Membrane expression of proteinase 3 is genetically determined

Isolated human neutrophils exhibit a bimodal membrane proteinase 3 (PR3) expression. PR3 is the main target antigen in Wegener granulomatosis (WG). Cells with low expression can be easily distinguished from cell subsets with high expression. In a recent study, a large neutrophil subset expressing membrane PR3 (mPR3+) was a risk factor for systemic ANCA-associated vasculitis. PR3 membrane expression patterns are quite stable in a given individual, raising the possibility of genetic variance. The aims of this study were: (1) to investigate the association of mPR3 expression and the risk of WG in an independent German cohort; (2) to test the hypothesis that mPR3 expression on neutrophils is genetically influenced; and (3) to investigate whether or not mPR3 expression is a function of intracellular PR3 content. mPR3 expression was assessed by FACS analysis in isolated human neutrophils. Neutrophil mPR3 expression was studied in 35 patients with WG, 15 patients with other inflammatory diseases, 125 healthy volunteers, and 27 (15 monozygotic and 12 dizygotic) pairs of twins. The intracellular PR3 content was assessed by intracellular flow cytometry and by Western blotting after separating mPR3 low and high expressing cells by FACSort. FACS analysis in a subset of 16 healthy subjects showed a highly conserved PR3 phenotype in two independent investigations >12 mo apart (r = 0.937). Patients with WG demonstrated a significantly higher percentage of mPR3+ neutrophils than healthy controls and patients with other inflammatory diseases. The mPR3+ percentage was highly correlated in MZ twins (r = 0.99) compared with DZ twins (r = 0.06). The intracellular PR3 content was not different in persons with low or high mPR3 expression, nor was the PR3 content different in cells with low or high mPR3 expression within a given individual. These data indicate that WG patients have a higher percentage of mPR3-expressing neutrophils. Furthermore, mPR3 expression is influenced by genetic variance. Finally, mPR3 expression is independent of intracellular PR3 content.     

Neutrophil membrane expression of proteinase 3 (PR3) is related to relapse in PR3-ANCA-associated vasculitis

Wegener granulomatosis (WG) is strongly associated with the presence of antineutrophil cytoplasm autoantibodies (ANCA) with specificity for proteinase 3 (PR3). Relapses of WG are frequently preceded by a rise of autoantibody titer and PR3-ANCA are able to activate primed neutrophils in vitro. Except being stored intracellularly and translocated to the cell surface upon neutrophil stimulation, PR3 can also be detected on the surface of non-stimulated neutrophils (membrane PR3 or mPR3), with an interindividual variability in percentages of mPR3(-)-positive cells and level of mPR3 expression. This study began with the hypothesis that the presence of PR3 on the surface of non-stimulated neutrophils enables interaction with PR3-ANCA and influences clinical manifestations of the disease. It analyzed mPR3 expression on neutrophils of 89 WG patients in complete remission and 72 healthy controls to evaluate whether the presence of PR3 on the surface of resting neutrophils is related to clinical manifestations of WG and/or to the susceptibility to develop relapses. The number of patients with a bimodal mPR3 expression on resting neutrophils did not differ between patients and controls. However, in WG patients, an increased percentage of mPR3(+) neutrophils and an elevated level of mPR3 expression compared with healthy individuals (P = 0.037) were found. Within the group of WG patients, an elevated level of mPR3 expression was significantly associated with an increased risk for relapse (P = 0.021) and with an increased relapse rate (P = 0.011), but not with the disease extent or particular manifestations at diagnosis or at relapse. These data support the hypothesis that PR3 expression on the membrane of neutrophils plays a role in the pathophysiology of PR3-ANCA associated vasculitis.     

A large subset of neutrophils expressing membrane proteinase 3 is a risk factor for vasculitis and rheumatoid arthritis.

It has been shown previously that proteinase 3 (PR3), a neutrophil intracellular protease that is the main antigen of antineutrophil cytoplasm (ANCA) autoantibodies, is present on the plasma membrane of a subset of freshly isolated neutrophils. This study shows that the size of this subset of membrane PR3-positive (mPR3+) neutrophils is a stable feature of a given individual, most likely genetically controlled. It ranges from 0 to 100% of neutrophils and allows us to define a new polymorphism in the healthy population, with three discrete phenotypes corresponding respectively to less than 20% mPR3 + neutrophils (mPR3low) or to a mean percentage of 47% (mPR3intermediate) and 71.5% (mPR3high) mPR3+ neutrophils. The frequency of the mPR3high phenotype was significantly increased in patients with ANCA-associated vasculitis (85% versus 55% in healthy subjects). The percentage of mPR3+ neutrophils was not affected by disease activity, relapses, or therapy, and did not reflect in vivo cell activation. In addition, mPR3+ phenotypes were normally distributed in cystic fibrosis patients, indicating that infection and/or inflammation per se do not lead to a high percentage of mPR3+ neutrophils. The frequency of the mPR3high phenotype was not related to anti-PR3 autoimmunization, since it was increased in vasculitic patients regardless of the ANCA specificity (anti-PR3, anti-myeloperoxidase, or unknown). Interestingly, the frequency of the mPR3high phenotype was also increased in patients with rheumatoid arthritis. It was normal in type I-diabetes, a T cell-dependent autoimmune disease. It is proposed here that a high proportion of membrane PR3-positive neutrophils could favor the occurrence or the progression of chronic inflammatory diseases.     

Increased membrane expression of proteinase 3 during neutrophil adhesion in the presence of anti proteinase 3 antibodies

We investigated membrane proteinase 3 (mPR3) expression during TNF-alpha-induced adhesion of neutrophils in the presence of anti-PR3 antibodies, a situation occurring during anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis. Three increasing levels of mPR3 expression were observed on the mPR3(+) neutrophil subset after stepwise cell activation. TNF-alpha activation without adhesion, TNF-alpha-induced adhesion, and adhesion in the presence of anti-PR3 mAb or human anti-PR3 ANCA resulted, respectively, in a two-, seven-, and 24-fold increase of mPR3 levels. In plasma, anti-PR3 antibodies poorly recognized suspended neutrophils, whereas they bound to mPR3 on adherent cells. mPR3 upregulation was also triggered by IL-8, formyl-methionyl-leucyl-phenylalanine (fMLP), and neutrophil adhesion to activated human umbilical vein endothelial cells. It involved beta2 integrins and Fcgamma receptor, because it was prevented by anti-CD18 antibodies and was not observed with anti-PR3 F(ab')(2). Furthermore, it was specific to anti-PR3 mAb, and no mPR3 upregulation was observed with anti-myeloperoxidase or anti-HLA-ABC mAb. Newly expressed mPR3 molecules, after TNF-induced adhesion, were mobilized from secretory vesicles (CD35(+)) and secondary granules (CD11b(+)). The adhesion- and antibody-dependent upregulations of mPR3 expression occurred with little azurophilic granule degranulation, no sign of apoptosis, and no further CD177 upregulation. In conclusion, this study describes an amplifying loop in polymorphonuclear neutrophil activation process, whereby ANCA are involved in the membrane expression of their own antigen during cell adhesion. This could explain the restriction of ANCA-associated vasculitis to small vessels, the main site of neutrophil adhesion.     

Membrane proteinase 3 expression and ANCA-induced neutrophil activation

BACKGROUND: Proteinase 3 is the major autoantigen in Wegener's granulomatosis (WG). Membrane PR3 expression is bimodal; low expressing cells (mPR3(low)) can be distinguished from cells with high expression (mPR3(high)) within a given individual. High mPR3 expression is a WG risk factor and is associated with relapse. However, no mechanisms for this important clinical observation have been provided. We tested the hypothesis that mPR3 expression, rather than the expression of other membrane molecules implicated in anti-neutrophil cytoplasmic autoantibodies (ANCA) activation, determines the robustness of the PR3-ANCA-mediated response. METHODS: mPR3(low) and mPR3(high) neutrophils from a given individual were separated by magnetic cell sorting. Superoxide was measured by the ferricytochrome assay, and Akt phosphorylation by Western blotting. Double staining and flow cytometry were used to assay Fc gamma-receptor and beta 2-integrin expression with respect to the mPR3 phenotype. Degranulation was measured via beta-glucuronidase activity, migration with fibronectin-coated transwells, and cell quantification by the myeloperoxidase (MPO) assay. RESULTS: PR3-ANCA-treated mPR3(high) versus mPR3(low) neutrophils showed more superoxide generation (33.7 +/- 15.2 nmol O(2) (-) to 14.6 +/- 8.4, P < 0.01), more degranulation (29%+/- 5 to 22%+/- 3, P < 0.05), and more PI3-K/Akt activation. In contrast, all responses in both mPR3 subsets were similar after other stimuli. We observed no differences in the beta 2-integrin, Fc gamma R IIa, and III expression with respect to the mPR3 subtype. Furthermore, we found no differences in the mobilization of PR3-containing granules and no differences in migration through fibronectin. CONCLUSION: The degree of neutrophil mPR3 expression has definitive functional consequences.     

Kinetics of peripheral blood CD34-positive cells and the optimum timing for harvesting peripheral blood stem cells during BEP chemotherapy in patients with testicular germ cell tumor

PURPOSE: Recently, high-dose chemotherapy with peripheral blood stem cell (PBSC) rescue has been developed for poor risk testicular germ cell cancer. In this study, we investigated the optimum timing for harvesting PBSCs with the use of bleomycin + etoposide + cisplatin (BEP) chemotherapy, which is a well known first-line regimen for the testicular cancer. MATERIAL AND METHOD: Peripheral blood CD34-positive cell ratios were measured during a total of 10 courses of BEP chemotherapy in 6 patients with metastatic germ cell cancer between 1996 and 1998. We performed 4 apheresis in 3 patients during this period. Recombinant human granulocyte-colony stimulating factor (rhG-CSF) was administrated from the day on which the neutrophil count decreased less than 1,000/microliter. RESULTS: The peripheral blood CD34-positive cell ratios became maximum (3.0-24.6%; average 10.0%) on the day 18 to 21 (median day 19) of BEP chemotherapy with rhG-CSF administration. The maximum ratios of peripheral blood CD34 positive cells were achieved when the number of leukocyte were 6,880-23,600/microliter and exceeded 6,000/microliter after the 18th day of BEP chemotherapy. The average number of collected CD34 positive cells was 9.5 x 10(6)/kg at a single apheresis, and 12.6 x 10(6)/kg per patient. CONCLUSION: Efficient hematopoietic progenitor cells were mobilized by BEP chemotherapy with rhG-CSF administration of first-line setting. Our results suggest that the optimum timing of PBSCs harvest is the day when the numbers of leukocyte exceed 6,000/microliter after the 18th day of BEP chemotherapy and the following day.     

Membrane proteinase 3 and Wegener's granulomatosis

Proteinase 3 (PR3) is found in neutrophil and monocyte lysosomal granules. Anti-neutrophil cytoplasmatic antibodies (ANCA) with specificity for PR3 are characteristic for patients with Wegener's granulomatosis. The interaction of ANCA with neutrophilic ANCA antigens is necessary for the development of ANCA-associated diseases. ANCA bind to membrane-expressed PR3 and induce full-blown activation in primed neutrophils. We discuss two different aspects of membrane PR3 (mPR3). The first aspect is the amount of PR3 and mechanisms controlling this issue. The second aspect is the presence of two neutrophil subsets that differ in the mPR3 expression phenotype.     

Expression of stem cell markers on mononuclear cells derived from heparinized cadaveric organ donors before and after disconnection from the respirator

We have attempted to evaluate the level of the earliest human hematopoietic cell marker expression (CD34, CD117, CD133, CD184) on cells obtained from heparinized cadaveric organ donors before and after disconnection from the respirator. Moreover, we compared various cell populations: (1) coexpressing CD34/CD117; (2) CD34/CD133; (3) highly enriched hematopoietic stem cells (CD34+CXCR4+CD45+); and (4) highly enriched tissue-committed stem cells (CD34+CXCR4+CD45-). Finally, we analyzed whether the level of hematopoietic stem cell marker expression depended on the age of the donor. The expression of the membrane receptors (CD34, CD45, CD117, CD133, CD184) was studied by flow cytometry. We observed that the proportion of mononuclear cells expressing these markers slightly decreased in bone marrow harvested after disconnection from the respirator compared with the samples obtained before disconnection. Moreover, the proportion of cells expressing CD117 antigen depended on age of the donor.     

Successful multilineage engraftment of human cord blood cells in pigs after in utero transplantation

BACKGROUND: Successful engraftment of human hematopoietic stem and progenitor cells (HSPCs) in a large animal may serve not only as a model to study human hematopoiesis but also as a bioreactor to expand human HSPCs in vivo. The aim of this study was to accomplish xenotransplantation of human HSPCs into pig. METHODS: Total mononuclear or CD34-positive HSPCs obtained from human cord blood were xenotransplanted percutaneously under an ultrasonographic guidance into preimmune pig fetuses. Peripheral blood and bone marrow (BM) cells of recipient pigs were collected and analyzed for the presence of human cells by a polymerase chain reaction to detect human specific Alu sequence on DNA extracted from those cells. Fluorescence-activated cell sorting (FACS) analysis was also performed to detect human hematopoietic cells. RESULTS: Transplantation of human cord blood cells into pig fetuses aged less than 52 days postcoitus resulted in a good engraftment rate. In one case, engraftment was detected up to 315 days posttransplantation by polymerase chain reaction. Human hematopoietic cells were detectable also by FACS in peripheral blood and BM. Furthermore, human CD34+ HSPCs were also observed in the BM of recipients. Those CD34+ cells in BM were sorted by FACS and subjected to further analyses. First, in vitro colony formation assay resulted in formations of multilineage colonies. Second, when they were transplanted into an immunodeficient mouse they were engrafted in the mouse. CONCLUSIONS: These data indicate an engraftment of human HSPCs in pig BM. In utero transplantation of human HSPCs into a preimmune pig fetus is useful to establish a pig reproducing human hematopoiesis.     

Major histocompatibility complex HLA region largely explains the genetic variance exercised on neutrophil membrane proteinase 3 expression

ANCA-associated vasculitides, a common cause of rapidly progressive glomerulonephritis, are influenced by genetic variance. Neutrophil membrane expression of the ANCA antigen proteinase 3 (PR3) is pathogenically important. A subset of membrane PR3-positive neutrophils can be distinguished from a membrane-negative subset in any given subject. The percentage of membrane PR3-positive neutrophils is genetically determined. In this study, 17 pairs of HLA-matched siblings were typed for their percentage of membrane PR3-positive neutrophils. The HLA-matched siblings showed a high concordance (r = 0.67, P < 0.05), similar to that seen in monozygotic twins. For testing of whether the HLA system influences membrane PR3 percentage, membrane PR3 typing and HLA typing of 51 unrelated patients with Wegener's granulomatosis and 49 normal control subjects was performed. Using two independent statistical methods, a group of 34 HLA antigens was found to predict a large fraction of the membrane PR3 phenotype in patients and control subjects. Certain major histocompatibility HLA antigens have been implicated to conflicting degrees in ANCA-associated vasculitides. However, in earlier studies, the contribution of the HLA system to the genetic variance of the disease was unclear. In this cohort, found was an association of Wegener's granulomatosis with the same group of HLA antigens that predicted for membrane PR3 percentage and a similar correlation with clinical parameters at initial presentation. The disease status in 80% of the patients and 82% of the control subjects could be predicted correctly on the basis of HLA typing by discriminate function analysis (P < 0.001). After removal of the predicted individual from the sample, this association remained significant (64 and 63% correct prediction; P < 0.001). The data suggest that a complex interaction of the entire HLA system is responsible for the genetic influence on membrane PR3 percentage and Wegener's granulomatosis.     

Gene delivery using human cord blood-derived CD34+cells into inflamed glomeruli in NOD/SCID mice

BACKGROUND: Bone marrow reconstitution using genetically-modified hematopoietic stem cells has been reported to confer resistance to inflammation and prevent renal injury in glomerulonephritis. Although this strategy has potentials for clinical use, taking hematopoietic stem cells from bone marrow is highly stressful for patients. In this regard, umbilical cord blood may be a useful alternative and, therefore, we focused on their suitability as a source of hematopoietic stem cells for transplantation-based therapy for glomerulonephritis. METHODS: CD34+ cells were obtained from human umbilical cord blood, retrovirally transduced with human beta-glucuronidase (HBG) gene, and transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After confirming the successful chimerism, these mice were treated with lipopolysaccharide (LPS), and local HBG expression in glomeruli was examined using immunohistochemical analysis, HBG bioassay, and Western blot analysis. RESULTS: Clonogenic assay showed that 88.4 +/- 5.9% burst-forming unit-erythroid (BFU-E), 79.7 +/- 11.4% in colony-forming unit-macrophage (CFU-M), and 81.1 +/- 14.1% in colony-forming unit-granulocyte (CFU-G), respectively, possessed the transgene after transfection, suggesting that precommited cells were susceptible to retroviral infection. Flow cytometric analysis revealed that 24.1 +/- 14.5% of bone marrow cells in these chimera mice expressed human lymphocyte antigen (HLA) 8 weeks after transplantation. Also, clonogenic assay showed that a sustained engraftment of human hematopoietic cells expressed HBG. CD14-positive cells were recruited into the glomeruli upon LPS treatment and they secreted bioactive HBG, suggesting that cord blood-derived CD34+cells may differentiate into monocyte lineage while maintaining the expression of the transgene. CONCLUSION: These data indicate that umbilical cord blood cells can be utilized as a source of hematopoietic stem cells for the transplantation-based therapy of glomerulonephritis.     

人脐血造血干/祖细胞的磁力搅拌悬浮培养及移植实验

目的 探讨磁搅拌大规模培养体系对人脐血造血祖细胞的扩增效果以及扩增的人造血祖细胞植入动物体内后的造血重建情况.方法 从新鲜抗凝脐血中分离出单个核细胞(MNC),以添加干细胞因子、酪氨酸激酶受体3配基及血小板生成素的无血清培养体系进行培养.静态扩增组的细胞置于T25培养瓶中培养,磁搅拌悬浮扩增组(磁搅拌扩增组)的细胞采用Celstir装置进行培养,培养体系为50～100 ml.培养7 d后进行细胞计数、集落培养检测和细胞表面分子表达的测定.以不进行培养者为对照组.非肥胖糖尿病重症联合免疫缺陷(NOD/SCID)小鼠在接受2.5 Gy的亚致死剂量X射线照射后分别从尾静脉输入上述静态扩增组、磁搅拌扩增组和对照组的MNC(5×106个),另设不移植的空白对照组.观察小鼠的存活情况,6周后处死存活小鼠,检测骨髓细胞中CD34+细胞、CD3+细胞、CD19+细胞、CD33+细胞及CD45+细胞的含量以及人特异的Cart-Ⅰ和Alu基因的表达.结果 经过7天的培养,磁搅拌扩增组的造血祖细胞扩增倍数为(2.8±0.45)倍,明显高于静态扩增组的(2.1±0.48)倍(P<0.01).磁搅拌扩增组形成的红系集落、粒-巨噬细胞集落数均明显高于静态扩增组(P<0.05).静态扩增组扩增后的CD34+细胞、CD34+CD38-细胞和CD133+细胞含量均高于磁搅拌扩增组(P<0.05),而CD184+细胞和CD62L+细胞含量低于磁搅拌扩增组(P<0.01).移植后6周,对照组、静态扩增组和磁搅拌扩增组分别有3、4、5只小鼠存活,三组间两两比较,6周存活率的差异无统计学意义(P>0.05).存活6周的小鼠,其骨髓中能检人特异性CD34+细胞,以及CD3+细胞、CD19+细胞、CD33+细胞及CD45+细胞,也检测到人Alu基因和Cart-Ⅰ基因的表达.结论 磁搅拌培养能大规模扩增脐带血造血祖细胞,扩增的细胞能植入x射线照射的NOD/SCID小鼠,并重建其多系造血.     

Phosphodiesterase inhibition attenuates stored blood-induced neutrophil activation: a novel adjunct to blood transfusion

BACKGROUND: Activated neutrophils play a central role in the pathogenesis of ARDS and multiple organ failure (MOF). Transfusion of packed red blood cells (PRBCs) is an independent risk factor in the development of ARDS and MOF. It has been postulated that factors present in the supernatant of PRBCs activate neutrophils. The magnitude of neutrophil activation is dependent on the age of the stored blood. Our laboratory and others have reported that pentoxifylline (PTX), a nonspecific phosphodiesterase inhibitor, decreases neutrophil activation. We hypothesized that adding PTX to PRBCs would attenuate blood transfusion-induced neutrophil activation. STUDY DESIGN: Peripheral blood was obtained from healthy human volunteers. Oxidative burst, CD11b, and CD35 expression were measured by flow cytometry using a whole blood preparation. Whole blood was incubated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) (1 microM) alone and 42-day-old PRBC supernatant + fMLP with or without PTX (2 mmol/L). RESULTS: N-formyl-methionyl-leucyl-phenylalanine alone caused a significant increase in neutrophil oxidative burst (100%). The exposure of whole blood to PRBC supernatants + fMLP led to a 1.3-fold increase in neutrophil oxidative burst as compared with fMLP alone, indicating that PRBC supernatants prime neutrophils for oxidative burst by 75%. More importantly, PTX decreased neutrophil oxidative burst by 114% in supernatant + fMLP-stimulated whole blood (p < 0.001). PTX decreased CD11b expression in both fMLP (p < 0.01) and fMLP+supernatant-stimulated whole blood (p < 0.05). Supernatant from PRBCs did not have an additive effect to fMLP alone on CD11b expression. N-formyl-methionyl-leucyl-phenylalanine-induced CD35 expression was downregulated by PTX. The addition of PRBC supernatant did not increase the already upregulated fMLP-induced CD35 expression. CONCLUSIONS: Our results suggest that adding PTX to PRBC supernatant markedly decreases neutrophil activation. The lack of successful treatment strategies to effectively modulate the inflammatory response after blood transfusion indicates the need for novel therapies. Because the deleterious effects of blood transfusion on end-organ injury and MOF are associated with neutrophil activation, the adjunct use of PTX to blood transfusion may have therapeutic potential.     

Stem cell mobilization in idiopathic steroid-sensitive nephrotic syndrome

Steroid-sensitive nephrotic syndrome (SSNS) is classically thought to be a T-cell disorder. The aim of this study was to examine whether or not thymus homeostasis was affected in SSNS. Mature and naive T cell recent thymic emigrants were quantified in the peripheral blood of nephrotic patients and controls. Because the generation of new T cells by the thymus ultimately depends on hematopoietic stem cells, CD34+ cells were also included in the study. Nineteen patients with SSNS during relapse, 13 with SSNS during proteinuria remission, and 18 controls were studied. Cell-surface markers (CD3, CD4, CD8, CD19, CD16, CD56, CD45RA, CD62L, CD34, and CD38) were analyzed by flow cytometric analysis. T-cell rearrangement excision circles (TRECs) were quantified in CD2+ cells by real-time polymerase chain reaction. Stroma cell-derived factor-1 (SDF-1) genotype and metalloproteinase-9 (MMP-9) plasma levels were also determined. Mature T cells (CD4+ and CD8+), circulating naive T cells (CD62L+ and CD3+ CD62L+), and recent thymic emigrants (CD45RA+) as well as TRECs, that measure thymus production, had a similar level in the three groups of patients. Conversely, CD34+ hematopoietic stem cells displayed a two-fold increase in SSNS patients during relapse either compared with controls or SSNS patients at remission. In addition, compared with controls, SSNS patients at remission displayed (1) a decrease in CD19+ cells (B cells) and (2) an increase in CD16CD56+ cells [natural killer (NK) cells]. In conclusion, thymus homeostasis is not significantly affected in nephrotic patients. Hematopoietic stem-cell mobilization at proteinuria relapse, as well as changes in B and NK cells during remission, suggest that SSNS might be due to a general disturbance of hematopoietic and immune cell trafficking.     

Expression of stem cell markers on fetal and tumoral human liver cells in primary culture

Hepatic stem cells can be identified by the expression of putative markers such as CD117 (c-kit), CD90 (Thy-1), CD34, and HLA-DR. We have identified populations expressing these markers in both fetal and tumoral human liver by flow cytometry, using monoclonal antibodies against CD90, CD117, CD34, and HLA-DR. In tumoral liver CD117+/CD90+ cells were found in decreasing number from the neoplastic (2.48 +/- 0.67) and peritumoral region (0.88 +/- 0.12) to the area of para-tumoral (normal) parenchyma (0.13 +/- 0.04). The CD117+/CD34+ cells showed the following distribution: 0.35 +/- 0.05% in the tumoral region, 1.01 +/- 0.23% in the peritumoral region and 0.35 +/- 0.01 in the para-tumoral region. Using the same markers on fetal liver cells we have also identified small populations of CD117+/CD90+ cells (0.28 +/- 0.07%) and CD117+/CD34+ cells (1.13 +/- 0.24%), presumably resident stem cells or hematopoietic stem cells. Immunomagnetic negative separation was then performed on fetal liver cells using monoclonal antibodies against specific markers of hematopoietic lineages such as CD3, 14, 16, 19, 22, and CD56 to eliminate this population. The remaining cells were then incubated with fluorescently labeled monoclonal antibodies against CD90 and CD117 and analyzed using fluorescence microscopy. As expected these markers were expressed on the majority of the selected cells (89.28 +/- 9.56%). Isolation using appropriate markers and initiation of primary cultures is a first step to the therapeutic use of fetal stem cells and for the study of adult liver stem cells involvement in carcinogenesis.     

Renal expression of matrix metalloproteinases in human ANCA-associated glomerulonephritis.

BACKGROUND: Expression of matrix metalloproteinases (MMPs) by infiltrating and intrinsic renal cells is increased in inflammatory conditions, and may correlate with disease activity of glomerulonephritis. We analysed renal expression of MMPs, tissue inhibitor of metalloproteinase-1 (TIMP-1) and markers of neutrophil and monocyte infiltration in renal biopsies of patients with active anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis. METHODS: Immunohistochemical expression of MMP-2, -3, -9, TIMP-1, the neutrophil- and monocyte-derived MMP activators cathepsin G, neutrophil elastase and myeloperoxidase (MPO), and the monocyte marker CD14 was determined in renal biopsies of active proteinase 3 (PR3)-ANCA (n = 7) and MPO-ANCA (n = 6) associated glomerulonephritis, and in normal renal tissue (n = 4). Double labelling experiments of MMPs and TIMP-1 were performed with MPO and CD68, labelling neutrophils and macrophages. RESULTS: MMP-2-, MMP-3-, MMP-9- and TIMP-1-positive cells were detected in ANCA-associated glomerulonephritis in glomeruli with active inflammation (cellular crescents or fibrinoid necrosis), only occasionally in normal appearing glomeruli, and not in sclerotic glomeruli and positive cells were found in the tubulo-interstitium. MMPs and TIMP-1 were expressed predominantly by MPO-and CD68-positive cells. In normal renal tissue, no expression was detected, with the exception of weak mesangial staining for MMP-2. In ANCA-associated glomerulonephritis, glomerular MMP-2, -9 and TIMP-1 correlated with glomerular cathepsin G expression, while the number of MMP-9-expressing cells per glomerulus correlated with the percentage of crescentic glomeruli. Tubulo-interstitial expression of MMPs correlated with all markers of neutrophil and monocyte infiltration, and interstitial MMP-9 and TIMP-1 expression correlated with renal function at the time of renal biopsy. CONCLUSIONS: Expression of glomerular and interstitial MMP-2, -3, -9 and TIMP-1 is increased in active ANCA-associated glomerulonephritis and correlates with inflammatory activity.     

动员自体骨髓干细胞对大鼠急性肾小管坏死的治疗

目的：观察粒细胞集落刺激因子联合干细胞因子动员骨髓干细胞的作用、骨髓干细胞是否具有向损伤肾组织归巢的能力及其在肾脏组织中的分布,初步探讨粒细胞集落刺激因子联合干细胞因子是否具有促进急性肾小管坏死修复的作用。方法：160只8～10周龄雄性SD大鼠随机分为4组：对照组,模型组、G-CSF＋SCF治疗组、G-CSF＋SCF对照组,检测：（1）外周血白细胞总数及单个核细胞中CD34＋细胞百分比的变化;（2）尿NAG酶检测;（3）肾脏组织病理学改变;（4）肾组织CD34＋细胞表达变化。结果：（1）G-CSF＋SCF治疗组和G-CSF＋SCF对照组外周血中白细胞数、CD34＋细胞百分比于第5天达高峰,与对照组、模型组相比,差异有统计学意义（P〈0.05）,以后逐渐下降;相应地,G-CSF＋SCF治疗组肾组织内CD34＋细胞较对照组、模型组也明显增多（P〈0.05）。（2）手术后第5、10、17天,G-CSF＋SCF治疗组尿NAG酶、肾脏病理学改变均明显好于模型组（P〈0.05）。第24天G-CSF＋SCF治疗组尿NAG酶、肾脏病理学改变基本恢复正常,而模型组仍异常。第31天各组间尿NAG酶、肾脏病理学改变其差异无统计学意义。结论：（1）粒细胞集落刺激因子和干细胞因子联合应用对缺血再灌注损伤诱发急性肾小管坏死大鼠的骨髓干细胞有显著的动员作用。（2）骨髓干细胞能在损伤的肾小管归巢和定居,并可能参与损伤肾组织的修复。（3）粒细胞集落刺激因子和干细胞因子联合应用能在一定程度上加速急性肾小管坏死后肾功能的修复。     

Transplantation of human peripheral blood stem cells into fetal rhesus monkeys (Macaca mulatta)

BACKGROUND: Methods for assessing engraftment efficiency have been explored in a primate xenogeneic model of in utero hematopoietic stem cell transplantation. METHODS: Human peripheral blood stem cells (PBSC) were obtained by leukapheresis from a human male donor after 4 days of administration of recombinant human granulocyte-colony stimulating factor (5 microg/kg/ day). PBSC were enriched for the CD34+ population with and without T-cell depletion. The resulting mononuclear cells consisted of two cell populations, one that was stem cell enriched (0.83% CD3+ cells, 95% CD34+; group 1) and one that was stem cell enriched and T-cell depleted (<0.03% CD3+ cells, 98% CD34+; group 2). Four fetal monkeys (two per group) received either two or four i.p. injections (approximately 5x10(6) cells/injection) via ultrasound guidance every other day over a 7-day period (gestational days 50, 52, 54, and 56). One fetus in each group also received i.p. recombinant human stem cell factor (25 microg/kg) and recombinant human granulocyte-colony stimulating factor (10 microg/kg) posttransplant every 10 days from gestational day 60-150. RESULTS: Four healthy newborns were delivered at term, and specimens were analyzed by polymerase chain reaction for the human Y chromosome (birth, monthly to 6 months; blood, marrow, progenitor assays). Polymerase chain reaction results were positive for all four newborns in all specimens assessed, and flow cytometric analysis for human CD45 in marrow showed engraftment ranging from 0.1-1.7%. There was no evidence of graft-versus-host disease in any of the animals. CONCLUSION: These studies show that (1) multilineage engraftment of human PBSC can be achieved in the fetal rhesus recipient, (2) the rhesus fetus appears to tolerate relatively high numbers of human CD3+ cells, and (3) healthy chimeric rhesus infants can be delivered at term after multiple in utero procedures.     

Identification of hematopoietic cell populations from the dermal papillae of human hair follicles

Hematopoietic stem cell (HSC) activity has been identified from the hair follicles (HFs) in mice; however, it has not been identified in human HFs. We used immunohistochemistry and flow cytometry to identify cultured dermal papilla (DP) cells expressing CD45 to test for hematopoietic activity in colony-forming assays of granulocyte/macrophage hematopoietic progenitors (CFU-GM). Occasional CD45-positive cells were detected in cultured DP cells. After in vitro stimulation with IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) for 7 days, about 1% of the cells were CD45-positive by flow cytometry analysis, an fifty-fold expansion in cell numbers. We further examined whether mesenchymal stem/progenitor cells reside in human dermal papillae. Cultured DP papilla cells incubated with monoclonal antibodies to remove the CD45 positive cells were induced into multilineage differentiation with the formation of CFU-GM. Our findings preliminarily indicate that human dermal papilla contain at least a CD45-positive hematopoietic cell population and a mesenchymal stem/progenitor cell population.     

Proteinase-3 mRNA expressed by glomerular epithelial cells correlates with crescent formation in Wegener's granulomatosis

BACKGROUND: Wegener's granulomatosis (WG) is characterized by systemic vasculitis with crescentic glomerulonephritis (CGN) and circulating autoantibodies directed against neutrophil cytoplasmic antigens (ANCA). Proteinase 3 (PR-3), a neutral serine proteinase in neutrophils implicated in the growth control of myeloid cells, has been identified as the target antigen for ANCA in WG. Since the kidneys are frequently involved in WG, we studied the in situ expression of PR-3 by renal parenchymal cells. METHODS: We assessed the expression of PR-3 in kidney biopsies of 15 patients with WG by immunohistochemistry (IHC) and in situ hybridization (ISH). Normal kidney tissue served as the control. RESULTS: We detected PR-3 mRNA and PR-3 protein in distal tubular epithelial cells (TECs) and glomerular epithelial cells (GECs) in normal kidney tissue and in CGN. Furthermore, a strong glomerular PR-3mRNA expression restricted to the site of cellular crescents was detected in patients with WG. The analysis of 144 glomeruli with cellular or sclerotic crescents revealed a positive correlation of glomerular PR-3mRNA expression with the percentage of cellular crescents per glomerulus. The capability of human TECs and GECs to synthesize PR-3 was confirmed by Northern blot and ISH on cultured cells. CONCLUSION: These data provide evidence that nonhematopoetic renal parenchymal cells express PR-3 and that glomerular expression of PR-3 is associated with crescent formation in WG. Our findings suggest that renal parenchymal cells may directly be involved in the pathogenesis of CGN in WG.