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.     

Monoclonal antibodies against human granulocytes and myeloid differentiation antigens

Monoclonal antibodies (MCA) were obtained by immunizing BALB/c mice with 99% pure granulocytes from normal donors or with a whole leukocyte suspension obtained from a chronic myelogenous leukemia (CML) patient, and then fusing the mouse spleen cells with a 315–43 myeloma cell clone. Four MCA were selected and studied using ELISA, immunofluorescence, cytotoxicity assays, and FACS analysis. Antibodies 80H.1. 80H.3. and 80H.5 (from normals) and 81H.1 (from CML) detected antigens expressed on neutrophils. Antibodies 80H.1 and 80H.3 (lgG) also reacted with monocytes but not with other blood cell subsets. Antibodies 80H.5 and 81H.1 (lgM) were cytotoxic and reacted strongly with most of the cells of the neutrophil maturation sequence. i.e., myeloblasts, promyelocytes, myelocytes, and mature granulocytes. Antibodies 80H.5 and 81H.1 also inhibited BFU-GM and CFU-E. Antigens recognized by 80H.3. 80H.5, and 81H.1 were expressed both on a proportion of cells from HL.60, KG.1, ML.1, and K562 myeloid cell lines, and on a proportion of blast cells isolated from patients with acute myelogenous leukemia. They were not found on lymphoid cell lines or lymphoid leukemia cells. These MCA recognize either late differentiation antigens expressed on mature neutrophils and monocytes (80H.1 and 80H.3) or early differentiation antigens (80H.5 and 81H.1) specific to the granulocytic lineage. They may be useful for a better definition of those antigens specific to hematopoietic stem cells and their relationship with normal or neoplastic hematopoiesis.     

Loss of the CD5+ and CD45RAhi B cell subsets in alcoholics

Chronic alcoholics are frequently immunodeficient, have polyclonal hypergammaglobulinaemia, and often have autoantibodies. Recent work in other diseases has shown that functional distinctions of possible relevance to autoimmunity and immunodeficiency can be found among the B cell subsets defined by differential expression of the surface markers CD5 and CD45RA. Therefore, we have evaluated the CD5,CD45RA B cell subsets of both chronic alcoholics without evidence of active liver disease (AWLD), and alcoholics admitted for acute alcoholic liver disease (ALD). Mean B cell numbers were normal in AWLD, but significantly reduced in ALD. Analysis of B cells by three-colour flow cytometry in 20 patients and 29 controls revealed a sharp decrease in the percentage of alcoholics’ B cells which were CD5⁺, 37·6% versus 16·3%, P<0·00001; absolute CD5⁺ B cell numbers were similarly reduced (58·9 cells/μl versus 20·9; P =0·0012). In addition to the loss of CD5⁺ B cells, there was a reduction in the percentage of B cells which are CD5⁻CD45RA^hi, leaving many patients with a B cell profile which was predominantly CD19⁺CD5⁻CD45RA^lo. This subset appears phenotypically similar to the IgM-producing CD5⁻CD45RA^lo subset described by others, and may be enriched for autoantibody-producing cells. One outlier patient was an ALD with 61% of B cells which were CD5⁺, which also is a profile consistent with increased autoantibody production.     

Coexistence of somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivity in some forebrain neurons

The indirect immunofluorescence technique was used to demonstrate the coexistence of somatostatin together with avian pancreatic polypeptide-like immunoreactivity within certain neurons of the rat forebrain. Numerous neurons containing these peptides were observed in the neocortex, hippocampus, olfactory tubercle, striatum, nucleus accumbens and lateral septum. In studies of serial sections stained alternately for these two peptides, and in restaining experiments, It could be determined that in many neurons in these areas these two peptides coexisted. In other brain areas such as the anterior periventricular hypothalamus, somatostatin cells were never found to contain avian pancreatic polypeptide-like immunoreactivity. Also, within the pancreas these two peptides were never found to coexist in the same cells. The findings represent a further example of the coexistence of more than one neuropeptide within a single neuron.     

A human B cell differentiation antigen selectively expressed on mature B cells identified by a monoclonal antibody (HB-2)

A monoclonal IgM antibody (HB-2), produced against a membrane antigen on the Raji, B cell line, reacted by indirect immunofluorescence with 2 to 40% of lymphoblasts from the B cell lines, Raji, Daudi, SN-1036, and SB but not with other types of cell lines, including pre-B, myeloid, melanoma, or T cells. HB-2 antibody reacted with 10 ± 3% of normal blood mononuclear cells, and was unreactive with monocytes, granulocytes, platelets, or erythrocytes. Two-color immunofluorescence revealed that HB-2 antigen expression was confined to cells bearing surface Ig. An interesting finding was the fact that 25% of plasmablasts induced by pokeweed mitogen also expressed the HB-2 antigen. However, pre-B and plasma cells from normal bone marrow did not express the HB-2 antigen either on their membrane surface or in their cytoplasm. Analysis of 85 leukemias revealed that the HB-2 antigen was expressed on acute and chronic B cell leukemias and Burkitt's lymphomas, but not on malignacies of pre-B, T, myelocytic, or plasma cells. The results suggest that expression of the HB-2 antigen is confined to mature B cells.     

Immunofluorescent evidence for exocytosis and internalization of secretory granule membrane in isolated chromaffin cells

Cultured bovine adrenal medullary chromaffin cells were stimulated with the secretogogues Ba2+ or carbamyl choline plus Ca2+ in the presence of a monospecific rabbit IgG fraction directed against bovine dopamine beta-hydroxylase. The anti-dopamine beta-hydroxylase was labeled either with fluorescent protein A or with a fluorescent second antibody to rabbit IgG. Stimulation produced a patchy cell surface distribution of fluorescence. There was no noticeable internalization of the fluorescence for up to 2 h. In similar experiments using fluorescent monovalent fragments (Fab) of the same monospecificidopamine-beta-hydroxylase IgG, a more uniform distribution of the fluorescence was observed. A few min after a 5 min period of stimulation with Ba2+, the fluorescence appeared to be on or near the cell surface; however, after 20 min or more it was distributed throughout the cytoplasm except that the cell nuclei were not labeled. Thus, dopamine beta-hydroxylase which appeared on the cell surface as a consequence of exocytosis was internalized in the presence of monovalent antibody fragments, but not in the presence of the divalent (polyclonal) antibody, presumably because endocytosis of dopamine beta-hydroxylase was inhibited by crosslinking of the dopamine beta-hydroxylase molecules. The internalized anti-dopamine beta-hydroxylase Fab fragments were found to reappear on the cell surface during a second secretory response. It is concluded that the interior of the chromaffin granule membrane, for which dopamine beta-hydroxylase is a marker, becomes exposed on the surface of the cell during secretion and that the membrane is then retrieved back into the cell where it can be re-used in a further secretory cycle.     

Detection of radiolabelled bone marrow cells bearing surface ace immunoglobulins by combined autoradiography and immunoperoxidase

A method is described for the simultaneous detection of radiolabelled bone marrow cells bearing surface immunoglobulins by combined autoradiography and immunoperoxidase. Bone marrow cells from normal CBA mice prelabelled in vivo with ¹²⁵IUDR or exposed in vitro to [³H]thymidine were incubated with rabbit anti-mouse immunoglobulins under capping conditions, washed, cytocentrifuged and treated with methanol and hydrogen peroxide to destroy endogenous peroxidase. Cells were then covered with peroxidase-conjugated goat anti-rabbit immunoglobulins, washed, treated with diaminobenzidine a and hydrogen peroxide and finally covered with autoradiographic stripping film and exposed for different times. Peroxidase-positive cells were typically capped and those radiolabelled had autoradiographic silver grains overlying the nucleus.     

Ontogeny of J chain expression in pig lymphocytes

The ontogeny of lymphocytes expressing J chain in the cytoplasm (J+) was studied in pig foetuses by the immunofluorescent technique. Peripheral blood lymphocytes were the first J+ cells in prenatal life. The spleen and lymph nodes contained J+ cells in the last days of gestation. J+ cells were found in the lamina propria of the gut and some glands of conventional but not of germ-free piglets. J chain was not detected on or in cell membranes at any developmental stage.     

HIV-1整合酶真核表达载体的构建及在Hela细胞中的表达和定位

目的构建重组基因表达载体pcDNA6/V5-HisA-IN并观察其在Hela细胞中的表达.对HIV-1整合酶表达以后在细胞里的定位情况进行研究.方法通过PCR扩增和定向克隆技术构建重组真核表达载体pcDNA6/V5-HisA-IN.以Lipofectamine2000介导转染Hela细胞.细胞用4%的多聚甲醛固定以后,经过PI对细胞核染色,整合酶抗体跟整合酶反应以后,用FITC标记的二抗进行孵育,用共聚焦显微镜观测整合酶在细胞中的表达情况.结果经过定向克隆和测序鉴定证实,重组真核表达载体pcDNA6/V5-HisA-IN构建成功.免疫荧光实验显示,Lipofectamine2000介导质粒转染Hela表达成功,整合酶主要定位在细胞核.结论 HIV-1整合酶真核表达载体构建成功,转染以后,整合酶表达并且主要定位在细胞核.