Observations on the Technique for the Study of Human Chromosomes-by the Culture of Leukocytes from Peripheral Blood

A critical analysis of the various features of blood leukocyte culture for the study of human chromosomes was carried out. The following observations were made: (1) Fasting blood was essential for effective separation of leukocytes. (2) These cells are easily obtained by differential centrifugation and RBC sedimentation. (3) Non-specific agglutination of leukocytes was prevented by the use of Eagle''s medium for suspension cultures. (4) No contamination occurred in a series of 50 leukocyte cultures to which antibiotics were not added. (5) Addition of an experimental Phaseolus vulgaris extract at concentration of 1 × 10^?4 to cultures resulted in a 12 to 15% mitotic index. (6) Desacetyl-methyl-colchicine (Colcemid) had optimal effect at concentration of 0.1 μg./ml. (7) Distilled water added to cell suspension in culture medium (5: 1) was an effective hypotonic agent.A simplified technique of leukocyte culture for chromosome preparations is proposed.     

Computer-assisted morphometry of the intracapillary leukocyte pool in the rabbit lung

Computer-assisted morphometry was performed to evaluate the number and cell characteristics of capillary and alveolar leukocytes in rabbit lungs. An image-processing system and a programmable spreadsheet program were used, which allowed morphometric analysis of a large reference area. Neutrophils represented the largest intracapillary leukocyte population (2.2×10⁷/ml parenchyma, which corresponds to an approximately 104-fold microvascular enrichment of this cell type related to cell counts calculated for the capillary blood volume). In addition, large numbers of intracapillary lymphocytes (1.7×10⁷/ml parenchyma; 47-fold enrichment) and monocytes (0.3×10⁷/ml parenchyma; 86-fold enrichment) were detected. The total count of pulmonary leukocytes thus approximated the total number of pulmonary endothelial cells; and the total circulating pools of the different leukocytes were surpassed by the corresponding lung capillary pools, 3.2-fold for neutrophils, 1.2-fold for lymphocytes and 4.8-fold for monocytes. In contrast, alveolar cell numbers ranged from 1–2% of the capillary counts for all types of leukocytes. We conclude that the rabbit lung microvasculature harbours large pools of immunocompetent cells, which may contribute to host-defense mechanisms at the gas-exchange area.     

Morphological and Developmental Studies of the Snake Trypanosome Trypanosoma hydrae Ayala, Atkinson, Vakalis, 1983 in Experimentally Infected Hosts and in Culture

Trypanosoma hydrae from the broad-banded watersnake, Nerodia fasciata confluens, underwent development in the freshwater leech, Placobdella parasitica. Epimastigotes and transitional stages were present only in the crop and gastric caecum. Only one metacyclic form was observed. The potential of leeches as vectors is discussed. Two broad-banded watersnakes were infected by inoculation with culture forms of T. hydrae maintained on NNN medium. Parasitemias varied from 10⁵/ml to 10⁶/ml with dividing forms seen only rarely. A broad and a slender form of T. hydrae are described from the peripheral blood of a watersnake 7 months after an experimental infection.     

A stromal cell line from rainbow trout spleen, RTS34st, that supports the growth of rainbow trout macrophages and produces conditioned medium with mitogenic effects of leukocytes

Summary A rainbow trout spleen cell line, RTS34, was developed from a long-term hemopoietic culture. This cell line consisted of a mixed stromal cell layer with an associated cell population of macrophage-like cells that formed proliferative foci and released nonadherent progeny cells into the culture medium. A stromal cell line, RTS34st, was isolated from the RTS34 cell line. RTS34st cultures contained cells with fibroblast-like and epithelial-like morphologies and showed enhanced [³H]thymidine incorporation in response to either FBS or rainbow trout serum. The combination of FBS and trout serum was synergistic. Conditioned medium from RTS34st stimulated thymidine incorporation by peripheral blood and head kidney leukocytes, but not by leukocytes from the spleen. In addition, RTS34st provided a hemopoietic inductive microenvironment for immature precursor cells, selectively supporting the growth of macrophage-like cells. Therefore, RTS34st appears useful for studying the different roles of the stroma in regulating hemopoiesis in fish.     

Growth and Immunologic Studies on the Culture Forms of Trypanosoma lewisi*

SYNOPSIS. Culture forms of Trypanosoma lewisi grown at 27 C in a diphasic blood agar medium resemble in structure the stage found in the invertebrate host. Cultures inoculated with approximately 1 × 10⁶ trypanosomes/ml attain maximum populations of 2–7 × 10⁷ organisms/ml after 5–6 days of incubation. The stationary phase persists 6–15 days. The decline of the population is of relatively long duration with approximately 1 × 10⁶ viable organisms/ml present after 90 days. Variations in growth were attributed to the preparation of defibrinated heated rabbit blood incorporated into the culture medium. With inocula of 3.0 × 10⁵ trypanosomes/ml there was a lag in growth not observed with larger inocula. Trypanosomes incubated at elevated temperatures had altered growth curves compared to organisms at 27 C. Agitation of cultures did not affect the growth or stationary phases, but hastened the population decline. Heated and unheated 5% (v/v) normal rat serum incorporated in the liquid phase of the medium altered the growth of the organisms. Heated serum caused a decrease in the population and an extended lag phase. The effects on growth were more marked with unheated serum suggesting that both heat-stable and labile components affect growth. Antisera from rats injected with live culture forms included in the liquid phase inhibited, while antisera from rats 24 days after infection with the blood stream forms had no effect on the growth of the culture forms. Antisera from rabbits immunized with sonicates of culture forms also altered the growth of the organisms in culture. Rabbit antisera prepared by immunization with sonicates of dividing and non-dividing blood stream forms had no effect on the in vitro growth. Antisera from animals immunized with rat blood and culture medium were also without effect. The immunologic implications of the data are considered and discussed.     

New Type of Colony-forming Cell located in the Pleural Cavity

A semi-solid agar culture system has been developed which supports the clonal growth of granulocytic and/or macrophage colonies from their specific progenitor cells^1,2. In the course of studies on the level of these progenitor cells in the peripheral blood of mice, whole blood was cultured in agar-medium. Cultures were prepared in 35 mm plastic Petri dishes and each culture contained 1 ml. of 0.3% agar in modified Eagle's medium. The media and general technique of agar culture have been described elsewhere³. In these experiments, 0.2 ml. of whole, unheparinized blood was taken directly from the axilla of anaesthetized 3 months old C57BL mice and added to 5 ml. of agar-medium, held liquid at 37° C. One ml. portions of this mixture were pipetted into four replicate culture dishes, allowed to gel and incubated for 7 days at 37° C in a fully humidified atmosphere of 10% CO₂ in air. Each culture contained 0.1 ml. of a 1:6 dilution of pooled sera from C57BL mice injected 3 h previously with 5 µg endotoxin to provide an adequate concentration of the specific colony-stimulating factor (CSF), required for granulocytic and macrophage colony formation.     

High-performance liquid chromatographic determination of diadenosine 5′,5‴-p1,p4-tetraphosphate with precolumn fluorescence derivatization and its application to metabolism study in whole blood

Diadenosine 5′,5‴-p¹,p⁴-tetraphosphate (Ap4A) was converted with chloroacetaldehyde to the fluorescent di-1,N⁶-ethenoadenosine derivative within 60 min at 80°C. It was separated by reversed-phase HPLC and detected fluorimetrically (excitation and emission wavelengths of 275 and 410 nm, respectively). The detection limit of Ap4A was ca. 0.2 μg/ml in plasma when 10 μl of the sample was applied to the column. The rate of degradation of Ap4A added to whole blood (5 μg/ml) was examined using this method. Half-lives (means ± S.E., n = 3) were 0.88 ± 0.30 min (in rat blood), 13.7 ± 3.6 min (in dog blood and 17.2 ± 1.4 min (in human blood). A marked species difference in the degradation rate of Ap4A in blood was observed.     

Isolation,X-Ray Structure and Biological Activity of Deacetyl-dihydrobotrydial Produced by Botrytis squamosa

Mitogenic substances on human peripheral blood mononuclear leukocytes were screened from culture filtrates of microorganisms newly isolated from soil and sea water by measuring [³H]- thymidine incorporation into the cells. Strong mitogenic activity was found in marine bacteria, particularly in marine vibrios. These mitogen samples exhibited neither hemagglutinating activity nor leukoagglutinating activity. They could scarcely stimulate murine lymphocytes.

Cell-cell interaction among leukocyte subsets in response to a bacterial mitogen was investigated using the most powerfully mitogenic sample (culture filtrate of strain H 52–2). A slight decrease in the mitogen response was observed on depletion of plastic surface adherent cells. Separation of T and non-T cells from each other by erythrocyte-rosette sedimentation resulted in a markedly diminished mitogen response. Considerable restoration of the mitogen response was obtained when T cells were mixed with mitomycin C-treated adherent cells or mitomycin C-treated non-T lymphocytes, or when non-T lymphocytes were mixed with mitomycin C-treated T cells.     

Chromosome aberrations induced in human peripheral blood lymphocytes using heavy particles from 10 B (n, ) 7 Li reaction

The relationship between α-particle dose and chromosome aberration yield was studied in human peripheral blood leukocytes cultured in vitro. The α-irradiation was produced from thermal neutron capture by boron, according to the nuclear reaction ¹⁰B (n, α)⁷Li. Blood samples containing 49 μg ¹⁰B per ml were exposed to the thermal neutrons in a reactor at a flux density of 2·10⁷n/cm²s. By subtracting the rad dose due to the reactor radiations alone from that due to both boron capture and the reactor radiations, the rad dose rate of heavy particles was estimated. The dicentric yield appeared to follow a linear response up to about 18 rad and then showed signs of “saturation”. Comparison with 250 kV X-ray data (doses up to 510 rad) gave an RBE of 22.97.     

In vitro proliferation of blood,spleen and thymus leukocytes from the turtle Mauremys caspica

Cell-mediated immune responses is commonly evaluated by cell proliferation assays. Mitogens are known to induce a vigorous proliferative response in lymphoid cells from mammals but relatively fewer studies have investigated mitogen-mediated lymphoproliferation in non-mammalian animals. In the present work, we incubated spleen, thymus and blood leukocytes with phytohaemagglutinin (PHA), concanavalin A (Con A), lipopolysaccharide (LPS) and pokeweed mitogen (PWM), by different times of incubation (96 and 120 h) and at different concentrations. Our results show that the optimal mitogen concentrations inducing proliferation on leukocytes from Mauremys caspica were 20 microg/ml PHA, 1 microg/ml Con A, 12.5 microg/ml LPS and 1/150 dilution PWM. The optimal time of incubation was dependent on the type of leukocytes (peripheral blood leukocytes, splenic leukocytes or thymic cells) and the mitogen utilized.     

Establishment of lymphocytoid cell lines in relation to number of leukocytes,surface area of monolayer formation,and feeding procedures

Summary Twenty-eight lymphocytoid cell lines were established within 6 months from the peripheral blood of a single healthy donor. Establishment seemed independent of feeding regimens. Establishment occurred if cultures in 100-ml glass bottles (surface area of 46 cm²) were initiated with a leukocyte population between 4.9×10⁷ and 6.8×10⁷ cells, and if cultures in 16-ml test tubes (surface area of 1.8 cm²) were initiated with a total leukocyte population between 4.9×10⁶ and 2.2×10⁷ cells. It is concluded that establishment is related to a optimal relationship between number of leukocytes and the surface area for possible monolayer formation. When the surface area is decreased, the initial number of leukocytes must be reduced. Establishment of a cell line did not occur with an initial total population below 4.9×10⁶ leukocytes. There is a positive correlation between initial leukocyte number, initial growth velocity, and the determination of establishment by subculture. This work was supported in part with funds from the Mary B. and L. H. Marshall Foundation. Hans W. von Heyden is supported by a Fellowship from the Deutsche Forschungsgemeinschaft. After September 1, 1972, the authors will be at the Medizinische Universit?t Klinik II, Tübingen, West Germany.     

Quantitative assessment of the adherence of normal human blood mononuclear cells to vascular endothelial cell monolayers

We have developed a method to reliably quantitate the in vitro adherence of ⁵¹Cr-labeled blood mononuclear leukocytes to cultured monolayers of vascular endothelial cells from human umbilical veins. Normal mononuclear leukocytes adhered to endothelial cells more than to cover glass at all studied time periods over 4 hr with major differences seen at 2 hr (9.7 ± 1.2% vs 3.7 ± 1.1%; P < 0.01). Only a minority of cells adhering to endothelium were esterase positive. Similar patterns of binding were seen using varying concentrations of suspended mononuclear cells (1–4 × 10⁶/ml) simulating that occurring in vivo in different clinical states. This approach shows promise for in vitro approaches to lymphocyte-vascular endothelial interactions in human immune/inflammatory disorders.     

Preparation of 111in leukocytes after hemolytic removal of erythrocytes

An optimized procedure is described for isolation and highefficiency radiolabeling of leukocytes using ¹¹¹In-oxine. The chief advantages over conventional methods include virtually no loss of leukocytes during washing and separation steps; a significant reduction in the time required to prepare leukocytes for radiolabeling compared to non-hemolytic preparations; a 28% increase in the average labeling efficiency obtained using ¹¹¹In-oxine; >95% cell viability as measured by the trypan blue exclusion test; elimination of contaminating red blood cells from the leukocyte pellet prior to labeling; and 80% survivability at 15 min post injection (measured as per cent of blood activity on leukocyte fraction).     

Generation of Nitric Oxide by Peripheral Blood Leukocytes and Platelets in Healthy Humans and Patients with Thermal Trauma

The generation of nitric oxide (NO) by human peripheral blood leukocytes and platelets has been studied in healthy subjects and patients with burns (with the affected area varying from 10 to 45% of the body surface). Differential centrifugation was used to isolate leukocytes and platelets from the blood. The leukocyte suspension was diluted with a complete medium to a concentration of 1 × 10⁷ cells/ml, and the platelet suspension, to 1 × 10⁸ cells/ml; the suspensions were then cultured for 15 h (37°C). The concentration of nitrite, an NO metabolite, was determined using the Griss reaction. The relative production of NO by leukocytes of healthy subjects and patients was 0.75 ± 0.06 and 2.93 ± 0.16 mol/l, respectively (p < 0.001), and its relative production by platelets of healthy subjects and patients was 2.15 ± 0.14 and 3.62 ± 0.13 mol/l, respectively (p < 0.01). The absolute generation of NO by leukocytes of healthy subjects and patients is 0.47 ± 0.05 and 3.02 ± 0.28 mol/l, respectively (p < 0.001), and its absolute generation by platelets of healthy subjects and patients was 7.70 ± 0.55 and 14.68 ± 0.84 mol/l, respectively (p < 0.001). Thus, the absolute production of NO by platelets is 16 times higher than the absolute production of NO by leukocytes of healthy subjects. Stress increases the generation of NO by both leukocytes and platelets. The absolute generation of NO by platelets in thermal trauma is positively correlated with the plasma content of fibrinogen in the patients.     

Prostaglandin E inhibition of mitogen stimulation in patients with multiple sclerosis

Kirbey et al have reported that leukocyte function from patients with multiple sclerosis is not suppressed by PGE₂, as are normal leukocytes. We examined the ability of PGE₂ (0.01–0.5 μg/ml) to suppress Phytohemagglutinin induced ³H-thymidine incorporation in peripheral blood lymphocytes from multiple sclerosis patients and normals. There was no difference in sensitivity between the two groups. There was also no difference in activity of the prostaglandin producing suppressor cell between the multiple sclerosis patients and controls.     

High-performance liquid chromatography determination of dipotassium clorazepate and its major metabolite nordiazepam in plasma

A rapid and sensitive high-performance liquid chromatographic method is described for the quantitative analysis of dipotassium clorazepate (CZP) and its major metabolite nordiazepam (ND) in fresh human and dog plasma. The method consists of two separate selective ND extractions from a plasma sample without and with conversion of all the CZP to ND. For quantitation, diazepam (DZP) is used as the internal standard. The chromatographic phase utilized in a reversed-phase Hibar® EC-RT analytical column prepacked with LiChrosolv RP-18 with a solvent system consisting of acetonitrile-0.05 M sodium acetate buffer, pH 5.0 (45:55). The UV absorbance is monitored at 225 nm using a variable-wave-length detector. The mean assay coefficient of variation over a concentration range of 20–400 ng per ml of plasma is less than 3% for the within-day precision. Recoveries of ND, DZP and CZP (as ND) are essentially quantitative at all levels investigated. The calibration curves of ND are rectilinear (r² = 0.99) from the lower limit of sensitivity (2 ng/ml) to at least 2000 ng/ml in plasma. Applicability of the method to CZP and ND disposition studies in the anaesthetized mongrel dog is illustrated. When the two separate selective nordiazepam extractions from plasma cannot be performed immediately after blood sampling, an extrapolation kinetic method is suggested for the estimation of CZP concentration. In all previous in vivo studies, CZP has been determined only with gas-liquid chromatographic methods.     

Trypanosoma acomys (Wenyon, 1909): Continuous Culturing with a Mouse Fibroblast Cell-Line (A9)

The continuous culturing of Trypanosoma acomys in the presence of a murine areolar-adipose cell line (A9) was possible for the 1st time. The trypanosomes were cultured at 37° C with A9 in DMEM supplemented with 20% heat inactivated fetal bovine serum, using an initial inoculum from primary cultures of lung or blood clots from infected spiny mice. The cultures were maintained for 115 days and underwent 15 passages before termination and cryopreservation. Using this culture system T. acomys subcultures were initiated from 3 different initial inocula (3 × 10⁴, 1.5 × 10⁵ and 7.4 × 10⁵ parasites/ml) and growth curves revealed that the lowest inoculum gave the best growth pattern. This inoculum yielded a population doubling time of less than 12 h for 4 days, a high peak density of 7 × 10⁶ parasites/ml and the most gradual decline compared to the other 2 inocula. Rosetting epimastigotes and nests of amastigotes were observed in close association with the feeder layer cells. Epimastigotes were the most predominant form in culture supernatants but other morphological forms observed included trypomastigotes and sphaeromastigotes.     

甲肝病毒在人血白细胞中体外连续传代和在分类白细胞中增殖的研究

1985年我们采用间接免疫荧光法(IF法)检测出甲肝患者外周血白细胞中有甲肝抗原(HAAg)存在,继而又将HAAg阳性白细胞直接种入PLC/PRF/5细胞,分离到两株甲肝病毒(HAV)NJ—3株和H—1株。为了弄清白细胞所携带的病毒究竟仅为吸附吞饮,抑或能在其中复制增殖,我们将分离到的HAV用正常人血白细胞进行体外增殖试验,现将结果报告如下。     

The protective effect of adenosine triphosphate-MgCl<Subscript>2</Subscript> on ischemia-reperfusion lung injury is leukocyte dependent

Adenosine triphosphate (ATP)-MgCl₂ attenuates ischemia-reperfusion (I-R)-induced lung injury in rats. A previous study indirectly suggests that Mg²⁺-dependent ecto-ATPases on the surface of leukocytes are responsible for the hydrolysis of ATP-MgCl₂ to adenosine, which then contributes to the protective effect of ATP-MgCl₂. This study investigated the role of leukocytes in I-R injury and the protective effect of ATP-MgCl₂ in our buffer-perfused isolated rat lung model. After isolating the lung blood flow of adult male Sprague-Dawley rats, the lungs were perfused through the pulmonary artery cannula with a physiologic salt solution containing human serum albumin. The protective effect of ATP-MgCl₂ pretreatment with or without leukocytes was investigated. Capillary permeability (K_fc), lung weight gain (LWG), lung wet weight/body weight ratio (LW/BW), lung lavage protein concentration (LPC) and pulmonary artery pressure (PAP) were measured. I-R produced a significant increase in K_fc, LWG, LW/BW, LPC, and PAP. The increases in these indices were significantly attenuated by pretreatment with ATP-MgCl₂ (1×10^–6 M) together with leukocytes (2.9×10⁶/ml in the perfusate) but not with ATP-MgCl₂ alone. Our data suggest that I-R-induced acute lung injury is not dependent on circulating leukocytes. Pretreatment with ATP-MgCl₂ plus leukocytes but not ATP-MgCl₂ alone had protective effects against I-R lung injury. Whether these findings occur in vivo could not be determined in this study. In our isolated lung red blood cell-free perfusate system, the protective effect of ATP-MgCl₂ requires the presence of leukocytes.     

Bird Erythrocytes in Experimentally Introduced Blood Clots, for Differentiation from Autogenous Mammalian Clots

In studies of the effect of snake venom on blood clotting, 5 ml of freshly drawn, unclotted dog blood was centrifuged to separate red cells from plasma. Chicken erythrocytes were added to the plasma to give 2 × 10⁶ cells/ml. The mixture was clotted by adding 1 ml of 0.1 M CaCl₂ to it, and clotting allowed to occur in an 8 mm (ID) glass tube. The 10 cm long clot so obtained was injected into the inferior vena cava of the experimental dog. Such introduced clots and their emboli could be readily recognized by the presence of nucleated erythrocytes.