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PubMed1989

Validation of the hemizona assay in a monkey model: influence of oocyte maturational stages.

The hemizona assay (HZA) was used in the monkey model to investigate sperm binding to the zona pellucida and to evaluate binding patterns according to various stages of oocyte development. Adult cynomolgus monkeys were superovulated with human menopausal gonadotropin. The oocytes were retrieved by laparoscopy, stored in salt solution, then cut into hemizonae by micromanipulation. Metaphase II oocytes showed significantly tighter binding than prophase I oocytes (P less than 0.001). Metaphase I oocytes showed intermediate binding, significantly different from the other groups (P less than 0.01). It is concluded that: (1) This study demonstrates the feasibility of HZA as a test to evaluate sperm/zona interactions in the monkey, using standards reported for human studies. (2) Oocyte meiotic competence seems to be accompanied by an increase in zona-binding properties.

Animals↗
PubMed1989

Infertility and bioethical issues of the new reproductive technologies.

The scientific breakthroughs resulting in the delivery of Louise Brown in 1978 have opened the floodgates for an ongoing bioethical discussion about medically assisted reproduction. The majority in our society has accepted in vitro fertilization as an ethically justifiable procedure for infertile couples. The concern persists, however, that new reproductive technology has started us on the course of a slippery slope with potentially dire consequences for the so-created children, the traditional family, and, indeed, for society as a whole. The moral status of the embryo is the central issue in debates about such reproductive developments as the "spare" embryo, embryo freezing, embryo donation, embryo research and micromanipulation. Conflicts of interests between the adult's desire to become a parent and the welfare of the offspring are at the root of moral objections raised against manipulation of human reproduction. Extracorporal conception with the possibility for various gamete donors has also brought the long-practiced procedure of artificial insemination by donor and the potential consequences for the child into the discussion. Surrogate mothering and surrogate gestational mothering force us to redefine the age old dictum mater certa est and can render the child a helpless pawn in parental, emotional, and legal strife. Over the ages, society has through firmly established values exerted control over reproduction and acceptance of the new member in the community. Sex without reproduction was a severe blow to the highly regarded societal belief in parenting as the epitomy of life goals. Reproduction without sex through various technically feasible collaborative means further jolts fundamental traditional values and mandates their re-evaluation. Ethical belief systems are by nature highly charged and fiercely defended. Thus, in a pluralistic society, a consensus on the question "What ought to be done of all that can be done with new reproductive technologies?" is probably unachievable. Heated controversies between interest groups constitute an additional psychological burden complicating the ethical ambiguities for some infertile couples who have to decide about using noncoital conception. The interdisciplinary group report by the ethics committee of the American Fertility Society published in the "Ethical Considerations of the New Reproductive Technologies" constitutes a wide framework of guidelines for rational consideration. It will, one hopes, help to formulate needed regulations because some segments of our society as well as many scientists and physicians in the field believe that not all that potentially can be done ought to be done.(ABSTRACT TRUNCATED AT 400 WORDS)

Ethics, Medical↗
PubMed1989

Effects of hemopoietic growth factors on stem cells in vitro.

The central feature of hemopoiesis is the lifelong, stable cell renewal. This process is supported by hemopoietic stem cells, which in the steady-state appear to be dormant in cell cycling. The entry into cell cycle of the dormant stem cells may be promoted by such factors as IL-1, IL-6, and G-CSF. Available evidence indicates that the effects of IL-1 on stem cells are indirectly mediated in part by IL-6 and G-CSF. Once the stem cells leave G0 and begin proliferation, the subsequent process is characterized by continued proliferation and differentiation. While several models of stem cell differentiation have been proposed, micromanipulation studies of individual progenitors suggest that the commitment of multipotential progenitors to single lineages is a stochastic (random) process. The proliferation of early hemopoietic progenitors appears to be supported by IL-3, IL-4, and/or GM-CSF. Once the progenitors are committed to individual lineages, the subsequent maturation process appears to be supported by late-acting, lineage-specific factors such as Ep (for erythropoiesis), G-CSF (for neutrophil production), and IL-5 (for eosinophilopoiesis). Thus, hemopoietic proliferation appears to be regulated by a cascade of factors directed at different developmental stages.

Animals↗
PubMed1989

Proliferative potential of degranulated murine peritoneal mast cells.

The fate of mast cells after degranulation was investigated. Purified peritoneal mast cells of WBB6F1-+/+ mice were sensitized with monoclonal anti-dinitrophenol (DNP) IgE antibodies and stimulated with DNP conjugated with human serum albumin. Mast cells were vitally stained with neutral red, and highly degranulated mast cells were identified under a phase-contrast microscope and individually picked up with the micromanipulator. When these highly degranulated mast cells were individually plated in methylcellulose, their potential to produce a cluster or a colony was comparable to that of morphologically intact mast cells. Moreover, when highly degranulated mast cells were injected into the skin of genetically mast cell-deficient WBB6F1-W/Wv mice, the proportion of injection sites at which mast cell clusters appeared was comparable to the value observed when morphologically intact mast cells were injected. The present result indicates that proliferative potential of mast cells is not reduced by their degranulation.

Animals↗
PubMed1985

Isolation and characterization of novel suppressor T cell clones from murine fetal thymus.

Murine fetal thymus from C57BL/6J (B6) and DBA/2J contains a cell population that suppresses CTL responses to alloantigens. This suppressor cell population was found to exist in high frequency in murine fetal thymus at the 14th day of gestation. The activity of this cell in the thymus declined rapidly with increasing time of gestation, and suppressor activity in the thymus was undetectable by the time of birth. On the other hand, suppressor activity could be detected in organ cultures of 14-day fetal thymus even after the organs were cultured for 14 or 21 days. Fetal thymocytes from B6 or DBA/2J mice were grown as long-term lines in interleukin 2 (IL 2)-containing medium. Clones of suppressor cells were derived from long-term cultures by micromanipulation. The clones had an average doubling time of 13 to 16 hr and were dependent on IL 2 for growth. The clones were 10- to 100-fold more efficient in suppressing CTL responses to alloantigens than day 15 fetal thymocytes. Analyses of cell surface molecules with the use of monoclonal antibodies and conventional anti-H-2 sera by radioactive binding assays showed that cloned suppressor cells from B6 fetal thymus were Thy-1 and Lyt-2+, and expressed little or no L3T4, Lyt-1, H-2K, H-2D, and class II molecules. The suppressor clones lacked the cytolytic activity of conventional CTL and also served as very poor target cells in CTL-mediated cytolysis. The suppressor function of the cloned cells was radiation-resistant, and this suppression could not be reversed by the addition of excess exogenous IL 2. The cloned cells suppressed CTL responses only when they were added within the first 48 hr of a 5-day culture period. Analyses of the antigen specificity of the suppressor cells showed that they suppressed CTL responses in a nonantigen-specific manner.

Animals↗
PubMed1987

Differentiation of blast cells from a Down's syndrome patient with transient myeloproliferative disorder.

A male neonate with Down's syndrome and congenital myeloproliferative disorder was studied. His blood picture showed the unique coexistence of leukocytosis with matured cells and a large number of blast cells. The in vitro proliferation and differentiation of blast cells into various lineages in the presence of phytohemagglutinin-stimulated leukocyte conditioned medium (PHA-LCM) was examined by using a liquid culture and a methylcellulose culture system. The differentiation of blast cells into myeloid cells was confirmed by specific cytochemical stainings, electron microscopy, and an immunologic study. No specific factors in the plasma of the patient promoted the proliferation or differentiation of blast cells. The cellular composition of colonies grown in methylcellulose culture from single blast cells was studied by a micromanipulation technique. High plating efficiency was observed. Of 136 cultures, 78 showed colony growth. Half of the blast cells were colony-forming cells that could proliferate and differentiate into basophils, neutrophils, eosinophils, macrophages, and erythrocytes in the presence of PHA-LCM. Using the blast cells with a high differentiation capacity to the basophil pathway, we studied the effect of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Recombinant GM-CSF support neutrophils, eosinophils, and macrophages but not typical basophils. These findings of the cell differentiation of blast cells into various kinds of cells in vitro were in agreement with the finding of neutrophilia, eosinophilia, basophilia, and thrombocythemia in this patient.

Cell Differentiation↗
PubMed1986

Inhibition of Plasmodium falciparum growth by IgG antibody produced by human lymphocytes transformed with Epstein-Barr virus.

Supernatants from Epstein-Barr virus (EBV)--stimulated B lymphocytes obtained from two adult Gambians who were partially immune to malaria markedly inhibited the growth of Plasmodium falciparum in vitro (55-95% inhibition). When 22 separate colonies were derived by micromanipulation from one of these primary cultures and their supernatants assayed, the degree of inhibition correlated with levels of IgG fluorescent antibody and total IgG. The inhibitory anti-P. falciparum IgG immunoprecipitated an antigen of mol. wt 195,000, identified as the major schizont surface glycoprotein by dual biosynthetic labelling with 3H-glucosamine or 35S-methionine. Other studies on the analogous schizont surface protein of rodent malarias have shown that this antigen stimulates protective immunity. Production of this inhibitory antibody by adult Gambians may therefore contribute to their immunity to malaria. Human antibodies produced by EBV-stimulated B lymphocytes may be used to identify other important P. falciparum antigens and have potential applications for immunotherapy.

Adult↗
PubMed1987

In vitro retroviral transfer of ras genes to single hemopoietic progenitors.

Recent studies have shown that retroviruses can serve as efficient vectors of exogenous genes that can be inserted and expressed in a variety of mammalian cell types. Several investigators have exposed total bone marrow populations to retroviruses in vitro and have demonstrated the presence of exogenous genes after inoculation into irradiated mice. Our approach was to identify individual pluripotent hemopoietic progenitors in vitro and to use these single cells as targets for retroviral gene transfer. This approach was made possible by our previous identification of in vitro colonies containing pluripotent, undifferentiated blast cells with very high secondary replating efficiencies. By using a monoclonal antibody to detect the product of the transferred gene, we were able to document infection of single multipotent cells and to quantitate the percentage of the progeny cells that expressed the transferred gene. Specifically, individual blast cells were obtained by micromanipulation, exposed to Harvey sarcoma virus, and ras gene expression was detected by immunofluorescence in individual colonies. A variety of types of p21-positive colonies were seen, including a macrophage (m)-neutrophil (n)-erythroid (E)-mast cell (mast)-megakaryocyte (M) colony, an mEmastM colony, an nmmast colony, mnE colonies, mn colonies, and m colonies. These results demonstrated that multipotent progenitors were recipients of exogenous genes and that these genes were expressed in the differentiated progeny. Initial experiments failed to demonstrate that the cells in the infected colonies were transformed. Retroviral infection of isolated blast cells may provide a unique method for studies of the effects of a variety of genes, including oncogenes, in hemopoietic cells.

Animals↗
PubMed1988

[Experimental study of single and combined coaxial exposure by high-peaked pulse wave CO2 and Nd:YAG laser on the brain: effect on the brain microvasculature in the subacute stage].

The effect of single or combined coaxial exposure by Nd: YAG and CO2 (pulse wave) laser on the brain at the subacute stage was examined in experimental animals. Soft X-ray microangiography and histological examination of the brain were performed 48 hours after laser exposure. The lasers in this study were pulse wave form CO2 of 2, 4 and 8 watts and YAG lasers of 10, 20 and 40 watts, employed separately or simultaneously using 130 YZ of Nihon Infrared Industries Company. Japanese white rabbits were anesthetized with pentobarbital. Bilateral fronto-parietal craniectomy were made, and the dura was removed. After intravenous injection of Evans blue, the lasers were employed to the cerebral cortex using a micromanipulator attached to the operation microscope. The spot size was 0.7 mm in diameter for CO2 laser and 1.2 mm for Nd: YAG laser. Forty-eight hours after exposure, microangiography was performed and brains were prepared for the histological examination. Histological examination and microangiogram of the brain after CO2 laser exposure revealed semilunar avascular area in the edematous layer surrounded with dilated vessels. Histological examination and microangiogram of the brain after Nd: YAG laser exposure revealed broad avascular or oligovascular zones in the surrounding edematous tissue, in which the surviving vessels were narrowed. Edematous zones were also shown in the subcortical portion. The histological examination and microangiogram after combined coaxial exposure of CO2 and Nd: YAG lasers revealed triangular avascular or oligovascular zones in the edematous tissue, in which the surviving vessels were narrowed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1978

Cytochemical and ultrastructural characters of human peripheral blood lymphocytes according to their surface markers.

The human peripheral blood lymphocytes are characterized by different surface markers: The B lymphocytes by the EAC rosettes and surface immunoglobulin and the T lymphocytes by the E-rosettes. Each lymphoid population has been studied for cytochemical parameters (Acid phosphatase, beta-glucuronidase) by light microscopy and for ultrastructural characteristics by micromanipulation and immunoelectronmicroscopy. It has been shown that the studied enzymes are markers for T cells with high affinity for sheep red blood cells and that the separation of lymphocytes by the rosette techniques leads to homogenous cell populations at the ultrastructural level. A classification of peripheral lymphocytes according to their immunocytochemical profile is discussed.

Acid Phosphatase↗
PubMed1988

[Endocytosis of the androgen-binding-protein (ABP) by the principal cells of rat epididymis].

The present study is based on the comparison between the radioautographic analysis of the fate of the androgen-binding protein purified from rat testes (HPLC) subsequently iodinated and injected into the epididymal lumen using a micromanipulator, and the biochemical analysis of the binding capacities of this molecule to soluble epididymal membrane extracts using HPLC and ultracentrifugation. The various experimental conditions used here allowed to demonstrate that ABP was internalized by the epididymal epithelium and to state that this internalization was not a non specific fluid phase endocytosis but a receptor-mediated-mechanism. Indeed, from a morphological stand point, the labeled ABP was associated rather with the membranes of the endocytic apparatus than with its content. In addition, from the two lumenal cell types able to resorb seminal fluid products, only the principal cells took up the labeled ABP. Our results clearly showed that this internalization was correlated with the presence of a 125I.ABP binding protein. Since the binding of this protein molecule to ABP was saturable and Calcium and pH dependent, it is strongly suggested that this molecule behaves as a receptor, the ligand (or one of the ligands) of which could be ABP.

Androgen-Binding Protein↗
PubMed1988

Production of two hemopoietic growth factors is differentially regulated in single T lymphocytes activated with an anti-T cell receptor antibody.

A method has been developed to measure the production by single activated T lymphocytes of two hemopoietic growth factors, granulocyte-macrophage CSF (GM-CSF) and multipotential CSF (multi-CSF or IL-3). When individual cells of the L3T4 (CD4)+ F23.1+ T cell clone E9.D4 were transferred by micromanipulation into wells coated with the monoclonal anti-T cell receptor antibody F23.1, up to 90% of cells produced CSF as detected by CSF-dependent hemopoietic cell lines. Production occurred in the absence of proliferation and did not require the addition of accessory cells or IL-2. Both the frequency of CSF-producing cells and the average production per positive cell depended on the density of the immobilized stimulating ligand, indicating that the response of each cell is not an all-or-none phenomenon but varies with the strength of stimulation. Individual cells of the clone varied over a 100-fold range in their total CSF titer with a mean value of about 0.05 U/cell. They also varied in their relative production of GM-CSF and multi-CSF. Thus, low producing cells secreted only GM-CSF whereas high producing cells also secreted multi-CSF. The failure of low producing cells to secrete multi-CSF was not genetically based since such cells could give rise to progeny that synthesized multi-CSF. These results suggest that the synthesis of these two lymphokines can be differentially regulated at the level of the single cell.

Antibodies, Monoclonal↗
PubMed1986

An assay for colony-stimulating factor (CSF) production by single T lymphocytes: estimation of the frequency of cells producing granulocyte-macrophage CSF and multi-lineage CSF within a T lymphocyte clone.

The frequency of cells producing hemopoietic colony-stimulating factors (CSF) in a murine T lymphocyte clone has been determined by using a simple microassay that does not require clonal expansion or the addition of accessory cells. When stimulated with concanavalin A (Con A), the clone LB3 produced both granulocyte-macrophage CSF (GM-CSF) and multi-lineage CSF (Multi-CSF), which could be detected by using the cell line FDC-P1, whose proliferation is dependent on the presence of either of these factors. Limiting dilution analysis of Con A-stimulated LB3 cells indicated a requirement for cell-cell contact for optimal production of CSF, which could be bypassed by preincubation of the cells at high density with Con A for 4 hr before dilution in the assay. Limiting dilution estimates of the frequency of CSF-producing cells among Con A-pretreated LB3 cells ranged from 20 to 50%. Direct measurement of CSF production by single Con A-pretreated cells isolated by micromanipulation revealed that 10 to 20% could secrete detectable CSF. However, when isolated Con A-pretreated two-cell and three-cell aggregates were assayed, 50 to 99% were positive, indicating that 30 to 80% of the cells in the aggregates secreted CSF. Assay of the supernatants from single cells and two-cell aggregates on both FDC-P1 cells and another cell line, 32D c13, which responds only to Multi-CSF, demonstrated that many cells produced GM-CSF only, and others varied in the relative quantities of GM-CSF and Multi-CSF produced.

Animals↗
PubMed1987

Genetically mast cell-deficient W/Wv mice as a tool for studies of differentiation and function of mast cells.

Genetically mast cell-deficient W/Wv mice are useful for the analysis of mast cell biology, especially as recipients of bone marrow cells and skin pieces. Inasmuch as suspension and clonal cultures of mast cells have been developed, we combined these in vivo and in vitro systems. Suspension-cultured mast cells had morphological and biochemical characteristics similar to those of mucosal mast cells (MMC). However, i.p. injection of such cultured mast cells gave rise to development of cells with characteristics similar to those of connective tissue mast cells (CTMC). When peritoneal cells of normal +/+ mice were cultured in methylcellulose, pure mast cell colonies appeared. Cells from individual mast cell colonies were divided and injected into the skin and stomach wall of W/Wv mice; CTMC developed in the skin and MMC in the stomach mucosa. This indicates the presence of a common precursor for CTMC and MMC. Morphology of such bipotent mast cell precursors was studied by using micromanipulation. About 4% of morphologically identifiable peritoneal mast cells may function as the bipotent precursors. Although W/Wv mice showed a defect in resistance against ixodid ticks, injection of suspension-cultured mast cells normalized the defect. The four examples mentioned above indicate that combinations of in vivo and in vitro systems increase the usefulness of W/Wv mice.

Animals↗
PubMed1987

A versatile chamber for microphysiologic studies with gas mixtures under high pressure.

A pressure chamber, designed for microelectrode recordings in isolated tissues or organs an capable of withstanding pressures up to 200 bar, is described. The versatility of the vessel allows a wide variety of experimental configurations and several types of studies. It features a complete access, easy visibility, interchangeable tissue bath and micromanipulator modules, as continuous perfusion and temperature control of the preparation under pressure. The ability to move micropipettes in 1-micron steps allows single unit recording on a variety of in vitro preparations at normal or elevated gas pressures. Successful physiologic tests, using the hippocampal slice preparation, are described. The results testify to the reliability of the system and to the usefulness of this in vitro model to study the gas pressure effects on isolated networks of mammalian CNS.

Air Pressure↗
PubMed1986

Use of a cell sorter for assays of cell clonogenicity.

A fluorescence-activated cell sorter has the capability of recognizing, categorizing, counting, and sorting cells, and it thus provides a convenient and rapid method for "micromanipulation" of known numbers of cells into appropriate vessels for cell viability or clonogenicity studies. Subsequent microscopic observation of the sorted cells allows discrimination between the separate processes of cell attachment (plating efficiency) and clonogenic growth (viability). Examples are presented showing the power of these techniques for studying the low-dose regions of radiation survival curves for Chinese hamster V79 cells grown as monolayers or spheroids, for cells irradiated under aerobic versus hypoxic conditions, and for cells of large spheroids exposed to Adriamycin, where the slow penetration of the drug results in a differential exposure to the various cell subpopulations.

Aerobiosis↗
PubMed1986

Development of mucosal mast cells after injection of a single connective tissue-type mast cell in the stomach mucosa of genetically mast cell-deficient W/Wv mice.

Mast cells may be classified into at least two phenotypically distinct populations: connective tissue-type mast cells (CTMC) and mucosal mast cells (MMC). Mast cells in the peritoneal cavity of mice are typical CTMC, whereas mast cells in the mucosa of the stomach show morphologic characteristics of MMC. We investigated whether CTMC may change to MMC. A single peritoneal mast cell of WBB6F1-+/+ mice was identified under the phase-contrast microscope, picked up with the micromanipulator, and injected into the stomach wall of genetically mast cell-deficient WBB6F1-W/Wv mice. The cells with histochemical and electron microscopical features of MMC developed in the mucosa, and those with histochemical features of CTMC in the muscularis propria. This directly demonstrates that a certain proportion of CTMC may function as a bipotent precursor for both MMC and CTMC.

Animals↗
PubMed1979

Subpopulations of Ig-secreting cells induced by peroxidase immunization: discrimination according to antibody storage and secretion.

Mice were injected in their hind footpads with peroxidase (PO) emulsified in Freund's complete adjuvant. The development of cells secreting anti-peroxidase antibody (Ab) and cells secreting immunoglobulins (Ig) were detected in the draining popliteal lymph nodes in the subsequent 35 days, using local haemolysis plaque assay with sheep red cell blood cells coated with either PO or anti-mouse Ig antibody. Plaque-forming cells (PFC) were isolated from the centre of plaques by micromanipulation and after appropriate treatment, were examined by electron microscopy for their intracellular Ab content and in corporation of [3H]-thymidine. Four subpopulations of Ig secreting cells were distinguished: (1) cells secreting Ig without Ab function and not containing intracellular Ab detectable between days 5 and 20; (2) cells secreting Ig without Ab function but containing Ab appearing on day 6 and present throughout the immune response; (3) cells secreting Ab and containing Ab; (4) cells secreting Ab, but without detectable intracellular Ab. These last subpopulations appeared on day 7 and were found in all subsequent assays. The analysis of the kinetics of these subpopulations suggest that cells secreting Ig without Ab function might be precursors of Ab secreting cells.

Animals↗