recipients of CD45RBhi T ceils as compared to control scid mice. The pattern of bands was also altered in colitic scid recipients of Bir Thl cells, but different from CD45RBhi T cell recipients. A predominam band was present in all normal scid mice and colitic Bir-Thl recipients but not in colitic CD45RBhi recipients. This band was identical to a mouse Helicobacter ganmani. Two bands were present in all colitic Bir-Thl and CD45RBhi recipients but not in control scid mice. One of them was identified as Helicobacter hepaticus plus others, probably Porphyromonas sp. and Dysgonomonas sp. The other band inchided Clostridinm indolis plus others, probably Porphyromonas sp. and Bacteroides sp. The pattern of bands present in colitic 11.-2 4- mice was different from either of adoptive scid recipients, with many fewer bands present compared to control 11-2 +/- mice. A dominant band present in IL-2-/- mice but not in control 11-2 +/- mice was identified as mouse Helicubacter rodemium sp. Conclusion. Chronic T cell-mediated colitis altered the composition of the enteric microbiota. Even though colitis was induced by C3H/HeJBir CD4 + T cells in each model, the alterations of the enteric microbiota appeared distinct in each instance. The differences in the pattern of colitis after adoptive transfer of CD45RBhi T cells vs. pathogenic Bir Thl cells might be related to the different shifts in the enteric microbiota in these two models.
M1145 Cholesterol is a Determinant of the Permeability Barrier to NH3 in Colonic Crypts Satish K. Singh, Selvi Krishnan BACKGROUND & AIM: The weak base, ammonia, is a fermentation product found at high levels (-20 raM) in the colon. NH3 freely crosses most membranes by diffusing via a lipid path, resulting in rapid intracelhifar alkalinization. However, we previously described a unique apical barrier to NH3 diffusion in rabbit proximal colonic crypts; this barrier appears to shield cells of the mid-crypt region from the acid-base extremes of the luminal compartment. As the apical lipid domain of colonic epithelial cells is highly cholesterol.enriched, we sought to determine if depletmg its cholesterol content would increase apical permeability to NH3in intact crypts. METHODS: We perfused single crypts dissected from rabbit proximal colon while monitoring intracelhilar pH (pHi) by digitally imaging the fluorescent dye BCECF. All crypts were himinaUy pre-perfused with the mucolytic dithiothreitol (1 raM) for 10 min followed by a 45 min perfusion with either a HEPES Ringer alone or one supplemented with 2% J3--cyclodextrin, an extracellular cholesterol acceptor known to deplete membrane cholesterol. RESULTS: In control experiments we again observed that exposing the crypt lumen to 20 mM NI-I4C[,pH 7.4 for up to 15 rains caused no change from baseline pHi. (7.04-+0.03 to 7.05-+0.02, mean-+ SEM, N = 5 8 cells from 7 cwpts) while a 20 ram NH,CI pulse applied from the basolateral side of the same crypt caused an expected acute alkalinization of -0.2 pH units; increasing lumen NI-hCI fivefuld (100 raM, pH 7.4) did not unmask an alkalinizatinn. However, in crypts pretreated with luminal I~-cyclodextrin, a rapid alkalinization was observed when 100 mM NI-hC1 was introduced into the lumen (7.22-+0.01 to 7.28-+0.02, N = 6 0 cells from 6 crypts), consistent with apical NH3 entry. Observed alkalinization was not blocked by bath or lumen amiloride (1 mM) suggesting that Na-H exchange was not homenstatically stimulated to alkalinize phi. Subsequent removal of luminal NH4C1 resulted in a prompt return to baseline pHi. No extravasation of the cdlimpermeant tracer, fluorescein-dextran (40,000 MW) was observed at the conclusion of experiments suggesting that crypt integrity and intercellular junctions remained intact. CONCLUSION: The apical borders of rabbit proximal colonic crypts possess art apical diffusion barrier to NH3 that is, at least in part, determined by apical membrane cholesterol content; thus, dietary, genetic and/or environmental factors that affect crypt cell cholesterol content may, in fact, profoundly affect colonic epithelial cell health.
Ml148 Lymphocytes are Required for the Development of Chronic Enterocolitis in Mice with a Myeloid Cell Specific Star3 Deficiency Wolfgang Reindl, Hans A. Lehr, Hermann Wagner, lrmgard Forster Stat3 is the key signal transducer downstream of cytokine receptors signalling through gpl30. Using the Cre~~ recombmase system, mice with a cell type specific disruption of the Stat3 gene in macrophages and neutrophils (lysozyme M (LysM)credStat3 ~~ were generated (Takeda et al. Immunity 10, 39-49). Analysis of these mice demonstrated that Stat3 is indispensable for ILIO signalling in macrophages and neutrophils. This signalling defect causes a spontaneous, chronic enterocolitis and impaired weight gain observed in LysMcre/Stat3 a~ mice. The hyperreactivity of myeloid cells is also the reason for the high susceptibility to endotoxin shock seen in these animals. To elucidate the significance of lymphocytes in the pathogenesis of colitis and endotoxin shock we crossed the conditional knockout mice to a RAG deficient background (LysMcre/Stat3 n~ RAG'). Peritoneal macrophages isolated from LysMcre/Stat3~~ and LysMcre/Stat3 "~ RAGr showed identical levels of IFN~/, IL-12 and NO production after activation with LPS. However, histology displayed no signs of colitis in LysMcre/Stat3 ~ RAG~ mice and their increase in body weight was similar to littermate controls. Despite the similar potential of myeloid cells to produce proinflammatory mediators, RAG~- LysMcre/Stat3 ~ - mice showed enhanced resistance to LPS challenge compared to LysMcre/Stat3 ~ mice. Nevertheless LysMcre/Stat3 r'~ RAG/- mice were far more susceptible to endotoxin than wildtype controls. These results demonstrate, that despite the fact that myeloid cells predominate in fully developed lesions of LysMcre/Stat3 n~ mice, T and/or B-lymphocytes play a key role in the initiation and perpetuation of colitis.
Ml146 M1149 Mesenteric C d l 9 § , Igm TM B Cells Play a Protective Function in Gcti2 T Cell Transferred Colitis Mouse Model Bo Wei, Peter Velazquez, Matthew Schrage, Tiffany Huang, Jonathan Braun
Nod2 is expressed by Myeloid Blood Dendritic Cells, mature Nod2 mutant and wild-type Blood Dendritic Cells induce similar T-cell phenotypes Henri Braat, Jan Van Den Bmnde, Esther De Jong, A. Van Bodegraven, Martien Kapsenberg, Daan Hommes, Sander Van Deventer
Commensal enteric microflora trigger abnormal mucosal CIM T cell reactivity in susceptible hosts leading to the development of inflammatory bowel disease. Genetic and adoptive transfer experiments, notably by Bhan, Mizoguchi and colleagues, have suggested a rote of B cells in mucosal immunologic homeostasis and IBD resistance. Our previous work demonstrated that enteric bacteria and GPCIVGai2 signaling is required for marginal zone B cell development. Impaired formation of these B cell subsets in restricted flora or Gai2-/mice is associated with increased susceptibility to immune colitis. Since MZ B cells are the predominant B ceil producer of ID 10, and a major component of resistance to Gram-positive bacteria, we predicted that MZ-like B cells might play protective role in IBD development. In current study, we evaluated the immunoregulatory action of splenic MZ B cells and the related mucosal B cell subset in mesenteric nodes (CD19+IgM h') on Gai2-/- colitis. Purified Gcti2-/- CD3 + T cells were transferred i.p. into Rag2-/- mice either alone, or with co-transfer of wildtype splenic MZ or mesenteric CD19+IgMh*B cells. Gai2-/- T cells alone induced
AGA
Abstracts
Introduction: Nod2 is an important pattern recognition receptor of the innate immune system which is constitutively expressed in dendritic cells. The Nod2 3020insC mutation is present within a subpopulation of patients with Crohn's Disease (CD) and might lead to dendritic cell dysfunction due to a shortening of the pattern recognition domain of the Nod2 protein. Mature dendritic ceils are potent APC's able to initiate T-cell specific immune responses indispensable of CD. Two peripheral subtypes of dendritic cells have been described: the myeloid- and plasmacytoid Blood Dendritic Cells (BDC) are characterized by BDCA-1 and BDCA-4 expression respectively. Aim: To assess functional differences in BDC from patients with and without the Nod?. mutation. Methods: Peripheral blood from three patients with and three patients without the homozygous Nod?. mutation was used to isolate BDCA-1 + and BDCA-4 + BDC by a magnetic separation procedure. The expression of Nod2
A-318