Organization of the Morphogenetic Field in Regenerating Amphibian Limbs

The amphibian limb is an example of a secondary embryonic fieldthat can be reactivated during larval or adult life so thatamputated parts are regenerated. Two major questions are: (1)what is the origin of the morphogenetic field of the regenerationblastema, and (2) what is the nature of this field and how doesit specify the spatial pattern of blastemal redifferentiation?Evidence is analyzed here which leads to the following propositions:(1) the field is represented in latent form by properties ofthe mature limb cells, and these properties are activated andinherited by the blastemal cells after amputation and dedifferentiation.At the same time, the inherited field is sensitive to the maturestump tissues and its spatial organization can be altered bya stump pattern alien to the one from which it was derived.(2) The properties of the mesodermal limb tissues representpositional values that are arranged in gradients along the proximal-distal,anterior-posterior and dorsal-ventral axes. These propertiesallow dedifferentiated mesodermal cells to change their positionalvalue to any value between their original one in the limb andthe value of any neighboring cell after creation of a discontinuity.The direction of change is always from proximal to distal inthe PD axis; it is uncertain as to whether change can take placeonly centripetally or both centripetally and centrifugally alongthe AP and DV axes. (3) Epidermal cells have the same positionalvalue everywhere in the limb and act as the distal and circumferentialboundaries up to which the mesodermal cells may change theirpositional values. The proximal boundary is represented by thelevel-specific properties of the mesodermal cells at the maximumextent of distal to proximal dedifferentiation. Normal regenerationcan then be visualized as occurring in the following way. Whendeletions are made in the limb pattern, cells with widely differentpositional values are confronted. During regeneration, blastemacells increase in number and continually interact with theirneighbors to adjust their positional values within the boundariesuntil discontinuities are eliminated. The multiple limbs resultingfrom rearrangement of stump tissue patterns can also be accountedfor by using these propositions. It is suggested that positionalinformation is encoded on the cell surface and/or in the extracellularmatrix.     

Stem cells, cell transplantation and liver repopulation

Liver transplantation is currently the only therapeutic option for patients with end-stage chronic liver disease and for severe acute liver failure. Because of limited donor availability, attention has been focused on the possibility to restore liver mass and function through cell transplantation. Stem cells are a promising source for liver repopulation after cell transplantation, but whether or not the adult mammalian liver contains hepatic stem cells is highly controversial. Part of the problem is that proliferation of mature adult hepatocytes is sufficient to regenerate the liver after two-thirds partial hepatectomy or acute toxic liver injury and participation of stem cells is not required. However, under conditions in which hepatocyte proliferation is blocked, undifferentiated epithelial cells in the periportal areas, called "oval cells", proliferate, differentiate into hepatocytes and restore liver mass. These cells are referred to as facultative liver stem cells, but they do not repopulate the normal liver after their transplantation. In contrast, epithelial cells isolated from the early fetal liver can effectively repopulate the normal liver, but they are already traversing the hepatic lineage and may not be true stem cells. Mesenchymal stem cells and embryonic stem cells can be induced to differentiate along the hepatic lineage in culture, but at present these cells are inefficient in repopulating the liver. This review will characterize these various cell types and compare the properties of these cells and the conditions under which they do or do not repopulate the liver following their transplantation.     

IL-1 secretion and membrane IL-1 expression by neonatal spleen cells during soluble antigen presentation

Antigen presentation and IL-1 production by neonatal spleen cells were studied in a murine model. The T-helper-cell line (D10-G4.1) (D10), which is specific for soluble antigen presented on syngeneic antigen-presenting cells and dependent on IL-1 for its proliferation, was used as an indicator cell for the ability of syngeneic neonatal or adult spleen cells to present antigen and produce IL-1. The antigen-presenting capacity of neonatal spleen cells is low as attested by D10 proliferation. During antigen presentation there is an augmentation of IL-1 production by the antigen-presenting spleen cell population. However, neonatal spleen cells do not respond to the same levels as do adult spleen cells. These reduced levels of secreted IL-1 cannot be attributed to a low potential for producing IL-1 as attested by the high levels of IL-1 made by these cells after induction by a crude IL-1 inducer factor (IL-1-IF) and by the stimulus of the IL-1-IF produced by D10 cells during antigen presentation by paraformaldehyde-fixed adult cells. The spontaneous expression of membrane IL-1 by neonatal cells is low. Membrane IL-1 levels on neonatal cells can be brought to adult levels by induction with IL-1-IF. Neonatal spleen cells have an impaired capacity to process and/or present soluble antigen. This impairment leads to a decreased stimulus of the T helper cell to produce inducer factors and thus a reduced level of IL-1 production by the neonatal cells during antigen presentation.     

Drosophila brain tumor metastases express both neuronal and glial cell type markers

Loss of either lgl or brat gene activity in Drosophila larvae causes neoplastic brain tumors. Fragments of tumorous brains from either mutant transplanted into adult hosts over-proliferate, and kill their hosts within 2 weeks. We developed an in vivo assay for the metastatic potential of tumor cells by quantifying micrometastasis formation within the ovarioles of adult hosts after transplantation and determined that specific metastatic properties of lgl and brat tumor cells are different. We detected micrometastases in 15.8% of ovarioles from wild type host females 12 days after transplanting lgl tumor cells into their abdominal cavities. This frequency increased significantly with increased proliferation time. We detected micrometastases in 15% of ovarioles from wild type host females 10 days after transplanting brat tumor cells into their abdominal cavities. By contrast, this frequency did not change significantly with increased proliferation time. We found that nearly all lgl micrometastases co-express the neuronal cell marker, ELAV, and the glial cell marker, REPO. These markers are not co-expressed in normal brain cells nor in tumorous brain cells. This indicates deregulated gene expression in these metastatic cells. By contrast, most of the brat micrometastases expressed neither marker. While mutations in both lgl and brat cause neoplastic brain tumors, our results reveal that metastatic cells arising from these tumors have quite different properties. These data may have important implications for the treatment of tumor metastasis.     

Phenotype, frequency, and EBV responsiveness of human marrow B and pre-B cells

We have purified subpopulations of B lineage cells from human adult (rib) bone marrow by cell sorting and panning. Limiting dilution analysis was then used for a clonal analysis of cells able to secrete IgG, IgA, or IgM spontaneously or after infection with EBV. Nonproliferating, high rate IgG or IgA producers occurred at frequencies of about one per 1000 marrow mononuclear cells. Their frequency and Ig production was unaffected by EBV, and they appeared not to express EBNA after exposure to EBV. These cells were Ia+, B1+, and over 85% expressed sIg of the IgM/D (up to 75%) and/or IgG/A isotypes (40 to 60%). B cells committed to the secretion of IgM represent 2 to 10% of marrow B lymphocytes. They were found to be Ia+/B1+/B2+/CALLA- and C3b receptor (CR3)-cells, and most (greater than 90%) required infection with EBV and proliferation to develop into IgM-producing lymphocytes. Thirty to 40% of these cells did not express Ig (H or L chain) on their surface, and therefore resembled pre-B cells at the beginning of the 4- to 5-wk culture period. Proliferating pre-B cells from adult human marrow have been described, but their conversion into IgM-producing cells has not been formally demonstrated. Although EBV induces IgM production, the expression of EBNA, and several rounds of cell division in these cells, the induction of stable (greater than 5 wk) growth transformation represents a rare event in these pre-B cells: in several thousand limiting dilution wells, not a single culture of sIg-cells showed stable growth transformation. The dichotomy between EBV-induced high-rate IgM responses and absent growth transformation discriminates activation and transformation as distinct aspects of EBV-induced B cell "responses", and suggests that cellular properties play critical roles for viral transformation. We propose a model in which cellular target genes for transforming sequences in the EBV genome are transiently expressed during B cell differentiation.     

Possibility of an artefact in determining hematopoietic stem cell proliferation by CFUs suicide methods

Spleen colonies in the irradiated mice are produced by both stem cells and by their more differentiated progeny. In the latter case the colonies are transitory, ceasing 10-11 days after cell injection. The transitory colonies may be the cause of systematic artifact during the determination of stem cell proliferation. It was shown in particular that the proliferation of stem cells after sublethal irradiation remains the same, while higher rates of suicide are determined by the death of the precursors of the transitory colonies. At the same time higher proliferation of stem cells is not artifact in lethally irradiated animals and is also detectable after exclusion of the effects of the precursors of the transient colonies.     

Fas/Fas ligand interactions promote activation-induced cell death of NK T lymphocytes

NKT cells are a versatile population whose immunoregulatory functions are modulated by their microenvironment. We demonstrate herein that in addition to their IFN-gamma production, NKT lymphocytes stimulated with IL-12 plus IL-18 in vitro underwent activation in terms of CD69 expression, blast transformation, and proliferation. Yet they were unable to survive in culture because, once activated, they were rapidly eliminated by apoptosis, even in the presence of their survival factor IL-7. This process was preceded by up-regulation of Fas (CD95) and Fas ligand expression in response to IL-12 plus IL-18 and was blocked by zVAD, a large spectrum caspase inhibitor, as well as by anti-Fas ligand mAb, suggesting the involvement of the Fas pathway. In accordance with this idea, NKT cells from Fas-deficient C57BL/6-lpr/lpr mice did not die in these conditions, although they shared the same features of cell activation as their wild-type counterpart. Activation-induced cell death occurred also after TCR engagement in vivo, since NKT cells became apoptotic after injection of their cognate ligand, alpha-galactosylceramide, in wild-type, but not in Fas-deficient, mice. Taken together, our data provide the first evidence for a new Fas-dependent mechanism allowing the elimination of TCR-dependent or -independent activated NKT cells, which are potentially dangerous to the organism.     

Chromatin sphingomyelin changes in cell proliferation and/or apoptosis induced by ciprofibrate

It has been shown that neutral-sphingomyelinase and sphingomyelin-synthase activities are present in chromatin and they modify the sphingomyelin (SM) content. The activity of the first enzyme is stimulated and the second inhibited, when the hepatocytes enter into the S-phase after partial hepatectomy, thus suggesting that ceramide may have a pivotal role in cell proliferation. An opposite function was attributed to ceramide in hepatocytes which undergo apoptosis after lobular ligature. In order to clarify this point, a model was developed in which the same liver cells undergo proliferation followed by induced apoptosis. To this purpose, the rats were treated for 7 days with ciprofibrate and then left without treatment for 4 days. During the treatment, the peroxisome enzyme markers increase their activity and the number of proliferating cells increases, reaching a maximum after 3 days of treatment, as shown by the number of cells positive for the proliferating cell nuclear antigen. At the same time, the chromatin sphingomyelinase activity reaches the maximum, while a similar increase is not found in the cytoplasm or in the isolated nuclei. On the contrary, SM-synthase activity is depressed in chromatin, but not in the nuclei in which a peak is shown after 3 days of ciprofibrate treatment. After drug withdrawal, the hepatocytes undergo apoptosis as confirmed by the increase of Bax and tissue transglutaminase (tTGase) expression; the chromatin SM increases as a consequence of an increase of SM-synthase activity. It can be hypothesised that chromatin SM may have a role in cell duplication by influencing the chromatin structure stability.     

Stem cell colloquy: conclusion

The stem cell data presented and discussed during the symposium raise the hope that important medical progress can be made in several fields: neuro-degenerative diseases, those linked to cellular deficit, some aspects of aging linked to cellular degeneration, and the treatment of cancers that may harm normal tissues at risk of being infiltrated by malignant cells. Three main types of stem cells are available. (i) Those present in normal adult tissue: contrary to what was believed, some data suggest that certain adult stem cells have a great plasticity (they can differentiate into cells different from those in tissues from which they were taken) and can proliferate in vitro without losing their properties. Nevertheless, their use faces several obstacles: in ill or elderly subjects, then these cells can be limited in number or not multiply well in vitro. In this case, auto-grafting of the cells cannot be used. They must be sought in another subject, and allo-grafting causes difficult and sometimes insoluble problems of immunological tolerance. (ii) Embryonic stem cells from surplus human embryos, obtained by in vitro fertilisation, which the parents decide not to use: these cells have a great potential for proliferation and differentiation, but can also encounter problems of immunological intolerance. (iii) Cells obtained from cell nuclear transfer in oocytes: these cells are well tolerated, since they are genetically and immunologically identical to those of the host. All types of stem cells can be obtained with them. However, they do present problems. For obtaining them, female oocytes are needed, which could lead to their commercialization. Moreover, the first steps for obtaining these cells are identical to those used in reproductive cloning. It therefore appears that each type of cell raises difficult scientific and practical problems. More research is needed to overcome these obstacles and to determine which type of stem cell constitutes the best solution for each type of disease and each patient. There are three main ethical problems: (a) to avoid the commercialization of stem cells and oocytes (this can be managed through strict regulations and the supervision of authorized laboratories); (b) to avoid that human embryos be considered as a mere means to an end (they should only be used after obtaining the informed consent of the parents; the conditions of their use must be well defined and research programs must be authorized); (c) to avoid that research on stem cell therapy using cell nuclear replacement opens the way to reproductive cloning (not only should reproductive cloning be firmly forbidden but authorization for cell nuclear transfer should be limited to a small number of laboratories). Overall, it appears that solutions can be found for administrative and ethical problems. Harmonisation of international regulations would be desirable in this respect, in allowing at the same time each country to be responsible for its regulations. A last ethical rule should be implemented, not to give patients and their families false hopes. The scientific and medical problems are many, and the solutions will be long and difficult to find. Regenerative medicine opens important avenues for research, but medical progress will be slow.     

Tumor formation by SV40-transformed human cells in nude mice: the role of SV40 T antigens

Human cells transformed in vitro by SV40 rarely form tumors in nude mice. We examined whether these cells as a group are inherently nontumorigenic or whether they are potentially tumorigenic but rejected by the athymic host, possibly by nonspecific immune mechanisms. SV80 and NG8 are SV40-transformed human cell lines that express all of the transformed properties, including anchorage-independent growth, but do not form tumors in adult nude mice after injection of as many as 10(8) cells. Both the SV80 and NG8 cell lines have SV40-specific transplantation antigens which crossreact with those present on SV40-transformed (but tumorigenic) rodent cells. We found that SV80 cells, though not NG8 cells, induced progressively growing lethal tumors if the cells are injected repeatedly into neonatal nude mice. Somatic cell hybrids between SV80 or NG8 cells and a highly tumorigenic cell line derived from a human tumor continue to express the virus-induced antigens and fail to form tumors in adult nude mice. These results strongly suggest that at least for some SV40-transformed human cells, the failure to form tumors in nude mice may be due to their expression of virus-induced transplantation antigens rather than the absence of tumorigenic potential.     

Interleukin-1-independent activation of human T lymphocytes stimulated by anti-CD3 and a Hodgkin's disease cell line with accessory cell activity

Antibodies directed against the human T cell receptor or the closely associated CD3 molecule stimulate polyclonal T cell proliferation via mechanisms that mimic a primary immune response. We have investigated the requirement for IL-1 production in anti-CD3 (OKT3)-mediated mitogenesis using a Hodgkin's disease cell line (L428) as the accessory cell. L428 cells did not produce detectable IL-1 following stimulation with lipopolysaccharide or phorbol ester (PMA), nor did they transcribe detectable levels of mRNA for IL-1 alpha or beta after such treatment. Despite their inability to produce IL-1, as few as 1 X 10(4) L428 cells reconstituted the proliferative response of accessory cell-depleted T cells to anti-CD3. Although larger numbers of non-rosette-forming (E-) cells were required for maximal responsiveness to anti-CD3, the maximal degree of proliferation was higher with E- cells than with L428 cells. L428-mediated T cell proliferation did not result from residual accessory cells in the responding population or an allogeneic effect since L428 cells were also capable of providing accessory cell activity for the anti-CD3-dependent generation of IL-2 by the Jurkat T cell line. Although the mechanism by which L428 cells provide accessory functions remains incompletely characterized, the ability of anti-HLA-DR F(ab')2 fragments to completely abrogate L428 and monocyte-mediated anti-CD3 mitogenesis, despite the addition of exogenous IL-1, provides evidence for the participation HLA-DR molecules in this response. These data indicate that anti-CD3-induced proliferation of unprimed human T lymphocytes can occur independently of IL-1 production by accessory cells and may involve the participation of HLA-DR molecules.     

DNA synthesis and content and the accumulation of total protein and hemoglobin during the differentiation of primary erythroid cells in chickens

Using cytophotometric and autoradiographic methods, it was shown that on days 2-3 of embryogenesis primary erythroid cells (PEC) divided actively. The distribution of erythroblasts (EB) according to their DNA content is not, however, typical of a proliferating population: it contains an unusually large number of 4c cells resulting from the cell cycle arrest at the G2 phase. It is established that reticulocytes (RC) do not divide and are arrested at G1 or G2 phases, since they do not incorporate 3H-thymidine after their formation is complete and their DNA contents are strictly confined to either 2c or 4c. All types of PEC include a large number of cells containing H2c DNA which is due either to the cell cycle arrest at the S phase, or to the formation of accessory nuclei. All PECs have much higher contents of hemoglobin and total protein than do adult hen erythrocytes (EC). Hemoglobin and total protein contents of H2c and accessory nuclei containing cells are much higher than those in 2c-cells. We have calculated that adult birds and embryos contain the same amount of hemoglobin per gram of weight, but the quantity of red blood cells in the former is ten times higher. A conclusion is drawn that proliferation and cytodifferentiation regulation mechanisms are directed, in primary erythropoiesis, to intense hemoglobinization of the cells, and, in adult erythropoiesis, to increasing their number. In both the cases homeostatic regulation of erythropoiesis works.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and myelin-associated glycoprotein) in regenerating adult mouse sciatic nerve

The localization of the neural cell adhesion molecules L1, N-CAM, and the myelin-associated glycoprotein was studied by pre- and postembedding staining procedures at the light and electron microscopic levels in transected and crushed adult mouse sciatic nerve. During the first 2-6 d after transection, myelinated and nonmyelinated axons degenerated in the distal part of the proximal stump close to the transection site and over the entire length of the distal part of the transected nerve. During this time, regrowing axons were seen only in the proximal, but not in the distal nerve stump. In most cases L1 and N-CAM remained detectable at cell contacts between nonmyelinating Schwann cells and degenerating axons as long as these were still morphologically intact. Similarly, myelin-associated glycoprotein remained detectable in the periaxonal area of the degenerating myelinated axons. During and after degeneration of axons, nonmyelinating Schwann cells formed slender processes which were L1 and N-CAM positive. They resembled small-diameter axons but could be unequivocally identified as Schwann cells by chronical denervation. Unlike the nonmyelinating Schwann cells, only few myelinating ones expressed L1 and N-CAM. At the cut ends of the nerve stumps a cap developed (more at the proximal than at the distal stump) that contained S-100-negative and fibronectin-positive fibroblast-like cells. Most of these cells were N-CAM positive but always L1 negative. Growth cones and regrowing axons expressed N-CAM and L1 at contact sites with these cells. Regrowing axons of small diameter were L1 and N-CAM positive where they made contact with each other or with Schwann cells, while large-diameter axons were only poorly antigen positive or completely negative. 14 d after transection, when regrowing axons were seen in the distal part of the transected nerve, regrowing axons made L1- and N-CAM-positive contacts with Schwann cells. When contacting basement membrane, axons were rarely found to express L1 and N-CAM. Most, if not all, Schwann cells associated with degenerating myelin expressed L1 and N-CAM. In crushed nerves, the immunostaining pattern was essentially the same as in the cut nerve. During formation of myelin, the sequence of adhesion molecule expression was the same as during development: L1 disappeared and N-CAM was reduced on myelinating Schwann cells and axons after the Schwann cell process had turned approximately 1.5 loops around the axon. Myelin-associated glycoprotein then appeared both periaxonally and on the turning loops of Schwann cells in the uncompacted myelin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mind the gap: Cells respond to tissue damage by changing orientation of cell divisions

Nature presents plenty of examples of cellular behavior that determines the shape of an organ during development, such as epithelial polarity and cell division orientation. Little is known, however, about how organs regenerate or how cellular behavior affects regeneration. One of the most exciting aspects of regeneration biology is understanding how proliferation and patterning are coordinated, since it means that cells not only have to proliferate but also have to do so in an ordered manner so that organs are reconstructed proportionally. Drosophila wing imaginal discs and adult wings are models used in different approaches to investigate this issue; they have recently been used to reveal that, after localized cell death, neighboring cells change their cell division orientation toward the damaged zone. During this process, cell polarity and spindle orientation operate in coordination with cell proliferation to regenerate proper organ size and shape.     

Mechanism of Staphylococcus aureus exotoxin A inhibition of Ig production by human B cells

Staphylococcus enterotoxins and toxic shock syndrome toxin 1 are members of a family of exoproteins that are produced by staphylococci and bind specifically to MHC class II molecules. Upon binding to MHC class II molecules, these exoproteins are potent stimulators of T cell proliferation via interaction with specific TCR V-beta segments of both CD4+ and CD8+ T cells. These exoproteins also directly stimulate monocytes to secrete IL-1 and TNF-alpha. Furthermore, these exoproteins have a profound inhibitory effect on Ig production by PBMC. We examined the effects of Staphylococcus enterotoxin A (SEA) on proliferation and Ig production of highly purified human B cells. Our results demonstrated that the binding of SEA to MHC class II molecules on B cells does not alter their ability to proliferate in response to Staphylococcus aureus Cowan strain I (SAC) or to produce Ig in response to SAC plus rIL-2. In contrast, the anti-DR mAb L243 inhibited both B cell proliferation and Ig production. Unable to determine a direct effect of SEA on B cell function, we investigated whether the capacity of SEA to inhibit SAC-induced Ig production by PBMC was T cell-dependent. Our results demonstrated that in the presence of T cells, under appropriate conditions, SEA can either function as a nominal Ag for stimulation of B cell proliferation and Ig production or induce T cell-mediated suppression of Ig production. SEA-induced Ig production required T cell help, which was dependent on pretreatment of the T cells with irradiation or mitomycin C; Ig production was not induced by SEA in the absence of T cells or in the presence of untreated T cells. Furthermore, SEA inhibited Ig production in SAC-stimulated cultures of autologous B cells and untreated T cells; pretreatment of the T cells with irradiation or mitomycin C abrogated SEA-induced inhibition of Ig production. Thus, T cell suppression of SAC-induced Ig production was dependent on T cell proliferation. Similar results were observed with both SEA and toxic shock syndrome toxin 1.     

Antigen nonspecific suppression of T cell responses by activated stimulation-refractory CD4+ T cells

Several classes of anergic T cells are capable of suppressing naive T cell proliferation and thereby limiting immune responses. Activated T cells, although not anergic, are transiently refractory to restimulation with Ag. We examine in this study whether activated refractory murine T cells can also suppress naive T cell responses. We find that they can, and that they exhibit many of the suppressive properties of anergic T cells. The activated cells strongly diminish Ag-mediated T cell proliferation, an activity that correlates with their refractory period. Suppression is independent of APC numbers and requires cell contact or proximity. Naive T cells stimulated in the presence of activated refractory cells up-regulate CD25 and CD69, but fail to produce IL-2. The addition of IL-2 to culture medium, however, does not prevent the suppression, which is therefore not solely due to the absence of this growth factor. Persistence of the suppressor cells is also not essential. T cells stimulated in their presence and then isolated from them and cultured do not divide. The suppressive cells, however, do not confer a refractory or anergic state on the target T lymphocytes, which can fully respond to antigenic stimulation if removed from the suppressors. Our results therefore provide evidence that activated T cells act as transient suppressor cells, severely constraining bystander T cell stimulation and thereby restricting their response. These results have potentially broad implications for the development and regulation of immune responses.     

Hemopoietic inductive envirnment necessary for an early stage in differentiation of thymic immune cells: effect of antilymphocytic serum

Treatment of adult mice with rabbit anti-mouse thymocyte serum decreased the number and frequency of alloantigen-sensitive units responsible for graft-versus-host reactions and prolonged the survival of skin allografts. Whereas alloantigen-sensitive units were suppressed directly in vitro, they were not apparently suppressed directly in vivo since the fall-off of their numbers and/or function did not occur during the first day after serum injection. Treatment of prospective recipients of thymus cell grafts impaired the production of alloantigen-sensitive units by transplanted primitive progenitors. Differentiation with proliferation of alloantigen-sensitive units was less affected. Similarly, treatment of prospective recipients of thymus cell grafts with antilymphocytic serum impaired the production of specific inducer cells responsive to sheep erythrocytes by transplanted more primitive cells, presumably antigenreactive cells. Production of new precursors of anti-sheep erythrocyte hemolytic plaque-forming cells by transplanted bone marrow was not affected. Thus, antilymphocytic serum impairs the generation of immunocompetent cells of thymic origin by altering a hemopoietic inductive environment necessary for an early stage in differentiation.     

Neural Control of Cell Cycle Events in Regenerating Salamander Limbs

Nerves, wound epidermis, and injury are indispensable for salamanderlimb regeneration, but their mechanism of action is not understood.A hypothesis has been presented (Tassava and Mescher, 1975)which suggests that injury is important to dedifferentiationand entry of limb stump cells into the cell cycle, nerves arerequired for one or more G₂ events in order that cells can proceedto mitosis, and the wound epidermis maintains the daughter cellsin the cell cycle. The resultant cells accumulate to form theblastema. Complete and partial denervation experiments, which attemptedto test this hypothesis, are discussed. Blastema cell cycleparameters, measured after complete denervation, did not varygreatly from innervated controls, even though denervated blastemaswere resorbed. Blastema cell cycle parameters of partially denervatedlimbs, which exhibited delayed regeneration, were likewise notlengthened when compared to completely innervated controls.These results are consistent with the view that after eithercomplete or partial denervation, some blastema cells continueto cycle and reach the M phase in the same time as controls.Other blastema cells block completely, never reach M, and arethen removed. A possible mechanism for resorption of denervatedblastemas is presented.     

Antigen presentation by neonatal murine spleen cells

The ability of murine neonatal spleen cells to present soluble antigen to T-helper cells and to produce growth factors in response to subsequent cellular interactions was studied. The T-helper-cell line (D10-G4.1) (D10), which is specific for the soluble antigen conalbumin presented on H-2-matched (H-2k) antigen-presenting cells, was used as cooperating and indicator cells in these cellular interactions. The D10 cells are TH2 T-helper cells which secrete the autocrine growth factor IL-4 and can also respond to exogenous IL-2 (T. R. Mosmann and R. L. Coffmann, Immunol. Today 8, 223, 1987). D10 cells require exogenous IL-1 for their proliferation and secrete, in addition to IL-4, IL-1 inducer factor and GM-CSF. The ability of neonatal spleen cells to present antigen and to stimulate D10 cells to produce IL-4 and proliferate is low. During antigen presentation there is an augmentation of IL-1 and IL-2 production by the antigen-presenting spleen cell population. However, neonatal spleen cells do not respond to the same levels as do adult spleen cells. The addition of exogenous IL-1 cannot repair the antigen presentation by neonatal cells. Experiments in which the antigen processing and presentation steps were separated from those requiring growth factor induction and secretion demonstrate that neonatal spleen cells are impaired in their ability to perform adequate antigen processing and presentation. The neonatal spleen cells are as competent as adult cells to cooperate with T-helper cells and secrete growth factors, provided antigen processing and presentation is performed by fully competent adult spleen cells. Experiments in which neonatal and adult antigen-presenting spleen cell populations were mixed, and others in which plastic adherent and nonadherent cells were separated, could not detect any suppressor mechanisms responsible for the low antigen presentation of neonatal cells. Thus, neonatal spleen cells are impaired in the initial stages of antigen processing and presentation. This impairment which leads to low levels of growth factor production is the major determinant in the ineffectual stimulation of T-helper cells by neonatal spleen cells.     

The cytological analysis of causes affecting the root cell length

The final length of the root cell is the result of a series of prooesses which represent a transition of the growing cell from its origin up to the completion of its elongation. These processes are associated on the one hand with cell proliferation, or, after the termination of proliferation, with the proceeding DNA synthesis, and with cell elongation on the other. The group of properties characterizing the growth region of the root as a cytologically heterogeneous complex, is at the same time the group of causes which affect the length of root cells. The individual cases documented in the paper point out the fact that the mechanisms regulating growth processes, have a locally limited action, often only within one single cell, and that they are simultaneously subordinated to the regulatory mechanism which controls the growth of the root as an entirety.