The influence of serum content in the medium on the survival of human bone marrow in suspension in vitro.

Samples of human bone marrow cells from patients with various diseases were cultivated in vitro by means of a simple stationary suspension method. Medium Eagle MEM with the addition of allogeneic serum and borosilicate glass were used. The cells survived significantly longer in samples with 50 per cent of serum than in samples with 30 per cent of serum only. Monocytes showed the longest survival (max. 95 days) in cultures with 50 per cent of serum. Myelocytes and segmented neutrophils as well as normoblasts survived till 45th day and plasmocytes till 66th day. Mitoses in monocytes were found till 50th day. Moreover so called "satellitosis" of plasmacytes around a macrophage was observed in cases with reactive plasmacytosis.     

In vitro studies of fetal rat fibroblasts after experimental fenoterol administration

White female rats were daily treated with Fenoterol in a dose of 1.8 mg/kg of body weight beginning with the 10th d till the end of gestation. Cell cultures were made by using the skin of mature foetuses. Fibroblast-like cells of primary and passaged cultures were examined. The slices were stained by Giemsa's reagent and examined with Brachet's reaction to ribonucleic acid. Histological reactions to acid phosphatase activity, succinic and lactic dehydrogenases demonstrated changes showing disturbed metabolism of the examined cells.     

Formation of the external zone of the median eminence of rats in the perinatal period

The formation of interrelations of the axons of neurosecretory cells and of ependyme cells with the capillaries of primary portal plexus in rats from the 14th day of embryogenesis till the 9th day of postnatal life was studied using the light and electron microscope methods. During the whole period under study, the basal processes of the ependyme cells reach the primary portal plexus of the capillaries. The terminals of the basal processes are usually separated from the endothelium of the capillaries by two basal membranes and enclosed pericapillary space. After the birth, some basal process penetrate in the pericapillary space and terminate on the endothelium. The surface of contact of the ependyme cell processes with the external basal membrane increases with the age, this being accompanied by the increase of pinocytotic activity. The neurosecretory axons are found in the median eminence already on the 14th day of embryogenesis, but by the 20th day only they reach the external basal membrane and penetrate sometimes in the pericapillary space. After the birth, the number of axons reaching the external basal membrane and the surface of contact between them increase gradually with, apparently, a concomitant intensification of the transport of neurohormones in the portal circulatory system of the hypophysial-hypothalamic complex.     

Establishment of a peritubular myoid-like cell line and interactions between established testicular cell lines in culture

Established cell lines and primary cultures derived from somatic cells of the testis have been used to study cell-cell interactions. Primary cultures of Sertoli cells or Sertoli-derived cell lines from the mouse (TM4) and rat (TR-ST) will aggregate when plated on monolayers of primary cultures of peritubular myoid cells or a rat (TR-M) cell line which has many properties of peritubular myoid cells. Time-lapse cinematography and scanning and transmission electron microscopy reveal that Sertoli cells formed aggregates after 1 day in coculture, display surface activity and move on the monolayer. When these aggregates touch one another, they rapidly combine. By the 4th day of culture, spherical aggregates are composed of 50 to 200 cells. They do not display surface activity or movement on the myoid monolayer. On the 5th and 6th day of culture most spherical aggregates have flattened to form dome-shaped aggregates in close association with the monolayer. Cells in the aggregates are characterized by long microvilli and some ruffles. In large aggregates, cells sometimes form close associations within the aggregates although junctions are seldom observed. Sertoli-derived cell lines will not aggregate on monolayers of Leydig-derived (TM3) or testicular endothelial-derived (TR-1) cell lines. Neither TM3 nor TR-1 cells will aggregate when plated on myoid monolayers. The TR-M cells produced an extensive extracellular matrix beneath the cells which contains collagen, an amorphous globular material resembling elastin and a fibrous noncollagenous component. Sertoli cells plated on this matrix will not aggregate. Thus the aggregation of Sertoli cells on myoid cell monolayers is cell type, but not species dependent and not determined solely by extracellular matrix components produced by TR-M cells.     

Primary cell cultures of leukosis P-388 as a possible model for the screening of antitumor substances]

A substrain of the ascitic leucosis P-388 was obtained as a result of interchanging passages of P-388 leucosis cells in the primary cultures and abdominal cavity of mice. The tumor cells of the ascitic leucosis P-388 multiplied in the primary suspended culutres as well as in vivo. The substrain lost its hemorrhagic properties. The content of DNA and RNA in the primary cultures of P-388 doubled every 16-18 hours and the number of the cells doubled every 24-26 hours. The cells of the adapted substrain of P-388 grew also in the semiliquid agarized medium forming compact colonies by the 4th-5th day of cultivation. The primary suspended cultures of P-388 were highly sensitive to cytostatics and in particular to vinblastin and kolchamine (alkaloids). In this connection they were recommended for prescreening antitumor compounds.     

The development of a kitten lung cell strain and its chromosomes

Summary The development of a line of epithelial cells derived from lung tissue of a 4-week old kitten (KL strain) with evidence regarding its chromosomal changes in vitro is described. The outgrowing cells from fragments in primary cultures in Eagle's medium plus 10% horse serum were scraped with a rubber policeman and dispersed with a syringe fitted with a No. 15 needle. The cell suspension was transferred into a T 30 flask. The floor of the T 30 flask was covered with avian plasma clot which was allowed to set for 10 minutes before adding the cell suspension. The appearance of the cells was epithelial-like at all stages of cultivation. The most frequent chromosome number in 6-day primary culture preparations was 38 (68%). Counts made from cells in the 4th, 9th and 33rd subcultures (64th, 83rd and 165th days from the date of primary culture) showed a spread of the chromosome numbers. In the latest observation, the 84th subculture (411th day), the most frequent chromosome numbers were 90 (22%) and 92 (26%). In addition, the mitotic activity of cells in the strain cultures was observed by phase microscopy.Tobacco Industry Research Committee Fellow.     

Biosynthesis of major plasma proteins in the primary culture of fat body cells from the silkworm, Bombyx mori

Plasma proteins termed "SP1" and "30K proteins" are synthesized by the fat body cells of the silkworm, Bombyx mori, in a sex- and stage-specific manner during larval development. We successfully established a primary culture of the fat body cells in order to investigate the regulatory mechanisms of plasma protein gene expression. The primary cultures of fat body cells contained at least two cell types: small oval cells, and large spherical cells. The cells adhered to and migrated on the cultured dish after plating. By the 7th day of cultivation, the cells clustered to form fat body-like structures, which were maintained for at least 3 months. Plasma proteins were actively synthesized in the primary cultures of the fat body cells isolated from the final instar larvae only when the cells tightly adhered to and clustered on the cultured dish. Immunocytochemical analysis revealed that only 10-15% of the clustered cells synthesized plasma proteins in our culture system, indicating that the primary culture comprises heterogeneous cells that are morphologically and functionally distinct. The patterns of SP1 syntheses in primary cultures faithfully reproduced their sex-dependency in vivo.     

Kinetics of oligodendrocyte-like cells in primary culture of mouse embryonic brain

An oligodendrocyte-like cell population was identified and its kinetic features were studied in primary cultures of mouse brain. Cells from telencephalon and diencephalon of 14-day-old embryos were dissociated before seeding on poly-l-lysine or collagen-coated glass coverslips. After some divisions and migrations, cells differentiated throughout the cultures; round and small overlying cells appeared after the 9th day in vitro. Their morphology, based on light microscopy, after May-Grunwald-Giemsa staining, scanning electron microscopy, and transmission electron microscopy, looked like that of oligodendrocytes. These cells are referred to here as oligodendrocyte-like cells. Their filiation, kinetics, and proliferation were studied by quantitative radioautography. Their density increased from the 9th to the 16th day in vitro. Two groups were found: large and clearly labeled oligodendrocyte-like cells and small dark unlabeled ones which undoubtedly derived from the first. Analogies between our observations in vitro and those carried out in vivo by several experimenters, suggest that in vitro these cells are probably oligodendrocytes.     

Growth of the eye and pigment epithelium of the retina during postnatal development of rats]

By the weight changes, the rat's eye grows during the whole life, whereas its scleral sector with the area equal to that of pigment epithelium of the retina grows most intensively between the 2nd and 5th days after birth. During this period, its weight attains half of the value characteristic of the scleral sector for one year old rats. This period of growth of the scleral sector coincides with the previously established peak of proliferative activity and appearance of first binuclear cells in the pigment epithelium. From the 5th day till the 12th month after birth, the weight of scleral sector increases twice and its area 6 times. This suggests that the mechanical tension of the scleral sector walls is one of the factors of growth of this eye part. On the basis of comparison of the scleral sector growth, changes in proliferative activity and number of polyploid cells in the pigment epithelium of the central zone of fundus oculi, the following periodization of the life cycle of cell population of the pigment epithelium is proposed: (1) from birth till the 15th day--period of principal determination (the number of binuclear tetraploid cells attains 80%), (2) from the 15th day till, at least, the 5th month--period of stabilization, (3) after 5 months--period of senescence characterized by the accumulation of highly ploid tri- and tetranuclear cells; its lower limit is not clearly defined.     

The development of mitochondrial oxidative enzymes in rat heart muscle.

Three different developmental patterns have been found in the heart muscle mitochondria: (a) Activity of inner membrane enzymes, succinate-cytochrome c reductase and rotenone-sensitive NADH-cytochrome c reductase, was found to increase rapidly after birth till the 25th day; no further increase was found till the 60th day. Both brances of the respiratory chain, i.e. NADH-dependent and flavoprotein-linked were found to develop in parallel. (b) Activity of retoenone insensitive-NADH cytochrome c reductase, an outer membrane enzyme, did not show any change during developement. (c) Activity of monoamine oxidase, another outer membrane enzyme, was found to increase after the 10th day of postnatal life and the increase in activity continued till the 60th day.     

Morphological characterization of stationary reticulum cells of the stromal in the mesenteric lymph node of the guinea pig

The large mesenteric lymph node taken from guinea pigs in a period of time ranging from the 10th day prepartum till the 26th day postpartum has been examined in order to study: the morphological features of the stromal stationary reticulum cells with particular regard to recognize their stages of development; the possible ontogenetic relationship between these cells during the maturation of the lymphoid tissue. Our data support the hypothesis that from local mesenchymal cells originates a pool of poorly differentiated reticulum cells that can give rise to stromal stationary reticulum cells (myofibroblast-like cells, fibroblast-like cells, pericyte-like cells and dendritic cells). These elements have a characteristic distribution pattern likely related to different local functional requirements.     

The role of dendritic cells in the initiation of immune responses to contact sensitizers. I. In vivo exposure to antigen

Twenty-four hours after skin painting mice with picryl chloride (PIC) there was a four- to fivefold increase in the numbers of dendritic cells (DC) isolated from the lymph nodes. These DC initiated primary proliferative and cytotoxic responses when added to cultures of normal syngeneic lymph node cells. The proliferative response was enhanced when the donors of the responding lymph node cells were sensitized with the same antigen. Contact sensitivity developed in syngeneic mice injected into the footpads with 30,000-50,000 DC from lymph nodes of mice painted with picryl chloride 1 day previously. Thus, 1 day after skin painting mice, there were dendritic cells in the draining lymph nodes which were able both to initiate primary stimulation of lymphocytes in vitro and to sensitize recipient mice to give specific delayed hypersensitivity reactions.     

Ultrastructural features of the developing hypothalamo-hypophysial axis in the rat

Summary Nerve fibres containing granular vesicles first appear in the median eminence of the rat on the 16th foetal day while secretory granules in the cells of the adenohypophysis are not present till the 17th foetal day. These observations suggest that the differentiation and early activity of pars distalis cells may depend on substances elaborated at nerve terminals in the median eminence. Although the loops of the primary plexus of portal vessels do not develop until the 4th postnatal day, substances released by nerve fibres in the neurohypophysis could reach the pars distalis through vessels already present at the 15th foetal day in the mesenchyme between the diencephalon and the adenohypophysis. This view is supported by the fact that the earliest cells to exhibit ultrastructural evidence of secretory activity are in the rostral pole of the pars distalis, the first region of the gland to become vascularized. The earliest granules to appear in the cells of the pars distalis correspond to those which are considered to contain mucoprotein hormones; somatotrophin type granules were seen only in postnatal tissues.The finding that, in the median eminence, the development of granular vesicles precedes that of agranular vesicles is discussed with reference to the times at which neurosecretory materials and monoamines become detectable in the region.We should like to thank Miss Ann Pearson, Mr. D. Burns, and Mr. J. Nailon for their technical assistance, and Mr. J. Simmons, F.R.P.S., for his help in the preparation of illustrations. This work was supported by grants from the National Health and Medical Research Council of Australia.     

Interaction of Staphylococcal Alpha Toxin and the Estrogenic Hormone Diethylstilbestrol

Previous work in this laboratory showed that diethylstilbestrol was capable of suppressing induced furunculosis in rabbits. The present study indicates that the synthetic estrogenic hormone diethylstilbestrol which is used for acne, estrogen deficiency, cancer, and other disorders, can reduce the cytolytic action of staphylococcal alpha toxin. The cytotoxic action of purified alpha toxin for tissue cultures was evaluated by use of such parameters as total and viable cell counts, glucose, and protein determination, and cytopathic effects (CPE) in the presence and absence of steroids. To 3-day-old primary rabbit baby kidney tissue cultures, 1 to 5 μg of diethylstilbestrol per ml was added; growth of tissue cultures in Eagles medium was continued till the 6th day, and then one tissue cytopathic dose per milliliter of alpha toxin was added, and the subsequent fate of tissue cultures was assayed. Such cultures yielded higher total and viable cell counts, utilized more glucose, and contained more protein than the control cultures. In control cultures, CPE was observed on the 3rd hr after the addition of alpha toxin, and it was complete in 24 hr, whereas in tissue cultures treated with diethylstilbestrol, the CPE was significantly reduced. The data presented in this study made possible the availability of a suppressor of the cytolytic action of alpha toxin and might be useful in assaying the action of alpha toxin in an in vitro inexpensive test system.     

Molecular characterization and evolution of arthropod-pathogenic Rickettsiella bacteria

We determined the 16S rRNA gene sequences of three crustacean "Rickettsiella armadillidii" strains. Rickettsiella bacteria overall appear to form a monophyletic group that diverged from Coxiella bacteria approximately 350 million years ago. Therefore, the genus Rickettsiella as a whole (not just Rickettsiella grylli) should be classified among the Gammaproteobacteria instead of the Alphaproteobacteria.     

Ultimobranchial body and parathyroid gland of the parrot Psittacula psittacula in response to experimental hypercalcemia

Psittacula psittacula when subjected to long term hypercalcemia by intramuscular injections of vitamin D2 (20,000 I.U.) on alternate days and by increasing dietary calcium, exhibit a rise in the serum calcium level after 10, 20 and 30 days of treatment as compared to their corresponding controls. The ultimobranchial cells show progressive hypertrophy up to 20th day of the treatment. From 20th day till the end of the experiment (30 days) these cells show feeble staining response. The parathyroid glands suffer from degenerative changes due to its inactivity under chronic hypercalcemia.     

汉坦病毒在原代人胚肾与人胚肺细胞中增殖及其特性的研究

近年来研究发现,汉坦病毒能在多种细胞株及人体细胞中增殖、适应.国内学者用人胚肺二倍体细胞(2BS)体外适应该病毒成功,但有关原代人胚肺、肾细胞的报道尚少.作者选用两株病毒及两种原代人胚细胞用于体外感染、观察,应用免疫荧光间接法及胶体金包埋前染色电镜技术对宿主细胞中增殖的病毒进行了动态观察及特异性定位研究,现报告如下.1 材料和方法1.1 细胞的分离和培养1.1.1 人胚细胞:取正常孕妇5—7个月水囊引产胚肾及肺组织,按常规方法分散细胞,5—7天后长满单层.1.1.2 非洲绿猴肾上皮细胞(VeroE6):由安徽省医学科学研究所倪大石惠赠.1.2     

初代和继代培养葡萄试管苗的内源nA、ZRs和ABA含量变化及其与生根的关系

葡萄品种皇家秋天与藤稔初代试管苗在第10天(根原基发生)时内源IAA和ABA含量达最低点后上升;ZRs含量达最高值后下降。皇家秋天继代试管苗的IAA在第8天(根原基发生)时达最低点,而后上升达稳定4／t;ZRs含量达最高值后下降;ABA含量降至最低点后急剧上升,在第10天(生根时)达最高值,持续4d后又下降。藤稔继代苗在第6天(根原基发生)时IAA含量最低,后上升,持续到第12天后又下降;ZRs含量达最高值后快速下降,至第12天达稳定状态;ABA含量达最低值后缓慢上升,第10天(生根)达最高点后又下降。     

Human T-cell cultures with selective autotumor reactivity

Summary T-cell cultures derived from the blood of 14 patients with solid tumors were propagated with T-cell growth factor (TCGF). The cultures were initiated from lymphocytes exposed to autologous tumor-biopsy cells. TCGF was added either immediately or 3–10 days later. In the former culture type the cell yield on day 7 was considerably higher. The cytotoxic potential of the cultured cells was assayed on two occasions, between days 7 and 10 and between weeks 5 and 8. Cells of all but two cultures had the potential to lyse autologous tumor-biopsy cells.On the population level, cytotoxicity was specific for autologous tumor in those cultures that were driven to growth with TCGF after the 3rd day. These lymphocytes did not lyse allogeneic tumor-biopsy cells. In contrast, all five cultures initiated in the presence of TCGF exhibited a broader cytotoxic potential, i.e., in addition to the stimulator autologous-tumor cells, they also lysed other targets. Another difference between the two culture types was their behavior toward K562. Tested on the 7th day they all lysed K562; however, this function declined in strength or disappeared later in the cultures exposed to TCGF after the 3rd day.Reexposure of the lymphocytes to autologous tumor-biopsy cells after 2 weeks of culture period, but not on the 7th day, induced DNA synthesis. This secondary response was specific inasmuch as allogeneic tumor cells had little or no effect.One of the autotumor restimulated cultures was tested for cytotoxic potential. It increased against the autologous but not against other tumors or K562 cells.     

Nerve fiber interrelationships with the ependyma of the 3d ventricle in rats in the perinatal period]

The localization and ultrastructure of nerve fibers in the wall and lumen of ventral region of III ventricle was studied from the 16th day of prenatal period till the 9th day of postnatal period in the Wistar rats. The nerve fibers were first found in the subependyme zone; between the ependyme cells and in the lumen of ventricle on the 18th day of development. The nerve fibers occur constantly in the lumen of ventricle beginning from the 3rd day of postnatal period. The dilatations of nerve fibers contain granules of 50 to 90 nm in diameter with the electron-dense center (carriers of monoamines and/or releasing hormonnes) granules with moderately osmiophilic contents and light vesicles of the same size. It is suggested that these structures reflect different stages of the release of neurohormones in the cerebrospinal fluid. The synapse-like contacts of nerve fibers with the ependyme cells are observed beginning from the 3rd day of postnatal period. Their role in the regulation of absorption of substances by the ependyme cells functioning is discussed.