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Biomedical subjects

W F Chen

Publications and source records attributed to W F Chen.

At least 73 records · Page 4Linked to original sources

The functional capabilities of cells leaving the thymus.

There has been a controversy for many years over the functional status of cells that leave the thymus (thymus migrants) to populate the peripheral lymphoid organs. Are they immunoincompetent like cortical thymocytes and so probably derived from them, or are they functionally mature like medullary thymocytes? Until recently the techniques used to assess putative thymus migrants have been indirect, but it is now possible to measure the function of recent thymus migrants directly. We used intrathymic injection of a solution of fluorescein isothiocyanate to label thymocytes, and used electronic cell sorting to purify the fluorescent cells that accumulate in the periphery over the following 3 to 4 hr. The migrants have been enriched from an original frequency of about 1:1000 in lymph nodes and spleen, to greater than 98% purity. These cells have been compared with normal peripheral T cells for proliferative and cytotoxic precursor activity in a high cloning efficiency, lectin-induced, limit dilution culture system and in an allospecific limit dilution system. The frequency of precursors of proliferative lymphocytes and cytotoxic lymphocytes and the size of the clones produced is the same for recent migrants and peripheral T cells. Thus by the criteria of proliferation and cytotoxic responses to mitogens and generation of allospecific CTL, thymus migrants, a few hours after their emigration from the thymus, are fully immunocompetent; we therefore see no evidence of a post-thymic precursor-type cell that requires major maturation steps after leaving the thymus.

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Development of large granular lymphocytes with anomalous, nonspecific cytotoxicity in clones derived from Ly-2+ T cells.

T cells cultured at limit dilution for 8 days in a concanavalin A-stimulated, filler-cell and growth factor-supported system produced cytolytic clones with high efficiency. These clones were not specific, lysing a wide range of targets, syngeneic and allogeneic, of tumor and normal cell origin. Lysis was a cell-mediated phenomenon but was not blocked by anti-Ly-2. One H-2-negative target was lysed, but one was resistant. Xenogeneic (human) tumor cells were not lysed. The cells in the clones were large, vacuolated, granular lymphocytes. They originated from single Ly-2+ responder cells and not from irradiated filler cells. Therefore, activated lymphocyte killers and other natural killer-like cells may be differentiated elements of the Ly-2+ T-cell lineage.

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The size of functional T-lymphocyte pools within thymic medullary and cortical cell subsets.

The frequency of all precursors of T cells capable of proliferation (PTL-p) and of all cytotoxic T-cell clones (CTL-p) was determined for different thymocyte subpopulations using a high cloning-efficiency, Con A and growth factor driven, limit-dilution assay and a lectin-mediated, non-specific cytotoxic readout. As shown previously, more than 99% of precursors were confined to the medullary-type fraction, isolated by fluorescence activated cell sorting as the 14-15% of thymocytes showing low binding of peanut agglutinin (PNA). However, 20-50% of medullary-type cells appeared incapable of responding in a culture system allowing all peripheral T cells to grow, suggesting that the absolute size of the functional pool was 7-12% of all thymocytes. The 3-4% cortisone-resistant fraction of thymus gave a high precursor frequency (PTL-p 1 in 1.3; CTL-p 1 in 6) and a high cloning efficiency per Thy 1 positive cell (80%) which was nevertheless below that of peripheral T cells. However, only 20-25% of the total thymic PTL-p and CTL-p could be recovered in this fraction. Functional precursors were therefore within both the cortisone-sensitive and the cortisone-resistant subgroups of medullary-type thymocytes. Attempts to induce function in PNA+ cortical-type thymocytes by increasing the level of T-cell growth factors in the cultures gave only a marginal increase, the bulk of small cortical cells remained functionally inert. However, the low frequency of precursors found in the PNA+ fraction (around 1% of that in PNA- thymocytes) was not entirely due to PNA- contaminants since a PNA+, high H-2 blast fraction, representing about 4% of all thymocytes, showed a significant, although still low, PTL-p and CTL-p frequency amounting to less than 1% of the total thymus precursor pool. The relevance of this minor subset is discussed.

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The Ly phenotype of functional medullary thymocytes.

The frequency of all precursors of T cells capable of proliferation (PTL-p), and of all precursors of cytotoxic T-cell clones (CTL-p), was determined for mouse thymic and peripheral T-cell subsets differing in Ly phenotype. A high cloning efficiency, concanavalin A (Con A) and growth factor driven limit dilution culture system was used. A lectin-mediated non-specific lysis readout was used for detecting cytotoxic clones. This approach provided a balance sheet of the overall distribution of functional cells regardless of specificity. Subsets of splenic T lymphocytes were isolated by fluorescence-activated cell sorting (FACS) after two-colour staining with monoclonal anti-Thy 1 and anti-Ly 2 antibodies. Both the Ly 1+2- and Ly 1+2% subsets responded by clonal proliferation, but cytotoxic activity was almost exclusively limited to the Ly 1+2% derived clones. Four subclasses of thymocytes were isolated by FACS after two-colour staining with peanut agglutinin (PNA) and monoclonal anti-Ly 2 antibody. These were PNA+Ly 2+, PNA+ Ly 2-, PNA- Ly 2+ and PNA- Ly 2-, representing 80, 5, 5 and 10% of total thymocytes, respectively. Their respective PTL-p frequencies were 1 in 333, 1 in 200, 1 in 5.3 and 1 in 3.2, values which included a significant activity loss on labeling and isolation. The slight activity in PNA+ cells may have been contaminants. The PNA- Ly 2+ subset formed larger clones than the PNA- Ly 2- subset. CTL-p frequency was 1 in 5 for PNA- Ly 2+ and 1 in 400 for PNA- Ly 2-. The few cytotoxic clones derived from the Ly 2- cells appeared to be genuine and not a result of contamination with Ly 2+ cells. Thus although both Ly subsets of medullary-type thymocytes were able to proliferate, the Ly 2+ subset contributed almost all of the cytotoxic activity of the unfractionated thymocytes. Medullary-type thymocytes display an Ly phenotype development and a level of functional maturation approaching that of peripheral T cells.

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Limit-dilution assay and clonal expansion of all T cells capable of proliferation.

A limit-dilution microculture system is presented in which almost all mature T cells, cultured at a level of about 1 cell/well, grow and expand to clones averaging 60,000 cells over an 8-9 day period. Cloning efficiency is 70-100%, so the set of the expanded clones is representative of the starting T-cell population. T cells of all Lyt phenotypes form clones of progeny cells. The system involves culture in flat-bottom microtitre trays, in the presence of concanavalin A as the initiating stimulus, together with appropriately irradiated spleen filler cells and a supplementary source of soluble T cell growth factors. The resultant clones may be screened for cytolytic function, as described in the accompanying paper. The system may be used to assay the level of T cells capable of expansion or precursor function (PTL-p) by using [3H]TdR uptake as a readout for the presence or absence of proliferating clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of complicating suppressor or helper effects.

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Leukocyte migration-inhibition agarose test in swine after vaccination and challenge with inactivated and activated pseudorabies virus.

Cell-mediated immunity to pseudorabies virus (PrV) was studied by means of the leukocyte migration-inhibition (LMI) agarose test. Migration of leukocytes from swine vaccinated with 10 TCID50 inactivate PrV and from normal control swine was not inhibited in the presence of specific PrV antigen. Cellular immune responses were detected in 80% of the vaccinated and 40% of the control group animals at the 1st week after challenge exposure. At the 2nd week, both groups of swine showed a peak level (p less than 0.001) immune response in the LMI test. This level of response remained until the 3rd week in the vaccinated group and the 2nd week in the control group. The averages of migration indices at the 1st, 2nd, 3rd and 4th weeks after challenge exposure were 0.74, 0.22, 0.60 and 0.89 in the vaccinated and 0.82, 0.29, 1.01 and 1.12 in the control group, respectively. Low concentrations of virus-neutralizing (VN) antibodies were present in 30% of the vaccinated swine at the 1st week, and 60% at the 2nd week after vaccination. Anamnestic antibody and primary VN antibody responses occurred after challenge in vaccinated and normal swine, respectively.

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The acquisition of receptors for peanut agglutinin by peanut agglutinin-negative thymocytes and peripheral T cells.

Using fluorescein-conjugated peanut agglutinin (PNA) and the fluorescence-activated cell sorter, we have shown that the cytotoxic T cells generated by culturing thymocytes (about 85% PNA+ cells) with Con A and medium conditioned by Con A-stimulated spleen cells were mostly PNA+. Cytotoxic T cells generated in parallel cultured of PNA- thymocytes or normal lymph node cells (in which the T cells are PNA-), however, were also mostly PNA+. The development of PNA+ cells from PNA- mature peripheral T cells was confirmed by using cultures of PNA- T cells separated on a fluorescence-activated cell sorter from lymph node cells. Immunization in vivo also resulted in the appearance of PNA+ T cells in the lymph node. These results indicate that receptors for PNA can no longer be regarded as unambiguous markers of T cell immaturity, or of the origin of a T cell from a PNA+ precursor. The possibility that a variable proportion of the PNA+ cells in the thymus may be generated by intrathymic activation of PNA- medullary thymocytes is discussed.

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Newcastle disease virus in Taiwan: II, Relationship between polykaryocytosis and virus virulence.

Fifteen selcted local isolates and five known Newcastle disease virus strains were examined for their cytopathic effects in chick embryo kidney (CEK) cells, egg-infectious units in chick embryos (CE), virulence by mean death time, intracerebral and intravenous pathogenicity indexes for CE and chicks, and ability to cause polykaryocytosis of fusion from within (FFWI) or fusion from without (FFWO) in CEK and BHK-21 monolayer cells. The capacity of the different virus strains to induce cell FFWI at 15 hr post-infection was related to their virulence for CE and chicks, but cell FFWO did not seem to be any relationship with the virulence of the strains.

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Colonic protection from dimethylhydrazine by a high fiber diet.

Results of this experiment showed that a high fiber bran diet had the ability to lower significantly the incidence of pathologic changes in the colon of mice receiving 1,2-dimethylhydrazine, a drug known for its specificity for causing carcinoma of the colon. This finding adds support to the hypothesis which clains that the addition of bran to the diet has the ability to lower the indicence of carcinoma of the large intestine.

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Isolation and identification of Marek's disease virus by fluorescent antibody technique.

Cell-associated herpesvirus related to Marek's disease (MD) was isolated from the direct culture of kidney cells of naturally infected chickens at Taoyuan or by inoculation of clinical specimens to chick kidney (CK) and chick embryo fibroblast cells. The virus isolates replicated in CK or chick embryo kidney cell cultures were identified to be MD by the fluorescent-antibody technique.

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Prevention of cerebral infarction in the dog by intact omentum.

Three groups of dogs underwent total occlusion of the right middle cerebral artery close to its junction with the internal carotid artery. Dogs that had omentum placed on the brain prior to occlusion of the middle cerebral arterial withstood this deprivation of normal cerebral blood flow in a statistically significant manner when compared with dogs that had no omental protection or had omental placement on the brain simultaneous with middle cerebral artery occlusion. These results raise the possibility that use of the intact omentum might be another technic for revascularization of the brain in patients with transient cerebral ischemic attacks or brain dysfunctions secondary to diminished cerebral blood flow who presently are not candidates for routine surgical procedures for cerebral revascularization.

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Intact omentum for ocular vascularization.

Twenty dogs had their intact omentum extensively lengthened by a series of surgical maneuvers. Transverse incisions were then made along the chest, shoulder, neck, and scalp which were undermined and connected to form a subcutaneous tunnel through which the omentum was brought up to the orbit. The lateral rectus muscle of the eye was divided and a scaleral flap developed along the lateral superior region of the eye which exposed the choroid upon which the omentum was secured. Subsequent studies demonstrated vascular connections between intraocular vessels and those of the omentum. Proof of the existence of these vascular connections was based upon fluorescent funduscopic, gross, and histologic evidence.

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