Immune Cytopenias after Ex Vivo CD34+-Selected Allogeneic Hematopoietic Cell Transplantation

Immune-mediated cytopenias (ICs), such as immune thrombocytopenia and immune hemolytic anemia, are among the adverse events after allogeneic hematopoietic cell transplantation (allo-HCT). Previous reports suggest that in vivo T cell depletion may increase the incidence of IC after allo-HCT. We evaluated whether a strategy that reduces functional donor T cells via ex vivo CD34⁺-selection associates with the development of IC in a cohort of 408 patients who underwent allo-HCT for hematologic malignancy. The cumulative incidence of IC at 6, 12, and 36 months after the 30-day landmark post-HCT was 3.4%, 4.9%, and 5.8%, respectively. Among 23 patients who developed IC, 7 died of relapse-related mortality and 4 of nonrelapse mortality. A median 2 types of treatment (range, 1 to 5) was required to resolve IC, and there was considerable heterogeneity in the therapies used. In univariable analyses, a hematologic malignancy Disease Risk Index (DRI) score of 3 was significantly associated with an increased risk of IC compared with a DRI of 1 or 2 (hazard ratio [HR], 4.12; P = .003), and IC (HR, 2.4; P = .03) was associated with increased risk of relapse. In a multivariable analysis that included DRI, IC remained significantly associated with increased risk of relapse (HR, 2.4; P = .03). Our findings show that IC events occur with relatively similar frequency in patients after ex vivo CD34⁺-selected allo-HCT compared with unmodified allo-HCT, suggesting that reduced donor T cell immunity is not causative of IC. Moreover, we noted a possible link between its development and/or treatment and increased risk of relapse.     

Establishment and Characterization of a Cell Based Artificial Antigen-Presenting Cell for Expansion and Activation of CD8+ T Cells Ex Vivo

Atificial antigen-presenting cells are expected to stimulate the expansion and acquisition of optimal therapeutic features of T cells before infusion. Here CD32 that binds to a crystallizable fragment of IgG monoclonal antibody was genetically expressed on human K562 leukemia cells to provide a ligand for T-cell receptor. CD86 and 4-1BBL, which are ligands of CO. stimulating receptors of CD28 and 4-1BB. respectively, were also expressed on K562 cells. Then we accomplished the artificial antigen-presenting cells by coupling K32, CD86/4-IBBL cell with OKT3 monoclonal antibody against CD3.named K32/CD86/4-lBBL/OKT3 cells. These artificial modified cells had the abilities of inducing CD8+ T cell activation. promoting CD8+ T cell proliferation, division, and long-term growth, inhibiting CD8+ T cell apoptosis, and enhancing CD8+ T cell secretion of IFN-Y and perforin. Furthermore, antigen. secific cytotoxic T lymphocytes could be retained in the culture stimulated with K32/CD86/4-1BBL/OKT3 cells at least within 28 day This approach was robust, simple, reproducible and economical for expansion and activation of CD8+ T cells and may have important therapeutic implications for adoptive immunotherapy. Cellular & Molecular Immunology.2007;5(1):47-53.     

Ex Vivo Gene Delivery to Hepatocytes: Techniques, Challenges, and Underlying Mechanisms

Gene delivery to primary hepatocytes is an important tool for a number of applications including the study of liver cell biology and pathology, drug screening, and gene therapy. Robust transfection of primary hepatocytes, however, is significantly more difficult to achieve than in cell lines or readily dividing primary cells. In this report, we investigated in vitro gene delivery to both primary rat hepatocytes and Huh7.5.1 cells (a hepatoma cell line) using a number of viral and non-viral methods, including Lipofectamine 2000, FuGene HD, Nucleofection, Magnetofection, and lentiviruses. Our results showed that Lipofectamine 2000 is the most efficient reagent for green fluorescent protein (GFP) gene delivery to primary rat hepatocytes (33.3 ± 1.8% transfection efficiency) with minimal adverse effect on several hepatic functions, such as urea and albumin secretion. The lentiviral vectors used in this study exhibited undetectable gene delivery to primary rat hepatocytes but significant delivery to Huh7.5.1 cells (>80% transfection efficiency). In addition, we demonstrated lentiviral-based and spatially defined delivery of the GFP gene to Huh7.5.1 cells for use in biological microelectromechanical systems.     

Comparative Analysis of Human-Derived Feeder Layers with 3T3 Fibroblasts for the Ex Vivo Expansion of Human Limbal and Oral Epithelium

Corneal transplantation with cultivated limbal or oral epithelium is a feasible treatment option for limbal stem cell deficiency (LSCD). Currently utilized co-culture of stem cells with murine 3T3 feeder layer renders the epithelial constructs as xenografts. To overcome the potential risks involved with xenotransplantation, we investigated the use of human-derived feeder layers for the ex vivo expansion of epithelial (stem) cells. Human limbal and oral epithelium was co-cultured with mouse 3T3 fibroblasts, human dermal fibroblasts (DF), human mesenchymal stem cells (MSC), and with no feeder cells (NF). Cell morphology was monitored with phase-contrast microscopy, and stem cell characteristics were assessed by immunohistochemistry, real-time PCR for p63 and ABCG2, (stem cell markers), and by colony-forming efficiency (CFE) assay. Immunohistochemical analysis detected positive staining for CK3 (cornea specific marker) and Iβ1 and p63 (putative stem cell markers) in all culture conditions. The level of Iβ1 and p63 was significantly higher in both limbal and oral cells cultured on the 3T3 feeder, as compared to the MSC or NF group (p<0.01). This level was comparable to the cells cultured on DF. Expression of p63 and ABCG2 in limbal and oral epithelial cells in the 3T3 and DF groups was significantly higher than that in the MSC or NF group (p<0.01). No statistical difference was detected between 3T3 and DF groups. The CFE of both limbal and oral cells co-cultured on 3T3 fibroblasts was comparable to cells grown on DF, and was significantly higher than that of cells co-cultured with MSC or NF (p<0.01). Epithelial cells grown on a DF feeder layer maintained a stem cell-like phenotype, comparable to cells grown on a 3T3 feeder layer. In conclusion, DF provides a promising substitute for 3T3 feeder cells during cultivation of xenobiotic-free corneal equivalents.     

Ex Vivo Expansion and Th1/Tc1 Maturation of Umbilical Cord Blood T Cells by CD3/CD28 Costimulation

One major limitation of UCBT is the lack of donor cells available for posttransplantation donor leukocyte infusions (DLI) to boost immunity or induce graft-versus-leukemia (GVL) activity. Starting from a ∼5% fraction of a UCB graft, we report the feasibility and biological characteristics of ex vivo expansion of frozen/thawed CB T cells by anti-CD3 and anti-CD28 antibody–coated Dynal beads in the presence of interleukin (IL)-2. We postulated that while undergoing expansion, UCB T cells may mature toward a Th1/Tc1 phenotype and acquire the potential for cytotoxicity. Whereas an almost 2-log expansion also led to the acquisition of IL-12Rα and an increase in Th1 characteristics, postexpansion lymphocytes produced less interferon-γ, tumor necrosis factor-α, and granzyme B; stored almost no perforin; and lacked cytotoxicity against allogeneic targets. Collectively, these suggest relative safety from acute/hyperacute graft-versus-host disease. CD8⁺ T cells expanded preferentially, whereas a higher rate of apoptosis in CD4⁺ T cells also promoted an inverted CD4/CD8 ratio. Most expanded T cells retained expression of CD27, CD28, and L-selectin but down-regulated CCR-7. In summary, UCB T cell proliferation sustained by CD3/CD28 co-stimulatory beads and IL-2 can lead to clinically relevant doses of DLI from a very small fraction of the UCB graft, although future strategies to reduce apoptosis may enhance their clinical potential.     

Effects of Flavonoids on the Resistance of Microsomes to Lipid Peroxidation In Vitro and Ex Vivo

Incubation of rat liver microsomes with preparations of grape flavonoids, dihydroquercetin, and silibinin increased their resistance to lipid peroxidation induced by NADPH-Fe2+. This was manifested in less pronounced accumulation of lipid peroxidation products and changes in activity of microsomal enzymes induced by lipid peroxidation. In vitro antioxidant activity of grape flavonoids markedly surpassed that of dihydroquercetin and silibinin. Addition of flavonoids into fodder led to moderate, statistically significant, and similar increase in the resistance of rat liver microsomes to ex vivo induced lipid peroxidation.     

鼠膝关节前交叉韧带和内侧付韧带的损伤拉伸研究

本研究是为了观察鼠膝关节前交叉韧带(Anterior cruciate ligament,ACL)和内侧副韧带(Medial collateral ligament,MCL)在拉伸损伤后的病理变化.股骨-ACL-胫骨和股骨-MCL-胫骨复合物取自20只雄性Wistar大鼠,复合物拉伸应变分别为10% 或20%,拉伸时间分别为10 min或30 min.将拉伸后的样本于10%的福尔马林中固定、石蜡包埋、切片,分别进行奥新兰-PAS及HE染色.结果发现未拉伸的对照组, ACL基质较MCL者含有更多的黏多糖; 当拉伸应变为10%时,ACL的大部分细胞被拉长呈纺锤形,并出现细胞核固缩,时间越长,核固缩现象越严重,MCL除细胞核改变外还伴有胶原的损伤;当拉伸应变为20%时,ACL出现核溶解及胶原撕裂,而MCL在拉伸不到20%时自胫骨连接处撕脱.拉伸后的 ACL和MCL样品从外观上依然完好而在显微镜下可明显看到细胞或胶原的损伤.韧带拉伸损伤首先发生于韧带细胞,进一步的拉伸会扩展到胶原纤维,细胞的严重受损对韧带的修复极其不利.     

Enterohemorrhagic Escherichia coli Colonization of Human Colonic Epithelium In Vitro and Ex Vivo

Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen causing gastroenteritis and more severe complications, such as hemorrhagic colitis and hemolytic uremic syndrome. Pathology is most pronounced in the colon, but to date there is no direct clinical evidence showing EHEC binding to the colonic epithelium in patients. In this study, we investigated EHEC adherence to the human colon by using in vitro organ culture (IVOC) of colonic biopsy samples and polarized T84 colon carcinoma cells. We show for the first time that EHEC colonizes human colonic biopsy samples by forming typical attaching and effacing (A/E) lesions which are dependent on EHEC type III secretion (T3S) and binding of the outer membrane protein intimin to the translocated intimin receptor (Tir). A/E lesion formation was dependent on oxygen levels and suppressed under oxygen-rich culture conditions routinely used for IVOC. In contrast, EHEC adherence to polarized T84 cells occurred independently of T3S and intimin and did not involve Tir translocation into the host cell membrane. Colonization of neither biopsy samples nor T84 cells was significantly affected by expression of Shiga toxins. Our study suggests that EHEC colonizes and forms stable A/E lesions on the human colon, which are likely to contribute to intestinal pathology during infection. Furthermore, care needs to be taken when using cell culture models, as they might not reflect the in vivo situation.     

体外细胞培养的加力装置及压力测量的研究

目的：在一定力学作用下,机体的器官、组织、细胞和生物大分子会发生相应的形态和功能改变,这对于维持正常生理功能具有重要作用。细胞力学是组织工程和细胞工程的基础之一,在离体培养过程中对细胞施加不同的机械力以研究应力对细胞的影响是细胞力学的一个重要研究领域,也是细胞力学的重要研究手段。本研究是为了模拟在体细胞的力学环境,实现在体外培养的条件下对细胞施加力的作用,设计了一种力加载装置和相应的压力检测模块。方法：力加载装置包括应力加载模块、细胞培养室、步进电机传动模块组成。计算机通过软件驱动步进电机控制活塞在培养室内直线往复运动,实现细胞培养室内压力大小、频率和持续时间的可控变化。应力检测模块可以实时监测培养室内压力大小的变化,并与预期参数对比后通过反馈系统调节各模块的运行,实现压力加载的精准控制。结果：系统压力加载的频率调控范围为0 Hz~1Hz,压力加载范围为-71 kPa~60 kPa。结论：该系统为研究压力对细胞的影响提供了一种简单、可行的方法,实验证明系统压力加载方式准确、可行,能对离体培养的细胞进行有效的压力加载。     

Ultrastructural Organization of Helicobacter pylori under Natural Conditions and during Ex Vivo Culturing

Ultrastructure of Helicobacter pylori were studied by transmission electron microscopy in freshly isolated and ex vivo cultured biopsy specimens of gastric antral mucosa from patients with duodenal ulcer.     

In Vivo and Ex Vivo Imaging Reveals a Long-Lasting Chlamydial Infection in the Mouse Gastrointestinal Tract following Genital Tract Inoculation

Intravaginal infection with Chlamydia muridarum in mice can ascend to the upper genital tract, resulting in hydrosalpinx, a pathological hallmark for tubal infertility in women infected with C. trachomatis. Here, we utilized in vivo imaging of C. muridarum infection in mice following an intravaginal inoculation and confirmed the rapid ascent of the chlamydial organisms from the lower to upper genital tracts. Unexpectedly, the C. muridarum-derived signal was still detectable in the abdominal area 100 days after inoculation. Ex vivo imaging of the mouse organs revealed that the long-lasting presence of the chlamydial signal was restricted to the gastrointestinal (GI) tract, which was validated by directly measuring the chlamydial live organisms and genomes in the same organs. The C. muridarum organisms spreading from the genital to the GI tracts were detected in different mouse strains and appeared to be independent of oral or rectal routes. Mice prevented from orally taking up excretions also developed the long-lasting GI tract infection. Inoculation of C. muridarum directly into the upper genital tract, which resulted in a delayed vaginal shedding of live organisms, accelerated the chlamydial spreading to the GI tract. Thus, we have demonstrated that the genital tract chlamydial organisms may use a systemic route to spread to and establish a long-lasting infection in the GI tract. The significance of the chlamydial spreading from the genital to GI tracts is discussed.     

Uncoupling the systemic circulation and the Ex Vivo circuit during experimental isolated liver perfusion

Performing an ex vivo liver perfusion as a transient liver support requires perfusing the liver with a flow of 1 ml/min per kg of liver, which could reach 25% of the cardiac output when a human liver is used. This high flow could be detrimental in patients with acute liver failure. Therefore, in an ischemic-induced liver failure pig model, we developed a circuit allowing low flows going out of and into the systemic circulation, whereas the flow going through the ex vivo liver is maintained at a high value. This was obtained by uncoupling the ex vivo circuit from the systemic circulation. Ex vivo liver perfusion was performed in a closed circuit with a flow averaging 1 ml/min per kg of ex vivo liver (700 to 800 ml/min, according to the weight of the livers we used). It was linked to the systemic circulation with input and output flows equal to that used during hemodialysis (200 ml/min). Compared with previously reported direct circuits, this perfusion system was well tolerated from a circulatory point of view. After the induction of ischemic liver failure, the ex vivo liver perfusion led to an increase in urea, branched amino acids to aromatic amino acid ratio, and fractional clearance of indocyanine green and galactose and to a decrease in ammonia and lactic acid. This system allowed the ex vivo liver to keep its clearing properties despite a low extracorporeal flow. It represents an extracorporeal circuit that could be used in place of the direct extracorporeal high-flow liver perfusion.     

Enhanced In Vivo Immune Responses to Bacterial Lipopolysaccharide by Exogenous CD40 Stimulation

The lack of specific T-cell help in immune responses to thymus-independent antigens results in weak, predominantly immunoglobulin M-mediated immunity with little or no memory. In the work presented here we show how the exogenous stimulation of CD40 by monoclonal antibodies can mimic T-cell help, resulting in enhanced immune responses which are protective against bacterial infection.     

Ex Vivo Programming of Antigen-Presenting B Lymphocytes: Considerations on DNA Uptake and Cell Activation

Plasmids used in DNA vaccination not only serve as a source of antigen, but also have an important adjuvant effect. This review focuses on recent advancements made in understanding how cells internalize DNA, and how internalized DNA activates immune response pathways. We also comment on the role of B cells in both of these processes.     

An Ex Vivo Study of the Biological Properties of Porcine Aortic Valves in Response to Circumferential Cyclic Stretch

Normal physiological mechanical forces cause constant tissue renewal in aortic valve leaflets (AVL) while altered mechanical forces incite changes in their structural and biological properties. The current study aims at characterizing the remodeling properties of AVL subjected to cyclic circumferential stretch in a sterile ex vivo bioreactor. The leaflets cultured were stretched at a maximum rate of 300%s(-1) corresponding to a 15% strain for 48 h. Collagen, sulfated glycosaminoglycan (sGAG), and elastin contents of the stretched, fresh, and statically incubated leaflets were measured. Cusp morphology and cell phenotype were also examined. AVLs exposed to cyclic stretch showed a significant increase in collagen content (p < 0.05) when compared to fresh and statically incubated AVLs. sGAG content was significantly reduced in the stretched AVLs (p < 0.05) when compared to the fresh leaflets and was comparable between stretched and statically incubated AVLs. There was no statistically significant change in elastin content in all the three groups of AVLs (p > 0.05). Native aortic valve morphology was well preserved in stretched leaflets. Immunohistochemistry and immunoblotting studies showed an increased expression of alpha-smooth muscle actin (alpha-SMA) in stretched leaflets while alpha-SMA expression was reduced in statically incubated AVLs when compared to the fresh leaflets. To conclude, circumferential cyclic stretch altered the extracellular matrix remodeling activity of valvular cells, and consequently the extracellular matrix composition of the AVLs. Most interestingly, the contractile and fibrotic phenotypic expression of valve interstitial cells was enhanced. These results show that circumferential cyclic stretch is a possible mediator for AVL remodeling activity.