Infection dynamics and virus-induced apoptosis in cell culture-based influenza vaccine production—Flow cytometry and mathematical modeling

Cell culture-based influenza vaccine manufacturing is of growing importance. Depending on virus strains, differences in infection dynamics, virus-induced apoptosis, cell lysis and virus yields are observed. Comparatively little is known concerning details of virus–host cell interaction on a cellular level and virus spreading in a population of cells in bioreactors. In this study, the infection of MDCK cells with different influenza A virus strains in lab-scale microcarrier culture was investigated by flow cytometry. Together with the infection status of cells, virus-induced apoptosis was monitored. A mathematical model has been formulated to describe changes in the concentration of uninfected and infected adherent cells, dynamics of virus particle release (infectious virions, hemagglutinin content), and the time course of the percentage composition of the cell population.     

Reshaping of T-lymphocyte compartment in adult prepubertaly ovariectomised rats: A putative role for progesterone deficiency

This study explores the role of ovarian hormones in the phenotypic shaping of peripheral T-cell pool over the reproductive lifespan of rats. For this purpose, 2-month-old prepubertally ovariectomised (Ox) rats, showing oestrogen and progesterone deficiency, and 11-month-old Ox rats, exhibiting only progesterone deficiency, were examined for thymus output, and cellularity and composition of major TCRαβ+ peripheral blood lymphocyte (PBL) and splenocyte subsets. Although ovariectomy increased thymic output in both 2- and 11-month-old rats, the count of both CD4+ and CD8+ PBLs and splenocytes increased only in the former. In the blood and spleen of 11-month-old Ox rats only the count of CD8+ cells increased. Although ovariectomy affected the total CD4+ count in none of the examined compartments from the 11-month-old rats, it increased CD4+FoxP3+ PBL and splenocyte relative proportions over those in the age-matched controls. The age-related differences in the cellularity and the major subset composition in Ox rats were linked to the differences in the ovarian steroid hormone levels registered in 2- and 11-month-old rats. The administration of progesterone to Ox rats during the seven days before the sacrificing confirmed contribution of this hormone deficiency to the ovariectomy-induced changes in the TCRαβ+ PBL and splenocyte pool from 11-month-old rats. The expansion of the CD8+ splenocyte subset in the 11-month-old Ox rats reflected increases in cellularity of memory and, particularly, naïve cells. This was due to greater thymic output of CD8+ cells and homeostatic proliferation than apoptosis in 11-month-old Ox rats when compared with age-matched sham-Ox control rats. The homeostatic changes within CD8+ splenocyte pool from 11-month-old Ox rats, most likely, reflected the enhanced splenic IL-7 and TGF-β mRNA expression. Overall, in adult female rats, circulating oestrogen and progesterone provide maintenance of T-cell counts, a diversity of T-cell repertoire, and the main T-cell subset composition in the periphery. Progesterone deficiency affects mainly the CD8+ lymphocyte compartment through increasing thymic CD8+ cell export and upsetting homeostatic regulation within the CD8+ splenocyte pool. These alterations were reversible through progesterone supplementation.     

Mitochondrion-dependent caspase activation by the HIV-1 envelope

Cells expressing the envelope glycoprotein complex (Env) encoded by the human immunodeficiency virus can fuse with cells expressing Env receptors (CD4 and CXCR4). The resulting syncytia undergo apoptosis. We developed a cytofluorometric assay for the quantitation of syncytium formation and syncytial apoptosis. Using this methodology, we show that caspase activation in syncytia is inhibited by pharmacological or genetic intervention on cyclin-dependent kinase-1, p53, and mitochondrial membrane permeabilization (MMP). Thus, transfection of fusing cells with the viral mitochondrial inhibitor of apoptosis encoded by cytomegalovirus, a specific inhibitor of MMP, prevented the mitochondrial cytochrome c release and abolished simultaneously the activation of caspase-3. Conversely, inhibition of caspases did not prevent MMP. These results indicate that Env-elicited syncytial apoptosis involves the intrinsic (mitochondrial) pathway.     

Semi-automated flow cytometric analysis of CD34-expressing hematopoietic cells in peripheral blood progenitor cell apheresis products

BACKGROUND: The measurement of CD34+ cells is the most important step in the quality control of peripheral blood progenitor cell apheresis products. For this purpose, flow cytometry is applied. Recently, a new test kit has been introduced for the enumeration of CD34-expressing cells, in combination with software support for semi-automation of data acquisition and analysis. STUDY DESIGN AND METHODS: This study evaluated the ProCOUNT kit. Ninety samples obtained from peripheral blood progenitor cell apheresis products from 39 patients with hemato-oncologic diseases were analyzed. For data acquisition and analysis, ProCOUNT software was used. Data comparison was performed with parallel measurements according to the International Society for Hematotherapy and Graft Engineering (ISHAGE) guidelines and the German reference protocol for analysis of CD34-expressing cells. RESULTS: Correlation of the German and ISHAGE techniques was excellent (r2 = 0.99). The initial correlation coefficient of ProCOUNT analysis with the German protocol was r2 = 0.89. In 21 (23.3%) of 90 ProCOUNT analyses, a warning message was encountered from the ProCOUNT software. Following manual reevaluation of these data with CellQUEST software, a correlation of r2 = 0.96 with the German protocol and r2 = 0.97 with the ISHAGE analyses was obtained. ANOVA testing revealed significant differences between ProCOUNT and ISHAGE techniques (p<0.05) and between ProCOUNT and the German protocol (p<0.05). No statistically significant difference between ISHAGE and German protocol was observed (p = 0.19). CONCLUSION: The ProCOUNT kit and software for semi-automated data acquisition and analysis represents a further step toward standardization of CD34 cell quantitation in peripheral blood progenitor cell apheresis products. However, the occurrence of software warnings is high, and analysis or data reevaluation by experienced staff is still mandatory. Therefore, currently there is no definite advantage of the kit and software over the existing guidelines for CD34+ analysis in peripheral blood progenitor cell grafts.     

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

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

A new flow cytometric method for simultaneous measurement of residual platelets and RBCs in plasma: validation and application for QC

BACKGROUND: Guidelines for the preparation of FFP in Austria and Germany require the quantification of residual RBCs in plasma. However, currently there is no validated method for enumeration of RBC counts as low as 1 x 10(9) per L. STUDY DESIGN AND METHODS: A new flow cytometric method was developed for QC, to simultaneously determine if fresh unfrozen plasma contains residual platelets and RBCs. This method uses test tubes (TruCount, Becton Dickinson Immunocytometry Systems) that contain a known number of brightly fluorescent polystyrene beads. Plasma is pipetted into these tubes and mixed with FITC-labeled anti-CD41 and PE-conjugated anti-glycophorin A MoAbs. Acquisition can be performed on a flow cytometer after an incubation period of 15 minutes. RESULTS: Individual standard curves for platelet counts revealed excellent correlation coefficients (r>0.998). Platelet counts were validated against simultaneous measurements by using a cell counter and a hemocytometer. While the flow cytometric method slightly overestimated platelet counts of >40 x 10(9) per L by 10 percent, its precision in the critical range was very good-that is, a deviation of platelets < or = 1 x 10(9) per L from target values, which was even better than microscopic enumeration. The limit of sensitivity was <0.5 x 10(9) per L of platelets. RBC counts were also validated against simultaneous measurements made with a second cell counter. Standard curves for RBC counts also revealed excellent correlation coefficients (r>0.997). The limit of sensitivity was <0.25 x 10(9) RBCs per L. Platelet counts in plasma obtained by plateletpheresis or plasmapheresis ranged from 3 to 60 x 10(9) per L. About 25 percent of all plasma samples had platelet counts greater than the allowed upper limit of 25 x 10(9) per L, while all plasma samples contained fewer RBCs than the upper limit of 6 x 10(9) per L. CONCLUSION: This newly developed method provides a simple, quick, precise, and easily reproducible tool for simultaneous measurement of residual platelets and RBCs in fresh plasma.     

Isolation and flow cytometric analysis of T-cell-depleted CD34+ PBPCs

BACKGROUND: To extend allogeneic HPC transplantation to a greater range of patients, the use of partially matched related donors is under development. Because of the inherently higher degree of histoincompatibility in such transplants, there is increased risk of GVHD as well as of graft failure. Ex vivo depletion of donor-derived T-lymphocytes from PBPCs is one of the most effective methods of preventing GVHD. Thus far, individual centers have used custom-developed procedures to deplete the graft of T cells that are responsible for alloreactivity, often employing relatively impure, nonstandardized reagents such as soybean agglutinin and complement. In addition, with improved methods of T-cell depletion, it has been difficult to accurately assess the number of T cells remaining. Because different centers have used different protocols to assay T cells, it has been difficult to reproduce and validate the results between institutions, and this has limited direct comparison of data between centers. STUDY DESIGN AND METHODS: A standardized approach for T-cell depletion was developed by using a Good Manufacturing Practice-manufactured magnetic cell separator (Isolex 300i, Nexell Therapeutics) and commercially available OKT3 antibody. T-cell depletion was performed on PBPCs from six haploidentical donors. RESULTS: CD34+ cell recovery was 47 percent (range, 31-63%) with a median purity of 94 percent (range, 75-99%) and median T-cell log depletion of 4.72 (range, 3.90-5.83). Because this high degree of depletion makes it challenging to accurately quantitate the remaining T cells, two highly sensitive flow cytometric protocols were developed, each of which accurately detects T cells with a sensitivity of 2 per 10,000 (0.02%). The purified CD34+ cells administered to the patients (dose range, 6.13-13.50 x 10(6)/kg) provided rapid neutrophil and platelet engraftment. CONCLUSION: With the Isolex 300i and a MoAb directed against T cells, a high degree of T-cell depletion is obtained. Sensitive, accurate, and reproducible assays have now been developed for T-cell enumeration in these highly purified cell populations.     

Adaptations in the structure and innervation of follicle-sinus complexes to an aquatic environment as seen in the Florida manatee (Trichechus manatus latirostris)

Florida manatees are large-bodied aquatic herbivores that use large tactile vibrissae for several purposes. Facial vibrissae are used to forage in a turbid water environment, and the largest perioral vibrissae can also grasp and manipulate objects. Other vibrissae distributed over the entire postfacial body appear to function as a lateral line system. All manatee vibrissae emanate from densely innervated follicle-sinus complexes (FSCs) like those in other mammals, although proportionately larger commensurate with the caliber of the vibrissae. As revealed by immunofluorescence, all manatee FSCs have many types of C, Adelta and Abeta innervation including Merkel, club, and longitudinal lanceolate endings at the level of the ring sinus, but they lack other types such as reticular and spiny endings at the level of the cavernous sinus. As in non-whisking terrestrial species, the inner conical bodies of facial FSCs are well innervated but lack Abeta-fiber terminals. Importantly, manatee FSCs have two unique types of Abeta-fiber endings. First, all of the FSCs have exceptionally large-caliber axons that branch to terminate as novel, gigantic spindle-like endings located at the upper ring sinus. Second, facial FSCs have smaller caliber Abeta fibers that terminate in the trabeculae of the cavernous sinus as an ending that resembles a Golgi tendon organ. In addition, the largest perioral vibrissae, which are used for grasping, have exceptionally well-developed medullary cores that have a structure and dense small-fiber innervation resembling that of tooth pulp. Other features of the epidermis and upper dermis structure and innervation differ from that seen in terrestrial mammals.     

Ex vivo microvesicle formation after prolonged ischemia in renal transplantation

INTRODUCTION: Patients with chronic renal failure suffer from dysfunction in coagulation. Kidney transplantation induces inflammatory reactions and thus activation of platelets. Activated platelets, in turn, form microvesicles by shedding. These microvesicles have been shown to have coagulant activities. Activated platelets in prolonged cold ischemia were associated with delayed graft function and inferior survival. We investigated ex vivo formation of microvesicles in kidney transplantation and the influence of cold graft storage on microvesicles. METHODS: 20 patients (47.4+/-10.6 years (mean+/-SD)) undergoing transplantation were included in the study after written informed consent. Dependent on cold preservation time of transplanted kidneys, recipients were allocated into two groups with 10.4+/-6.1 h (group 1) and 23.7+/-3.8 h (group 2) preservation time, respectively. Blood samples were drawn before anesthesia, 12 h, 2, 7 and 14 days after transplantation. To evaluate microvesicle release, samples were activated with thrombin-receptor-activating-peptide-6 (TRAP) or adenosine-di-phosphate. Microvesicles were counted as percentage of platelets smaller than a predetermined size in flow cytometry. RESULTS: Platelet derived ex vivo microvesicle formation was significantly higher up to 48 h after transplantation when stimulated with TRAP in group 1. Platelet count was significantly higher compared to baseline values in the short-term ischemia group but not with long-term ischemia. Creatinine was significantly lower at study end compared to baseline with no differences between both groups. CONCLUSIONS: Lower platelet microvesicle formation after ex vivo stimulation with TRAP was associated with longer graft ischemia time. This may be a sign of former activation of platelets which could influence graft function and survival.     

流式细胞计的多参数分选技术

多参数分选技术是多参数流式细胞计一项重要应用，此技术不仅提高分离细胞的纯度，而且可以得到更多的细胞信息及更多的不同性质细胞亚群，这无疑对更细地分离细胞，进一步研究了不同亚群的细胞性质是很有意义的。参照一些国外文献及经验，应用ＦＡＣ４４０流式细胞计，通过对人外周血细胞分析及分选，介绍了这项技术和流式细胞计多参数分选细胞方法，并详细地讨论了影响分选纯度的一些关键步骤，提供了多参数分选的一些数据供使用参