Cellular Composition and Proliferation Levels in the Hematopoietic Tissue of Black Scorpionfish (Scorpaena porcus L.) Head Kidney and Spleen During the Spawning and Wintering Periods

To identify cells and analyze proliferative activity of hematopoietic tissue, black scorpionfish head kidney and spleen cells were characterized by light microscopy and flow cytometry. Hematopoiesis of black scorpionfish head kidney was formed by the following series: erythropoietic, granulopoietic, lymphopoietic, and thrombopoietic. Flow cytometric analysis allowed dividing blood cells in hematopoietic organs into subpopulations differing by size, granularity, and proliferative activity. Three distinct subpopulations were observed during the wintering period. The number of small low-granulated cells, identified as lymphocytes and thrombocytes, was 41% ± 4% in both wintering and spawning fish. Proliferating subpopulation of blast (high-granulated) cells amounted to about 36% of the total cell count with 50% ± 5% of proliferating cells; the largest low-granulated cells (10% of total cells) comprised maturing white blood cells, monocytes, and macrophages. The spawning period was accompanied with an increase of maturing neutrophils and enhancement of blast cell proliferation. In the spleen three distinct subpopulations were observed. The subpopulation of small low-granulated cells comprised lymphocytes and thrombocytes similar to the head kidney and amounted 33% ± 4%. Other cells with larger diameter were identified as red blood cells. No proliferation was observed during the wintering period in the spleen, however, spawning induced cell proliferation of erythroblasts (small granulated cells) with the number of dividing cells 84% ± 1%. Anat Rec, 302:1136–1143, 2019. © 2018 Wiley Periodicals, Inc.     

A method to study apoptosis in eosinophils by flow cytometry

The aim of this study was to develop a simple flow cytometric procedure to study eosinophil apoptosis. Eosinophils were isolated from the peripheral blood of healthy, non-allergic individuals and then cultured in basal culture medium. The cells were examined after 24, 48 and 72 h for forward- and side scatter (FS-SSC) pattern, staining with FDA, PI, and anti-CD95, and light microscopic appearance. After culture for >24 h, two populations with different FS-SSC-patterns appeared, referred to as A and B. Population A consisted of living, FDA-positive eosinophils. The eosinophils in population B showed a lower FS scatter than those in population A and a staining pattern with PI indicating the presence of hypodiploid DNA. Anti-CD95 demonstrated a significant staining of the eosinophils in population B, which increased after 2 days in culture. The cells were sorted using a FACS-Scan cell sorter and by Annexin V-coated magnetic beads to permit separate analyses of PI-staining pattern, DNA electrophoresis, and light microscopic examination of the cells in population B. The present study suggest that it is possible to discriminate between apoptotic and living eosinophils using the FS-SSC pattern and the PI-staining pattern obtained by flow cytometry.     

Blood cells of the gilthead seabream (Sparus aurata L.): Light and electron microscopic studies

The peripheral blood cells of the gilthead seabream (Sparus aurata L.) were studied by light and transmission and scanning electron microscopic methods. Acidophilic erythroblasts and mature erythrocytes, round, oval, and fusiform thrombocytes, neutrophils, acidophils and basophils, lymphocytes, plasma cells, and monocyte-macrophages were characterized. A comparison of our light and electron microscopic results was carried out. The results were discussed with those for other fish species and the main modifications from the common vertebrate haematological pattern observed being considered.© Willey-Liss, Inc.     

Morphological observations of the thrombocyte of white-bellied sea eagle Haliaeetus leucogaster

The white-bellied sea eagle, Haliaeetus leucogaster, is a large territorial raptor species associated with coastal regions, lakes and river systems. It has an extensive distribution from the western coast of India throughout the Indo-Malaysian region, Papua New Guinea and Australia. Blood samples of the white-bellied sea eagle housed at Nakhonratchasima Zoo, Nakhon Ratchasima province in northeastern Thailand, were collected. Morphological observations of the thrombocytes were examined using scanning electron and transmission electron microscopy. The results revealed the following information: thrombocytes of white-bellied sea eagle were oval to rod-shaped with a rough membrane and the presence of a spread monolayer. Within the cytoplasm of the white-bellied sea eagle, thrombocytes were vesicles of varying sizes. During blood clot formation, the thrombocytes spread their membrane and used pseudopodials to attach to red blood cells causing blood cell clumping to occur. This study indicates that the morphology and activity of the white-bellied sea eagle thrombocytes differs from other non-mammalian thrombocytes.     

The use of flow cytometry to discriminate avian lymphocytes from contaminating thrombocytes

Evaluation of peripheral blood mononuclear cells (PBMC) in avian species by flow cytometry is complicated by the presence of large numbers of nucleated thrombocytes. With light scattering properties similar to those of lymphocytes, variations in the proportion of thrombocytes in PBMC can result in apparent shifts in percentages of lymphocyte subpopulations. We have applied a dual-labeling procedure for flow cytometric analyses to exclude thrombocytes from the analyzed population by labeling with K55 monoclonal antibody (MAb), which differentially labeled leukocyte and thrombocyte populations. Flow cytometric analyses with K55 labeled chicken PBMC differentiated leukocytes into three populations according to their intensity of fluorescence. Using the Kl MAb, the K55-low population was shown to consist of thrombocytes. Dual-labeling with K55 MAb and MAb specific for B lymphocyte, CD4 or CD8 markers indicated that the K55 intermediate population consisted of lymphocytes. Therefore, concentrations of CD4+ and CD8+ T lymphocytes could be determined from the lymphocyte fraction by gating specifically on the K55 intermediate cells. Selecting cross-reactive chicken MAbs that included K55, this protocol was shown to identify CD4+ and CD8+ T lymphocytes in PBMC of another avian species, the endangered Attwater's prairie chicken.     

Flow cytometric differentiation of avian leukocytes and analysis of their intracellular cytokine expression

A flow cytometric method for the identification of chicken blood leukocyte subpopulations and thrombocytes was developed. An anti-chicken CD45 phycoerythrin-labelled antibody was used to separate leukocytes from red blood cell nuclei. Leukocytes and thrombocytes were identified using a combination of their CD45-positivity and their typical side scatter properties. The identity of the CD45-positive cells was confirmed by sorting the subpopulations and subsequent light microscopic evaluation. In these differentiated cell populations, intracellular expression analysis of the proinflammatory cytokines interleukin-1β and interleukin-6 was subsequently optimized on whole blood after in vitro stimulation with lipopolysaccharide from Escherichia coli strain O127:B8.     

Effect of ketamine on apoptosis by energy deprivation in astroglioma cells using flow cytometry system

Apoptosis is a programmed, physiologic mode of cell death that plays an important role in tissue homeostasis. As for the central nervous system, ischemic insults can induce pathophysiologic cascade of apoptosis in neurophils. Impairment of astrocyte functions during brain ischemia can critically influence neuron survival by neuronglia interactions. We aimed to elucidate the protective effect of ketamine on apoptosis by energy deprivation in astrocytes. Ischemic insults was induced with iodoacetate/ carbonylcyanide m-chlorophenylhydrazone (IAA/CCCP) 1.5 mM/20 microm or 150 microm/2 microm for 1 hr in the HTB-15 and CRL-1690 astrocytoma cells. Then these cells were reperfused with normal media or ketamine (0.1 mM) containing media for 1 hr or 24 hr. FITC-annexin-V staining and propidium iodide binding were determined by using flow cytometry. Cell size and granularity were measured by forward and side light scattering properties of flow cytometry system, respectively. An addition of ketamine during reperfusion increased the proportion of viable cells. Ketamine alleviated cell shrinkage and increased granularity during the early period, and ameliorated cell swelling during the late reperfusion period. Ketamine may have a valuable effect on amelioration of early and late apoptosis in the astrocytoma cells, even though the exact mechanism remains to be verified.     

The use of Ficoll-Hypaque double density gradients in the separation of avian granulocytes from other cell types for the purpose of cell flow cytometric analysis

Avian peripheral blood and embryonic spleen cells were prepared for cell flow cytometry. The Ortho Spectrum III was the flow cytometer used in these experiments. The major objectives were to identify the location of lymphocytes and granulocytes in the cytogram displayed by flow cytometry, to develop a technique which would allow the collection of granulocytes relatively free of other cell types and to characterize the cell cycle within these cell populations. The cytogram of fresh avian cells developed in the Ortho Spectrum III revealed three characteristic cell clusters. Peripheral blood or embryonic spleen cells were separated on a Ficoll-Hypaque double density gradients into two distinct layers and a pellet. Light microscopic examination revealed the top layer of cells to be primarily lymphocytes while the middle layer of cells was granulocytes. Presentation of the cells from these layers to the Ortho Spectrum III revealed that granulocytes made up Cluster 3 while lymphocytes were included in the other clusters. The Ortho Spectrum III was employed to determine the presence of G1 (pre-DNA synthesis), S (DNA synthesis), and G2/M (post-DNA synthesis and mitosis) phases of cells in Clusters 1 and 2 and Cluster 3. While all the cells from peripheral blood were in G1, the embryonic spleen revealed cells in G1, S and G2/M in both Clusters 1 and 2 and Cluster 3.     

Establishment of embryonic cell line from sea bass (Lates calcarifer) for virus isolation

A continuous cell line was established from blastula stage embryos of sea bass (Lates calcarifer). The sea bass embryonic cells were maintained in Leibovitz's L-15 supplemented with 15% fetal bovine serum. The embryonic cell line was sub-cultured more than 70 passages over a period of 1.5 years and is designated as Sahul Indian sea bass embryonic (SISE) cell line. The cells were able to grow at temperatures between 25 and 32 degrees C with an optimum temperature of 28 degrees C. The growth rate of sea bass embryonic cells increased as the FBS proportion increased from 2 to 20% at 28 degrees C with optimum growth at the concentration of 15 or 20%. Polymerase chain reaction products were obtained from embryonic cells and blastula of sea bass with primer sets of microsatellite markers of sea bass. Four fish viruses were tested on this cell line to determine its susceptibility to these viruses and this cell line was found to be susceptible to IPNV VR-299 and nodavirus, and the infection was confirmed by cytopathic effect (CPE) and RT-PCR. Further, this cell line was characterized by immunocytochemistry using confocal-laser-scanning microscopy (CFLSM), transfection with pEGFP-N1, proliferate marker (BrdU).     

Isolation of side population cells from ginbuna carp (Carassius auratus langsdorfii) kidney hematopoietic tissues

Side population (SP) cells, characterized by a specific Hoechst dye efflux pattern by flow cytometry were isolated from kidney hematopoietic tissues of ginbuna carp (Carassius auratus langsdorfii). The hematopoietic activity of SP cells was evaluated by the repopulation and multilineage potential using an in vivo transplant system of ginbuna carp (donor) and ginbuna-goldfish hybrids (recipient). In a flow cytometric (FCM) analysis, a small and distinct population of ginbuna SP cells displayed efficient effluxes of Hoechst 33342 was virtually identical to the efflux observed in mammalian SP cells. The frequency of the ginbuna SP cells was 0.17+/-0.08% in the kidney hematopoietic cells. Morphologically, SP cells were composed of small lymphocyte-like cells having a thin-layered cytoplasm and a round nucleus. These characteristics of ginbuna SP cells were very similar to those of mammalian SP cells. Since cyprinid fish have two hematopoietic sites, the head (anterior) and body (posterior, trunk) kidney, the distribution of SP cells were examined in head and body kidney. The proportion of SP cells were 0.33+/-0.15% in the body kidney, but near 0% in the head kidney. After the ginbuna SP cells were injected into ginbuna-goldfish hybrids, the major types of donor-derived cells (erythrocytes, neutrophils, basophils, monocytes, thrombocytes, T and B lymphocytes) were detected in the recipient blood over a long period (9-16 months post-transplantation). In ginbuna carp, SP cells reside in the body kidney and contain primitive populations of hematopoietic stem cells (HSCs).     

Migration of mononuclear cells in the modified Boyden chamber as evaluated by DNA quantification and flow cytometry

In vitro migration of mononuclear cells in the modified Boyden chamber was evaluated using flow cytometry and DNA quantification (Hoechst 33258) of all adherent and nonadherent cells. The effects of different membrane pore sizes, cell concentrations and incubation times were studied. Pore sizes of 3 and 5 microm resulted in a reduction in the number of nonadherent cells compared with a pore size of 8 microm. Reducing the incubation time from 60 to 40 and 20 min resulted in too few migrating monocytes for analysis by flow cytometry. Flow cytometry showed that both monocytes and lymphocytes migrated and adhered to the membrane when using peripheral blood mononuclear cells (PBMC) to study monocyte migration. Migration of lymphocytes under these conditions is a novel observation. A substantial number of migrated cells could be identified by flow cytometry and quantified by DNA measurement as nonadherent below the membrane. Samples of synovial fluid (n = 49) and plasma (n = 133) as chemoattractants analysed in triplicate resulted in mean coefficient of variation (CV) values of 11 and 9%, respectively. Variation from assay to assay on the same day, using N-formyl-methionyl-leucyl-phenylanine (fMLP) 10(-7) M as chemoattractant resulted in a CV of 13%. Day-to-day variation, using fMLP 10(-7) M as chemoattractant and the same well on three different days, resulted in a CV of 21%. These results were obtained using a pore size of 5 microm, a PBMC concentration of 3 x 10(6)/ml and 60 min of incubation. The combination of DNA quantification and flow cytometry thus allowed characterization and quantification of subsets of migrating adherent as well as nonadherent mononuclear cells.     

Lernanthropus kroyeri (Van Beneden and Hesse 1851) parasitic Copepoda (Siphonostomatoidae, Lernanthropidae) of European cultured sea bass Dicentrarchus labrax (Linnaeus 1758) from Corsica: ecological and morphological study

Lernanthropus kroyeri (Copepoda, Siphonostomatoida: Lernanthropidae) is a gill parasite found on the European sea bass Dicentrarchus labrax. During a survey of sea bass of Corsican fish farms, we studied the biology of this parasite under culture conditions. We first chose to conduct a scanning electron microscopic study to give additional information about the lifestyle of the parasite. Our examinations made it possible to reveal some unreported superficial structures including details not described previously. Specializations associated with the tegument, in particular, sensory structures and anchoring systems were studied to understand the mechanisms of survival and dispersal of the species. Patterns variation of parasites communities was examined by taking into account environmental factors, such as temperature or salinity, and physiological parameters related to host. The relation between parasites and location of fish was also studied to quantify the importance of site influence on parasite communities. Prevalence and abundances of the infections in different culture systems, fish stocks, and sampling seasons are given. L. kroyeri was significantly present during spring and summer, coinciding with a period of increasing temperature. Significant differences were found grouping data by host size, with higher infection levels in the larger-sized fish.     

Binding of mouse monoclonal antibodies to human leukaemic cells via the Fc receptor: a possible source of 'false positive' reactions in specificity screening

Mouse monoclonal antibodies (MoAbs) of different classes and subclasses directed against antigens not expected to be present on human cells have been screened by indirect immunofluorescence using flow cytometry for binding to human non-lymphocytic leukaemic cells and normal peripheral blood leucocytes. Antibodies of the IgG2a and IgG3 subclass, but not of the IgG1, IgG2b or IgM class bound to the blast cell and monocyte populations in a peripheral blood mononuclear cell preparation from a patient with acute myelomonocytic leukaemia. F(ab')2 fragments of an anti-salmonella antibody of IgG2a subclass failed to bind, indicating that the results were not due to cross-reactivity with antigens of the cell membrane, thus implicating the Fc region in binding. Furthermore, binding was partly blocked by inclusion of 10% heat-inactivated normal rabbit serum in the assays. IgG2a bound to varying degrees to the leukaemic cell populations in seven of the nine non-lymphocytic leukaemic specimens tested, but no binding to normal peripheral blood mononuclear cells or granulocytes was detected. The results emphasize the importance of including appropriate controls when screening MoAbs for binding to various types of human cells.     

Experimental transmission of<Emphasis Type="Italic"> Cryptosporidium molnari</Emphasis> (Apicomplexa: Coccidia) to gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.) and European sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> L.)

Cryptosporidium molnari was experimentally transmitted to gilthead sea bream (Sparus aurata) and European sea bass (Dicentrachus labrax) by oral infection with infected stomach scrapings. The infection was also cross-transmitted from infected gilthead sea bream to sea bass by cohabitation. The course of the infection was assessed after necropsy by three microscopic diagnostic methods and their sensitivity was compared. At the end of all the experiments the prevalence of infection reached 100%. In the oral experiments, both fish hosts appeared infected as early as 7 days post exposure (p.e.), but gilthead sea bream exhibited a higher intensity of infection and infection proceeded at a faster rate than in sea bass. The cellular host reaction was stronger in sea bass than in sea bream, whereas the histopathological effect was lower in the former. Transmission could be favoured by cannibalism among cohabiting fish. This is the first report on piscine Cryptosporidium transmission. The implications for the aquaculture industry are discussed.     

Viral erythrocytic infection in sea bass: virus purification and confirmative diagnosis

Summary The viral erythrocytic infection (VEI) of sea bass (Dicentrarchus labrax) is evidenced by the presence of inclusion bodies in the erythrocytes and erythroblasts. Virus-like particles (VEIV) about 135–150 nm in diameter were observed in erythroblasts of affected fish. No culturable virus could be recovered from the organs and blood of VEI-affected fish, after inoculation of different fish cell lines. Histochemical staining revealed the presence of RNA but not of DNA in the inclusion bodies. Specific, rabbit hyperimmune sera were prepared after extraction and purification by gel chromatography of viral material from pelleted blood cells of infected fish, and evaluated by immunocytochemical assays. Electron microscopic observations revealed the presence of enveloped particles of about 125–150 nm in the antigenic preparation. The antisera specifically reacted to inclusion bodies, cellular membranes and nuclei of VEI-affected erythrocytes in immunofluorescent and peroxidase-antiperoxidase assays. Immunoelectron microscopy in ultrathin sections of head kidney samples from VEI-affected fish showed recognition of inclusion bodies and virus particles. Confirmative diagnostic procedures could be established through the use of specific anti-VEIV sera, which enabled differentiation between similar syndromes described in other fish species.     

Nonspecific cytotoxic cells in fish (Ictalurus punctatus). VI. Flow cytometric analysis

Nonspecific cytotoxic cells (NCC) from channel catfish (Ictalurus punctatus) were enriched using flow cytometry (EPICS V, 753). Parameters of forward angle and 90 degrees light scatter were utilized to sort cytolytically functional populations of NCC. Fullbright (GR. II) 9.75 microns diameter fluorescent polystyrene microspheres were used for optical alignment. Cells obtained from the anterior kidney, spleen and peripheral blood were analyzed by one and/or two parameters of forward angle light scatter (FALS) and log integral 90 degrees light scatter (L90 degrees LS). Populations identified from FALS or L90 degrees LS histograms were sorted and cells were tested for cytotoxicity against NC-37 target cells. Anterior kidney contained at least four different sized populations of NCC. Spleen cells were sorted by FALS and four different populations of NCC detected. Peripheral blood contained little NCC activity. Enrichment of NCC activity from anterior kidney tissue was compared using Percoll density gradient separation and flow cytometry. Three to four fold higher cytotoxic activity was obtained from sorted cells compared to the population obtained from 45.5% Percoll. Additional experiments to determine the enrichment of sorted NCC were conducted by analyzing the number of enriched NCC required to kill an individual NC-37 target cell. Approximately 1.5-2.0 NCC could lyse a chromium-51 labelled target cell. These studies demonstrated that NCC could be enriched by size and granularity discrimination using flow cytometry. Effector:target cell ratios of 10:1 and lower produced significant lysis when sorted cells were analyzed.     

Phagocytic defence mechanism in sea bass (Dicentrarchus labrax L.): An ultrastructural study

Background: The ultrastructure of the phagocytic process in fish has not been established in spite of the significant morphofunctional differences detected in the fish immune system with respect to the basic immunological pattern in vertebrates. We report the ultrastructure of the bacterial phagocytic defence mechanism in sea bass (Dicentrarchus labrax L.). Methods: Head-kidney, blood, and peritoneal exudate leukocytes were challenged with Aeromonas salmonicida and Escherichia coli and processed for transmission electron microscopic study. Results: Macrophages challenged with bacteria showed changes in the cell outline, in the chromatin pattern, and in the ultrastructural features of the cytoplasm as a consequence of an activation process. The phagocytic process consists of the following: (1) Bacteria-macrophage contact. One or more spot contacts between the bacterial wall and the phagocyte membrane are observed. (2) Bacteria engulfment. Slight depressions, membrane invaginations, or cytoplasmic processes are formed at the phagocyte surface. Macrophage processes occasionally surround the bacteria, overlaping and roaming parallel, or a single, long pseudopod encircles a bacterium several times. (3) Endocytic vesicle formation. Macrophages show one or more bacteria inside membrane-bound cytoplasmic vesicles. (4) Phagolysosome formation. Some dense granules (lysosomes) fuse with the endocytic vesicle. (5) Intracelular killing/digestion. Bacteria inside the endocytic vesicles are observed both virtually intact or damaged at different digestion stages. Conclusions: Sea bass macrophages possess the mechanisms necessary to both engulf and kill bacteria. Cellular and subcellular events in the morphology of phagocytosis and lysosomal dissolution of bacteria fit the general pattern described for mammals. © 1994 Wiley-Liss, Inc.     

Cell surface characterization of amastigotes of Trypanosoma cruzi obtained from different sources

The present study analyses the morphology and the exposition of surface carbohydrates and the Ssp4 antigen of amastigote forms of Trypanosoma cruzi (Y strain) obtained from three different sources: (a) intracellular, isolated from infected Vero cells 3 days after infection, (b) extracellular, isolated from the supernatant of Vero cells 15 days after infection, and (c) axenic, obtained by incubation of tissue culture trypomastigotes in LIT medium, at 37 °C for 4 days. No morphological differences were observed by light microscopy among these amastigotes. Transmission electron microscopy of thin sections showed a thick cell coat easily observed on the plasma membrane of axenic amastigotes. Carbohydrate-containing sites on the surface of the three different amastigotes were analysed using lectins, agglutination assays and flow cytometry. Mannose and/or glucose residues were found on the surface of all populations, but intracellular amastigotes showed the highest number. A small group of cells from the different populations expressed galactose and N-acetyl-glucosamine residues. The presence and distribution of the Ssp4 antigen in the different amastigote populations were evaluated using FITC and gold-labelled antibodies, and observed with an electronic programmable individual cell sorter and transmission electron microscopy. Ssp4 antigen was present on the membrane lining the flagellar pocket and on the cell surface, as well as inside the cytoplasmic vesicles of the host cell. Flow cytometry analysis of different amastigote populations showed that intracellular amastigotes presented the highest percentage of Ssp4-expressing cells. Received: 6 April 1997 / Accepted: 23 September 1997