Differential responses to growth factors by normal human mesothelial cultures from individual donors

A significant interindividual variation in the growth rates is found in normal cultured human mesothelial (NHM) cells derived from different donors. This variation is observed when the mesothelial cells are incubated in medium containing serum and when the potencies of several separate growth factors are measured by using defined media. Depending on the donor, gamma-interferon and interleukin-2 can be toxic, have no effect, or stimulate the growth rate of NHM cells. Cultured NHM cells can be induced to multiply by growth factors that are released by activated macrophages. Thus, interindividual variation in NHM cell growth control could play a role in the pathogenesis of mesothelioma for a person exposed to asbestos.     

Epoxide hydrolase activity in human blood mononuclear leukocytes: individual differences in native and mitogen-stimulated cells

Epoxide hydrolase (EH; EC 3.3.2.3) activity was measured in whole-cell sonicates of native and cultured peripheral blood mononuclear cells (PBMCs) from 19 healthy unrelated Caucasian donors (age, 28-55 years). We used 1,2-epoxy-3-(p-nitrophenoxy)propane (0. 34 mM) as the substrate and, for the diol assay, a quantitative HPLC method with spectrophotometric detection. One portion of the PBMCs was frozen immediately, while the other portion was PHA-stimulated and cultivated for 36 h. In native leukocytes, the EH activity varied from 2.2 to 8.2 pmol/min per 10(6) cells (3.8-fold), the mean+/-SD was 5.6+/-1.4 pmol/min per 10(6) cells. In most of the samples from different donors, the specific activity increased in cultivation, varying from 2.4 to 15.4 pmol/min per 10(6) cells (6. 3-fold), the mean+/-SD being 8.5+/-3.8 pmol/min per 10(6) cells. From a methodological point of view, enzyme measurement in native cells is simple to perform and may provide a better index of the specific activity, as the accuracy of electronic cell counting is better for cell samples taken before than after cultivation. The differences in the EH activity of PBMCs indicate that significant interindividual variation may occur in the detoxification of epoxides produced in the human lymphocyte test systems commonly used for genotoxicity screening of chemicals in vitro. Further studies are needed to determine the extent to which the reproducibility and thus also the sensitivity of such assays could be improved by analyses carried out to control the donors for their EH phenotype.     

An improved method of cell culture system from eye stalk,hepatopancreas, muscle,ovary, and hemocytes of <Emphasis Type="Italic">Penaeus vannamei</Emphasis>

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO₂. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO₂ supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.     

Cryobiophysical characteristics of genetically modified hematopoietic progenitor cells

The freezing responses of hematopoietic progenitor cells isolated from normal donors and from donors with mucopolysaccharidosis type I (MPS I) were determined using cryomicroscopy and analyzed using theoretical models for water transport and intracellular ice formation. The cells from donors with MPS I used in this investigation were cultured and transduced with a retroviral vector for the alpha-l-iduronidase (IDUA) enzyme in preclinical studies for human gene therapy. The water transport and intracellular ice formation (IIF) characteristics were determined at different time points in the culture and transduction process for hematopoietic progenitor cells expressing CD34 antigen from donors with MPS I and from normal donors. There were statistically significant changes in water transport, osmotically inactive cell volume fraction, and permeability between cells from different sources (normal donors vs donors with MPSI) and different culture conditions (freshly isolated vs cultured and transduced). Specifically, Lpg and Ea increased after ex vivo culture of the cells and the changes in permeability parameters were observed after as little as 3 days in culture. Similarly, the IIF characteristics of hematopoietic progenitor cells can also be influenced by the culture and transduction process. The IIF characteristics of freshly isolated cells from donors with MPS I were statistically distinct from those of cultured and transduced cells from the same donor. The ability to cryopreserve cells which are cultured ex vivo for therapeutic purposes will require an understanding of the biophysical changes resulting from the culture conditions and the manner in which these changes influence viability.     

Fat body and hemocyte contribution to the antimicrobial peptide synthesis in <Emphasis Type="Italic">Calliphora vicina</Emphasis> R.-D. (Diptera: Calliphoridae) larvae

Antimicrobial peptides accumulated in the hemolymph in response to infection are a key element of insect innate immunity. The involvement of the fat body and hemocytes in the antimicrobial peptide synthesis is widely acknowledged, although release of the peptides present in the hemolymph from the immune cells was not directly verified so far. Here, we studied the presence of antimicrobial peptides in the culture medium of fat body cells and hemocytes isolated from the blue blowfly Calliphora vicina using complex of liquid chromatography, mass spectrometry, and antimicrobial activity assays. Both fat body and hemocytes are shown to synthesize and release to culture medium defensin, cecropin, diptericins, and proline-rich peptides. The spectra of peptide antibiotics released by the fat body and hemocytes partially overlap. Thus, the results suggest that insect fat body and blood cells are capable of releasing mature antimicrobial peptides to the hemolymph. It is notable that the data obtained demonstrate dramatic difference in the functioning of insect antimicrobial peptides and their mammalian counterparts localized into blood cells’ phagosomes where they exert their antibacterial activity.     

Establishment of hematopoietic tissue primary cell cultures from the giant freshwater prawn Macrobrachium rosenbergii

The giant freshwater prawn Macrobrachium rosenbergii is one of the most important aquaculture species in Southeast Asia. In this study, in vitro culture of its hematopoietic tissue cells was achieved and characterized for use as a tool to study its pathogens that cause major farm losses. By transmission electron microscopy, the ultrastructure of the primary culture cells was similar to that of cells lining intact hematopoietic tissue lobes. Proliferating cell nuclear antigen (PCNA) (a marker for hematopoietic stem cell proliferation) was detected in some of the cultured cells by polymerase chain reaction (PCR) testing and flow cytometry. Using a specific staining method to detect phenoloxidase activity and using PCR to detect expression markers for semigranular and granular hemocytes (e.g., prophenoloxidase activating enzyme and prophenoloxidase) revealed that some of the primary cells were able to differentiate into mature hemocytes within 24 h. These results showed that some cells in the cultures were hematopoietic stem cells that could be used to study other interesting research topics (e.g. host pathogen interactions and development of an immortal hematopoietic stem cell line).     

柞蚕核型多角体病毒载体在培养细胞和休眠蛹中的基因表达效果

柞蚕核型多角体病毒（AnpeNPV）作为基因表达载体在柞蚕培养细胞（AnPe细胞）和柞蚕蛹中已经成功地表达出了外来基因,并生产出了大量蛋白质。本文比较了AnpeNPV与苜蓿尺蠖核型多角体病毒(AcMNPV)、家蚕核型多角体病毒(BmNPV)和美国白蛾核型多角体病毒(HycuNPV)基因表达载体在培养细胞和昆虫活体组织内的β-半乳糖苷酶基因表达效果。结果显示,5×105个细胞中β-半乳糖苷酶的最高酶活性分别是AnpeNPV在AnPe细胞为40.9 units/ml （TC-100培养液,FBS10%）和59.9 units/ml（SF-900Ⅱ培养液）,AcMNPV在Sf9细胞为72.4 units/ml（TC-100,FBS10%）和66.4 units/ml（SF-900Ⅱ）、在High5细胞为326 units/ml（EX-CELL 405培养液）,BmNPV在Bm4细胞为15.1 units/ml（TC-100,FBS10%）,HycuNPV在SpIm细胞为68.6 units/ml（SF-900Ⅱ）。活体组织内β-半乳糖苷酶的最高酶活性分别是柞蚕雌蛹为14.3 units/g、雄蛹为11.7 units/g,家蚕幼虫是10.1 units/g。实验证明AnpeNPV/AnPe的外来基因表达水平与AcMNPV/ Sf9和HycuNPV/SpIm相似、比BmNPV/ Bm4高、不及AcMNPV/ High5;AnpeNPV/柞蚕蛹,其雌蛹比BmNPV/家蚕5龄幼虫的外来基因表达效果好、雄蛹与之无明显差异,说明AnpeNPV基因表达载体无论是在培养细胞还是昆虫活体组织中均可与其他NPV基因表达载体相媲美。柞蚕蛹由于可以机械化、大规模地操作,显示对于大量生产蛋白质具有更好的应用前景。     

Ultrastructural and functional characterization of circulating hemocytes from Plutella xylostella larva: Cell types and their role in phagocytosis

The hemocytes of different types encountered in the diamondback moth Plutella xylostella larvae of each instar and the development of the differential hemocytes counts were herein presented. Hemocytes classes/populations characterized based on their affinity with fluorescent dye (acridine orange) and ultrastructural differences comprised the prohemcoytes (<10–16%), plasmatocytes (22–65%), granulocytes (25–72%), oenocytoids (<1–9%), and spherulocytes (<1%). Prohemcoytes were the smallest cells with a comparatively tremendous nucleus. Plasmatocytes and granulocytes occupied the main proportion of total cell numbers. Oenocytoids were in a most stable presence, i.e. rotund in a diameter of 10 μm and with a nucleus deviated from the central location; however, sometimes with two nuclei which were adjoining with each other. Spherulocytes were rare and only could be observed occasionally. Ultrastructural investigation revealed that hemocytes in the diamondback moth larvae were of the typical model as in the Lepidoptera insect larvae. It is interesting to find that the cell which could phagocytize bacteria in vitro was granulocyte, not the other types of hemocytes, although plasmatocyte was usually declared to participate in this reaction in various previous studies.     

3H-thymidine paper strip modification of the lymphocyte transformation test. III. Whole blood lymphocyte mixed culture using a cryopreserved stimulating cell mixture

In comparison to the classical mixed lymphocyte culture (MLC) method a MLC technique using whole blood cultures of responding cells and a pool of prepared stimulating cells from 10 healthy blood donors is described. The reproducibility of cryopreservation of stimulating lymphocytes was proven by means of cell electrophoresis.     

The relative abundance of hemocyte types in a polyphagous moth larva depends on diet

Hemocytes are crucial cells of the insect immune system because of their involvement in multiple immune responses including coagulation, phagocytosis and encapsulation. There are various types of hemocytes, each having a particular role in immunity, such that variation in their relative abundance affects the outcome of the immune response. This study aims to characterize these various types of hemocytes in larvae of the grapevine pest insect Eupoecilia ambiguella, and to assess variation in their concentration as a function of larval diet and immune challenge. Four types of hemocytes were found in the hemolymph of 5th instar larvae: granulocytes, oenocytoids, plasmatocytes and spherulocytes. We found that the total concentration of hemocytes and the concentration of each hemocyte type varied among diets and in response to the immune challenge. Irrespective of the diet, the concentration of granulocytes increased following a bacterial immune challenge, while the concentration of plasmatocytes and spherulocytes differentially varied between larval diets. The concentration of oenocytoids did not vary among diets before the immune challenge but varied between larval diets in response to the challenge. These results suggest that the resistance of insect larvae to different natural enemies critically depends on the effect of larval diet on the larvae’s investment into the different types of hemocytes.     

Evidence for an H-Y Crossreactive Antigen in Invertebrates

A sex specific antigen which crossreacts with the mammalian H-Y antigen has been identified on the cell surface of hemocytes from the lobster ( Homarus americanus ) and the gonadal cells of three insect species. The hemocytes from the male lobster, the testicular cells from the male beetle ( P. cornutus ), and the ovarian cells from two Orthopteran species ( L. maderae and D. punctata ) specifically absorbed H-Y antibodies. The specificity of H-Y antibody absorptions by cells from only one sex, suggest that an ancestral H-Y-like antigen may be present in invertebrates which could be engaged in sexual (cellular) recognition events.     

Gene Expression Signature of Normal Cell-of-Origin Predicts Ovarian Tumor Outcomes

The potential role of the cell-of-origin in determining the tumor phenotype has been raised, but not adequately examined. We hypothesized that distinct cells-of-origin may play a role in determining ovarian tumor phenotype and outcome. Here we describe a new cell culture medium for in vitro culture of paired normal human ovarian (OV) and fallopian tube (FT) epithelial cells from donors without cancer. While these cells have been cultured individually for short periods of time, to our knowledge this is the first long-term culture of both cell types from the same donors. Through analysis of the gene expression profiles of the cultured OV/FT cells we identified a normal cell-of-origin gene signature that classified primary ovarian cancers into OV-like and FT-like subgroups; this classification correlated with significant differences in clinical outcomes. The identification of a prognostically significant gene expression signature derived solely from normal untransformed cells is consistent with the hypothesis that the normal cell-of-origin may be a source of ovarian tumor heterogeneity and the associated differences in tumor outcome.     

Ontogenesis of hemocytes in the stick insect Bacillus rossius (Rossi) (Phasmatodea,Bacillidae) studied with an anti-hemocyte monoclonal antibody

The monoclonal antibody BrH1, specific for hemocytes of the stick insect Bacillus rossius, was employed to study the appearance of hemocytes during embryogenesis. Laid eggs were collected for eight weeks, and cryosections were probed with the antibody. First positive cells were detected at the fifth week, and increased in number onwards. No peculiar differences were observed in the overall morphology between embryonic and non-embryonic hemocytes.     

The Heat Shock Response of Carrot : Protein Variations between Cultured Cell Lines

We have defined several parameters surrounding the heat shock response of cultured cells of carrot (Daucus carota L.) and have found that these cells exhibit a typical “higher plant” heat shock response. In particular, the resolution of the heat shock proteins (hsps) by two-dimensional polyacrylamide gel electrophoresis (PAGE) has revealed a pattern of proteins very similar to the hsps from soybean; specifically, the low molecular weight class is composed of approximately 15 to 20 different polypeptides which likely represent different members of a small gene family. In addition, we have compared the (2-D) PAGE profiles of hsps isolated from several different cultured cell lines currently maintained in our laboratory and have found notable differences in the low molecular weight hsps between cell lines. Some of the differences appear to be quantitative, while others may be qualitative. Each of the cell lines was derived from a different seedling of the same seed stock of the same cultivar; thus, genetic differences should be minimized. In addition, two of the cell lines, which show clear differences, were initially derived from a single parental line, and thus arose from a single genetic stock. Possible explanations for the cell line differences observed here are either partial aneuploidy or modified gene regulation resulting from molecular changes during the time in culture (i.e. somaclonal variation). These observations serve to highlight the potential for variation that exists in cells in culture even for such a highly conserved response and gene set as the heat shock genes.     

Estrogen receptor beta yield from baculovirus lytic infection is higher than from stably transformed Sf21 cells

The production of estrogen receptors (ER) in cultured insect cells is advantageous because these cells are relatively easy to culture and they perform post-translation modifications necessary for protein stability and function. There are three options for protein expression in insect cells: transient transfection, lytic baculovirus infection, or transfection followed by selection to create stable cell lines. Stable transfection has been promoted to be advantageous for the production of recombinant proteins because no re-infection is required, which might provide better lot-to-lot reproducibility in protein production. In this paper, we demonstrate that lytic baculovirus infection of Sf21 cells yields approximately tenfold more bioactive ERβ than cells stably transformed with pIZ/V5-His plasmid under OpIE2 promoter. We provide the first evidence that stable expression of recombinant human ERβ decreases the proliferation of Sf21 cells by inhibition of cell replication in a ligand-independent manner. These results mirror findings in breast cancer cells showing that an increase in ERβ expression decreases cell proliferation. We conclude that baculovirus infection of Sf21 cells is better for human ERβ production than stable-transformation of Sf21 cells.     

Umbilical cord derived mesenchymal stromal cells in microcarrier based industrial scale culture sustain the immune regulatory functions

Mesenchymal stromal cells (MSCs) have been isolated from numerous sources and are potentially therapeutic against various diseases. Umbilical cord-derived MSCs (UC-MSCs) are considered superior to other tissue-derived MSCs since they have a higher proliferation rate and can be procured using less invasive surgical procedures. However, it has been recently reported that 2D culture systems, using conventional cell culture flasks, limit the mass production of MSCs for cell therapy. Therefore, the development of alternative technologies, including microcarrier-based cell culture in bioreactors, is required for the large-scale production and industrialization of MSC therapy. In this study, we aimed to optimize the culture conditions for UC-MSCs by using a good manufacturing practice (GMP)-compatible serum-free medium, developed in-house, and a small-scale (30 mL) bioreactor, which was later scaled up to 500 mL. UC-MSCs cultured in microcarrier-based bioreactors (MC-UC-MSCs) showed characteristics equivalent to those cultured statically in conventional cell culture flasks (ST-UC-MSCs), fulfilling the minimum International Society for Cellular Therapy criteria for MSCs. Additionally, we report, for the first time, the equivalent therapeutic effect of MC-UC-MSCs and ST-UC-MSCs in immunodeficient mice (graft-versus-host disease model). Lastly, we developed a semi-automated cell dispensing system, without bag-to-bag variation in the filled volume or cell concentration. In summary, our results show that the combination of our GMP-compatible serum-free and microcarrier-based culture systems is suitable for the mass production of MSCs at an industrial scale. Further improvements in this microcarrier-based cell culture system can contribute to lowering the cost of therapy and satisfying several unmet medical needs.     

Infection-Induced Interaction between the Mosquito Circulatory and Immune Systems

Insects counter infection with innate immune responses that rely on cells called hemocytes. Hemocytes exist in association with the insect''s open circulatory system and this mode of existence has likely influenced the organization and control of anti-pathogen immune responses. Previous studies reported that pathogens in the mosquito body cavity (hemocoel) accumulate on the surface of the heart. Using novel cell staining, microdissection and intravital imaging techniques, we investigated the mechanism of pathogen accumulation in the pericardium of the malaria mosquito, Anopheles gambiae, and discovered a novel insect immune tissue, herein named periostial hemocytes, that sequesters pathogens as they flow with the hemolymph. Specifically, we show that there are two types of endocytic cells that flank the heart: periostial hemocytes and pericardial cells. Resident periostial hemocytes engage in the rapid phagocytosis of pathogens, and during the course of a bacterial or Plasmodium infection, circulating hemocytes migrate to the periostial regions where they bind the cardiac musculature and each other, and continue the phagocytosis of invaders. Periostial hemocyte aggregation occurs in a time- and infection dose-dependent manner, and once this immune process is triggered, the number of periostial hemocytes remains elevated for the lifetime of the mosquito. Finally, the soluble immune elicitors peptidoglycan and β-1,3-glucan also induce periostial hemocyte aggregation, indicating that this is a generalized and basal immune response that is induced by diverse immune stimuli. These data describe a novel insect cellular immune response that fundamentally relies on the physiological interaction between the insect circulatory and immune systems.     

Differences in the number of hemocytes in the snail host Biomphalaria tenagophila, resistant and susceptible to Schistosoma mansoni infection

The relationships between schistosomiasis and its intermediate host, mollusks of the genus Biomphalaria, have been a concern for decades. It is known that the vector mollusk shows different susceptibility against parasite infection, whose occurrence depends on the interaction between the forms of trematode larvae and the host defense cells. These cells are called amebocytes or hemocytes and are responsible for the recognition of foreign bodies and for phagocytosis and cytotoxic reactions. The defense cells mediate the modulation of the resistant and susceptible phenotypes of the mollusk. Two main types of hemocytes are found in the Biomphalaria hemolymph: the granulocytes and the hyalinocytes. We studied the variation in the number (kinetics) of hemocytes for 24 h after exposing the parasite to genetically selected and non-selected strains of Biomphalaria tenagophila, susceptible or not to infection by Schistosoma mansoni. The differences were analyzed referred to the variations in the number of hemocytes in mollusks susceptible or not to infection by S. mansoni. The hemolymph of the selected and non-selected snails was collected, and hemocytes were counted using a Neubauer chamber at six designated periods: 0 h (control, non-exposed individuals), 2 h, 6 h, 12 h, 18 h and, 24 h after parasite exposure. Samples of hemolymph of five selected mollusks and five non-selected mollusks were separately used at each counting time. There was a significant variation in the number of hemocytes between the strains, which indicates that defense cells have different behaviors in resistant and susceptible mollusks.