General similarities and differences in the postradiation death of various animal species and of man

It was found in various animal species and man that an ordered internucleosome fragmentation of DNA is characteristic of lymphoid cells dying in the interphase. Both in vivo and in vitro, the postirradiation DNA degradation in thymocytes of rodents and piglets preceded the increase in the permeability of their plasma membrane. The in vivo kinetics of death of lymphoid cells from the thymus and spleen is similar in rodents and piglets. Rat thymocytes died in vitro earlier than thymocytes of piglets, calves and man which was evidently associated with a worse adaptive capacity of the latter to cultivation conditions.     

Plasmodium berghei: infective exoerythrocytic schizonts in primary monolayer cultures of rat liver cells.

Suspensions of rat liver cells which included hepatic exoerythrocytic schizonts (HEX) of Plasmodium berghei were used to initiate primary monolayer cultures of rat hepatic cells. These cell suspensions were prepared by using an enzymatic method for the dissociation of the livers of rats that had been infected with sporozoites of P. berghei 3 to 10, 18 to 28, and 29 to 36 hr prior to the liver dissociation procedure. These cell suspensions included HEX which were infective for recipient rodents when inoculated intraperitoneally into the recipients. HEX were considered to have been successfully maintained if they retained their infectivity for rodents following the cultivation period. The relative number of infective HEX present in the liver cell suspensions before and after cultivation was determined by use of an infectivity assay. Using this infectivity assay, it was observed that less infective HEX were present in the cell population following cultivation than were present before cultivation. Infective HEX were recovered from culture in experiments in which the time in vitro ranged from 3 to 44 hr. Twelve of fifteen (80%) attempts to maintain infective HEX in culture for 21 to 28 hr were successful, while one of eight (12.5%) attempts to maintain HEX in culture for 36 to 48 hr were successful. Thus, these experiments have provided an 80% success rate for maintaining HEX for a period equivalent to over 50% of the incubation period of HEX of this parasite. This technique should be sufficient for studying in vitro the factors which influence the development of HEX, as well as for testing methods of causal prophylaxis.     

Physical separation of hemopoietic stem cells from cells forming colonies in culture

Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.     

Immunomodulation by Histoplasma capsulatum products; polyclonal activation and mitogenic effects

The presence of reactive spleen cells to sheep red blood cells (SRBC) in nonimmunized BALB/c mice injected with histoplasmin, the culture filtrate of Histoplasma capsulatum, was monitored for 21 days following inoculation. Polyclonal activation, as evidenced by a sharp increase in the number of anti-SRBC rosetteforming cells (RFC), as well as an enhanced response to heterologous non-cross-reactive erythrocytes from other species, was found in the spleens of these rodents on Days 11 to 13. Elimination of B-cell-derived RFC by the addition of complement indicated that the erythrocyte-binding cells consisted of both T- and B-lymphocytes. An immunosuppressive effect was detected if histoplasmin was injected 2 days before the antigen (SRBC), but could be reversed by injecting the filtrate 30 min prior to the antigen, as is found with polyclonal activators displaying immunosuppressive activity. Histoplasmin also had a mitogenic effect on lymphocyte obtained from the spleen, bone marrow, and thymus similar in magnitude to that produced by lipopolysaccharide (LPS) and concanaval in A. The biological significance of these findings is discussed.     

Establishment and characterization of rough-tailed gecko original tail cells

Some of lizard species have the ability to lose their tail in order to defend against predators and regenerate the new tail. Lizard’s regenerated tail has attracted scientists’ attention for unraveling the regeneration process, but less information is known about the cellular characterization and cell growth properties of original tail. This research aimed to report cell culture and banking process of rough-tailed gecko or Cyrtopodion scabrum’s original tail cell sample from inner tissue without skin using tissue explant technique. For banking reports, it is essential to analyze this cells’ potential to proliferate, to investigate biological aspects such as cell culture features, differentiation and chromosome number and to report its species identification and quality control. To achieve optimal growth conditions, three different temperatures for incubation including 18, 23 and 37 °C and two different media including DMEM and L-15 were applied. The expanded cells were studied for their potential to adipose and osteoblast differentiation. Results indicated that lizard’s original tail cells could be successfully obtained by explant technique. The cells demonstrated fibroblast like morphology with population doubling times of approximately 24?±?0.5 h. Karyotyping analysis showed a distribution of 2n?=?40 chromosome number for this cell line. The comparison of different incubation media and temperatures showed that cell growth is equally optimal in all mentioned conditions according to growth curves. Adipose and osteoblast differentiation was obviously observed in these cells which confirms the hint of stem-ness in the produced mixed cells. According to cell banking policies, produced cells were also checked for bacterial, fungal, yeast and mycoplasma contaminations and no contamination was observed. Multiplex PCR for identification of species confirmed the species of lizard with no cross-contamination with other cells in the cell bank. Establishment of authenticated and well-characterized lizard’s original tail cell line will provide a valuable source for subsequent in vitro regenerative research and molecular studies which are not feasible in in vivo methods. This finding will allow us to get an opportunity to create and preserve a new collection of lizard cell lines in the future.     

In Vitro Reconstitution of Anti-Sheep Erythrocyte Antibody Response of T Cell-Depleted Spleen Cells by Allogeneic T Cells or by Factors Derived from Them

Restoration of the impaired antibody response to sheep erythrocytes (SRBC) in cultures of mouse spleen cells, which were deprived of thymus-derived lymphocytes (T cells) by treatment with anti-mouse brain-associated θ (BAθ) antiserum and complement, was studied by adding a small portion of syngeneic or allogeneic normal spleen cells in vitro. Allogeneic spleen cells had a far greater effect than syngeneic spleen cells on the restoration, as far as the normal spleen cells added were able to recognize the alloantigens on the anti-BAθ serum-treated spleen cells (bone marrow-derived lymphocytes). Treatment of the allogeneic spleen cells with mitomycin C did not affect their activity in the restoration of the impaired antibody response. The possibility that the role of T cells in the antibody response to SRBC may be replaced by a nonspecific mediator derived from T cells reacting with allogeneic cells was proven by the finding that supernatant of the mixed allogeneic spleen cell cultures restored the impaired anti-SRBC antibody response of the T cell-depleted spleen cells. The effect of such culture supernatant on the restoration of the antibody response was greatest when it was added to the T cell-depleted spleen cell cultures one day after cultivation with SRBC, suggesting that the effectiveness may result from triggering of the proliferation and differentiation of antibody-forming cell precursors, which have already reacted with the antigen, to antibody-forming cells.     

Determination of lymphokine-induced cytolytic activity of macrophages by measuring the 3H-thymidine residue in prelabeled tumor cells

A technique of macrophage-activating factors (MAF) detection by the in vitro determination of macrophage (Mph) antitumour cytolytic activity by 3H-thymidine residue in the pre-labelled neoplastic target cells (TC) is suggested. Peptone-induced Mph were cultivated for 20-22 hours in the presence of crude supernatants from concanavalin-A-stimulated spleen cells or from the secondary mixed lymphocyte culture. 3H-thymidine-labelled cells of mastocytoma P815 were then added to the washed Mph. The lysis was measured in 48 hours according to the isotope remainder in the non-acid-soluble fraction. The optimal conditions for MAF detection have been selected. Parallel MAF testing with this particular method and with a standard technique using labelled 51Cr TC made it possible to conclude that the method suggested is more sensitive because it permits a combined cultivation of Mph and TC for a relatively long time period.     

The effect of immobilized fibronectin on karyotypic variability in the skin fibroblast cell subline of indian muntjac

The numerical and structural karyotypic variability has been investigated in the Indian muntjac skin fibroblast cell subline MT on cultivating cells on the fibronectin-coated surface. In cell subline MT, cultivated on the fibronectin-coated surface for 1 and 2 days, the character of cell distribution for the chromosome number did not change. In 3, 4 and 8 days, the character of cell distribution for the chromosome number changed. These changes involve a significant decrease in frequency of cells with modal numbers of chromosomes, and an increase in frequency of cells with lower chromosome numbers. Many new additional structural variants of the karyotype (SVK) appear. The observed alterations seem to be due to both disturbances of mitotic apparatus and selection of SVK, which are more advantageous to changed culture conditions of the cell population. Detachment of cells from the fibronectin-coated surface, followed by a 1 day cultivation on a hydrophilic surface, commonly used for routine cell cultivation, does not restore the control cell distribution for the chromosome number, but cultivation in these conditions for 5 days restore control distribution. The frequency of chromosomal aberrations on cultivation on the fibronectin-coated surface for 3 and 4 days significantly increases, mainly at the expence of dicentrics (telomeric association). On prolongating the time of cultivation up to 8 days on the fibronectin-coated surface the frequency of chromosomal aberrations approaches the control value. Structural instability of chromosomes at cultivation on the fibronectin-coated surface demonstrates nonspecific reaction of "markerless" cell lines to unfavourable factors of the environment. We discuss possible reasons of differences in the character of karyotypic variability in cell lines of the Indian muntjac skin fibroblasts on cultivating on laminin and fibronectin.     

Effect of laminin on karyotypic variability in cell line of Indian muntjac skin fibroblasts

The numerical and structural karyotypic variability has been investigated in the Indian muntjac skin fibroblasts cell line M and karyotypic variant of this line M' on cultivation on a laminin 2/4 coated surface. In cell line M, cultivated on the laminin-coated surface for 4 and 14 days, and in karyotypic variant M', cultivated for 2, 4 and 14 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with model numbers of chromosomes, and an increase in frequency of cells with lower chromosome numbers. As a result, new modal chromosome numbers form. The frequency of cells with 4 chromosomes increases significantly; as a rule, such cells are absent in the control cell variants. Many new additional structural variants of the karyotype (SVK) appear. Detachment of cells M' from the laminin-coated surface followed by a 2 day cultivation on a hydrophilic surface, commonly used for routine cell cultivation, does not restore the control cell distribution for chromosomal number. The frequency of chromosomal aberrations on cultivation of the laminin-coated surface does not change relatively to controls. The observed alterations seem to be due to both disturbances of mitotic apparatus and selection of SVK, which are more advantageous to changed culture conditions of the cell population.     

Staining Air Dried Protoplasts for Study of Plant Chromosomes

The air drying technique used in mammalian cytology was applied to isolated plant protoplasts for study of chromosomes. For cultured celery cells, this technique resulted in good spreads of metaphase chromosomes with high resolution. Mitotic chromosomes of Brassica species are relatively small, poorly stained by common stains, and difficult to spread by the squash technique. In this study, however, the chromosomes of B. carinata in callus culture were spread well and stained clearly with Giemsa staining solution. The chromosome preparations by the present techniques should also be amenable to chromosome banding studies in plants.     

Staining air dried protoplasts for study of plant chromosomes

The air drying technique used in mammalian cytology was applied to isolated plant protoplasts for study of chromosomes. For cultured celery cells, this technique resulted in good spreads of metaphase chromosomes with high resolution. Mitotic chromosomes of Brassica species are relatively small, poor stained by common stains, and difficult to spread by the squash technique. In this study, however, the chromosomes of B. carinata in callus culture were spread well and stained clearly with Giemsa staining solution. The chromosome preparations by the present techniques should also be amenable to chromosome banding studies in plants.     

Further studies on generation of macrophages in in vitro cultures of mouse spleen cells and its inhibition by the capsular polysaccharide of Klebsiella pneumoniae.

Although the number of macrophages detected in cultures of mouse spleen cells at the start of the culture was very small, it markedly increased during further incubation. Macrophages were generated not only from the glass-adherent cell fraction of spleen cells, but also from the nonadherent cell fraction obtained after removal of adherent cells either by incubating in glass petri dishes or by passing through a glass bead column. The generation of macrophages from the nonadherent cell fraction occurred even when it was separated as late as 48 hr after the start of the culture. The phagocytic activity of macrophages newly generated from the nonadherent cell fraction was relatively weak, but it was activated during further incubation. Based on these results, the maturation process of macrophages can be divided into at least the following four stages; glass-nonadherent nonphagocytic precursor cells, glass-adherent nonphagocytic precursor cells, immature macrophages with low phagocytic activity, and mature macrophages with full phagocytic activity. The addition of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) to cultures of spleen cells markedly suppressed the generation of macrophages. The suppressive effect of CPS-K depended on its dosage, and the minimum concentration of CPS-K showing a definite effect was 0.05 μg/ml. CPS-K inhibited further generation of macrophages in either the nonadherent or adherent cell fraction at any time after the start of the culture. The suppressive effect of CPS-K on the generation of macrophages could not be reversed by simple washing of spleen cells which had been kept in contact with CPS-K for 3 hr. There was no evidence which showed that CPS-K exhibited direct cytotoxic effects on spleen cells in the culture.     

Formation of Mononuclear Phagocyte (Macrophage) Colonies by Mouse Spleen Cells in Liquid Culture

When spleen cells of the adult mouse were tested for the formation of mononuclear phagocyte (macrophage) colonies by the liquid culture technique with an incubation period of 7–8 days, about 100 macrophage colonies were produced from 1 × 10⁶ cells. The number of macrophage colonies appearing after 2 days of incubation was small, but thereafter increased progressively up to at least 8 days. In the later stages of incubation (after day 6) large colonies consisting of more than 100 cells appeared. Macrophage colonies in the early stages consisted almost solely of macrophages. On day 6 significant numbers of small round mononuclear cells with no detectable phagocytic activity were seen in the center of large colonies, and by day 8 marked crowding of these cells had occurred. The peripheral region of the large colonies consisted mainly of macrophages and the intermediate region of middle-sized round or slightly stretched cells with weak phagocytic activity. Approximately two-thirds of the colony-forming cells still remained after glass-adherent cells were removed from the spleen cells by passing over a glass-bead column. In cultures of glass-nonadherent cells macrophage colonies were not generated in the early stage. The number of colony-forming cells did not change significantly even after actively phagocytic cells were rigorously removed from the spleen cells. In addition, no macrophage colonies were generated in cultures of spleen cells treated with mitomycin C.     

The influence of a substrate including extracellular matrix proteins on karyotypic variability in two cell lines of Indian muntjac skin fibroblasts

The influence of cell-culture conditions on numerical and structural karyotypic variability has been investigated in two Indian muntjac skin fibroblast “markerless” cell lines, M and MT. The cells were cultivated on a substrate consisting of extracellular matrix proteins (ECMs) synthesized by human mesenchymal stem cells (SC5-MSC). The character of cell distribution for the chromosome number of the cell line M changed after cultivation for 1 and 4 days as compared to control cells, which were cultured on a hydrophilic surface without ECM coating. These changes involve a significant decrease in frequency of cells with the modal numbers of chromosomes and an increase in frequency of cells with lower chromosome numbers. Many new types of additional structural variants of the karyotype (SVK) appear. The MT cell line, differing from M line in the number of homologous chromosomes, exhibited a character of cell distribution similar to that of the M line for the chromosome number for only 1 day after cultivation on the ECM substrate, but not after 4 days under the same culture conditions, when no difference from the control cells was observed. Further cultivation of MT cells for 8 days did not change the character of cell distribution for the chromosome number relative to the control variant. The observed alterations seem to be due to disturbances in the correct chromosome-segregation process, which were caused by an abrupt shift in the cell-culture conditions. Analysis of the structural karyotypic variability revealed a significant increase in frequency of chromosomal aberrations in the M cell line for 1 and 4 days in culture on the ECM substrate as compared to the control cells. The frequency of dicentric chromosomes (telomeric associations) was increased and constituted more than 50% of all chromosome aberrations. No increase in frequency of chromosome aberrations was observed for MT cells cultured under the same conditions. It can be suggested that the differing by the karyotypic structure, but the genetically identical cell lines have different response to the substrate. In contrast to the M line, in the MT line, a fast normalization of numerical karyotypic characteristics and no enhancement of structural karyotypic variability takes place. This provides a possibility to cultivate an MT cell on the given protein substrate while maintaining a balanced karyotypic structure characteristic of MT cell line.     

Geographic distribution patterns of small mammals in Swaziland in relation to abiotic factors and human land-use activity

Swaziland is a small, topographically diverse southern Africa country whose mammalian fauna has been poorly studied. The distributions of small mammals in Swaziland were surveyed at 39 localities. A total of 15 species of rodents and ten species of insectivores were captured during the survey. The number of species of insectivore captured at a locality was positively correlated with mean annual rainfall, while the number of rodent species captured was negatively correlated with altitude. The number of rodent species captured was lower on Swazi Nation Land compared with privately owned land or protected land (reserves). This was probably due to the radical habitat alteration that had taken place on Swazi Nation Land, inter alia overgrazing by livestock, cultivation of maize and deforestation. The effect of this habitat alteration on the number of rodent species was more pronounced in high-lying areas of Swaziland. This may have been due to the fact that a large number of the rodent species inhabiting high-lying areas require thick, tall grassland habitats, whereas many of the low-lying species prefer more open habitats with less grass cover. Since grazing acts to reduce grass cover, it is suggested that the species inhabiting high-lying areas would be more affected by overgrazing, than low-lying species.     

In vitro activation of the in vivo colony-forming units of the mouse yolk sac.

Experiments were performed to investigate the presence of colony-forming units (CFU) in the mouse embryonic yolk sac during the developmental period in which the yolk sac is the sole hemopoietic organ. Injection of yolk sac cell suspensions from normal embryos into syngeneic, lethally irradiated adult recipients evoked a very low number of spleen colonies. However, prior cultivation of yolk sacs in vitro caused a dramatic increase in the spleen colony-forming capacity--as high as 84-fold--following 48 hours in culture. The yolk sac origin of the spleen colonies was confirmed by: (a) Chromosomal marker analysis; (b) dose-response analysis; (c) demonstrating that the above colonies were not of endogenous origin induced by the mere injection of grafted cells. We conclude that the yolk sac contains many precursors of colony-forming cells which though undetectable by immediate grafting apparently become activated in culture by an as yet unknown induction process.     

The splenic marginal zone in humans and rodents: an enigmatic compartment and its inhabitants

The role of the spleen in B memory cell development and maintenance is attracting increased attention. Studies in mice and rats have indicated that memory functions are associated with large B cells residing in the marginal zone (MZ) of the spleen. Although the cellular composition of the MZ is relatively well known in these species, controversies exist about the function of MZ B cells, their dependence on the presence of the spleen and the stage at which their development branches from that of recirculating follicular B cells. Additional confusion has arisen with respect to MZ B cells in humans, because the microscopic anatomy of the human splenic MZ differs decisively from that of rodents. Several recent publications indicate that the functional and migratory properties of human MZ B cells may be species-specific. The hypothesis derived from these publications and from our immunohistological observations implies that at least a major number of human splenic CD27⁺ MZ B cells are migratory. Phenotypic data suggest a recirculation pathway between the spleen and mucosal tissues in humans.     

Stability of C-band heterochromatin polymorphism in 2 transplantable human cell lines differing in sensitivity to Coxsackie virus B3

Heterochromatin of chromosomes is studied by means of a C-banding technique for the J-96 line of human cells, which is susceptible to enteroviruses and for the J-41 cell line derived from this culture and possessing high specific resistance to Coxsackie B viruses. The data obtained demonstrate stability of variform C-heterochromatin of chromosome pairs 1, 9 16 and certain marker chromosomes in the course of long-term cultivation.     

Long-term proliferation of mouse thymic epithelial cells in culture

Summary Epithelial cells from the normal mouse thymus were successfully cultivated on tissue culture plastic when plated with lethally irradiated support cells of the LA7 rat mammary tumor line. As the irradiated LA7 cells slowly decreased in number the thymus cells proliferated concomitantly to form a confluent monolayer. The cells now in culture have been subcultured 8 times, have doubled in number at least 30 times, and are still proliferating vigorously. The culture technique also supported clonal growth from a single cell, and nine clones have been isolated. The colony-forming efficiency of thymic cells plated at low concentrations was about 8%. These cultures were never overgrown by fibroblasts. The thymus cells were characterized as epithelial by the presence of cytoplasmic keratin and numerous desmosomes and tonofilaments. They were shown to be mouse cells by immunocytochemistry with species specific antibodies, by isoenzyme analysis, and by karyology. The cells stained when reacted with antibodies to tubulin, vimentin, and actin, but not with antibodies to Thy-1.2, Lyt-1, Lyt-2, Ia, or H-2 proteins. More than 85% of the cells had a normal mouse diploid chromosome number of 40. This culture technique opens the way for future studies of T-cell education with homogeneous thymic epithelial cell populations both in vitro and after reimplantation into genetically defined strains of mice. This work was supported by the Veterans Administration, Washington, D.C.     

Effect of cultivation conditions on the karyotype structure of a cell subline of the kangaroo rat kidney

Variations in cultivation conditions were found to exert influence on the distribution of cells for chromosome number by changing the modal class. The change of the HMEM medium for the EMEM medium during 2-6 passages results in the appearance of a new modal class with 16 chromosomes. The change in the chromosome number is preferably due to the loss of one X chromosome within the main structural variant of the karyotype (MSVK). On the other hand, the change of the HMEM medium for the F12 medium during 4-6 passages does not affect the cell distribution for the chromosome number. A comparative analysis of the total frequency of the MSVK cells and that of MSVK cells of the modal class showed that the karyotypic changes took place in all the variants, both in the modal class and beyond it due to other additive SVK. An exception is the variant NBLD (change of HMEM for the F12 during 6 passages). In this case chromosome changes occur mostly in the modal class, primarily due to the redistribution of chromosomes in groups. In all the variants there is an insignificant frequency of chromosomes, morphologically different from the MSVK. This confirms the findings according to which chromosomal changeability in the NBLD may be associated mostly with the change in the number of homologous chromosomes rather than with chromosomal aberrations. The frequency of chromosomal aberrations is the same in all the variants examined. The dependence of karyotypic characteristics on culture media mentioned above indicate that care should be taken in choice of culture conditions for permanent cell lines.