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Biomedical subjects

S Fong

Publications and source records attributed to S Fong.

At least 127 records · Page 7Linked to original sources
PubMed1981

Solid-phase selection of human T lymphocyte subpopulations using monoclonal antibodies.

This report describes a novel solid-phase technique for the positive selection of human T lymphocyte subsets labeled by indirect immunofluorescence with monoclonal anti-bodies. Fluorescein labeled normal human T cells or a T cell line were fractionated on plastic culture dishes precoated with affinity chromatography purified anti-fluorescein antibodies. Cell binding was specific for fluorescein, and was both time and temperature dependent. Bound cells were eluted at 37 degrees C with fluorescein-L-lysine. The eluted cells were enriched with highly viable and functional human T cell subsets. Thus Leu3 monoclonal antibody selected cells were shown to provide helper activity in the pokeweed mitogen induced IgM and IgG immunoglobulin secretory response of autologous B cells. The Leu2 antibody selected T cells suppressed both IgM and IgG secretory responses. In addition, studies with the monoclonal antibody 1G11, which binds to an antigen expressed on acute lymphocytic leukemia (T-ALL) cells and the T-ALL derived cell line RPMI-8402, demonstrated that this solid-phase technique can be used to select for cells which are present at low frequencies in a mixed population. It thus provides a simple and reproducible means for the preparative isolation of lymphocyte subsets associated with autoimmune and neoplastic disease for functional and biochemical analysis.

Animals↗
PubMed1981

Fractionation of human lymphocyte subpopulations on immunoglobulin coated Petri dishes.

This report describes a simple solid-phase technique for the positive selection of lymphocytes labeled with fluoresceinated antibodies. B lymphocytes were labeled with fluoresceinated anti-human Ig or monoclonal anti-human Ia (L243), and then were bound to plastic culture dishes coated with affinity purified goat anti-fluorescein specific antibody. Bound cells were eluted at 37 degrees C with 1 mM fluorescein-L-lysine phosphate-buffered saline. Functionally the eluted Ig positive cells responded to pokeweed mitogen (PWM) by in vitro secretion of IgM, as measured by radioimmunoassay of culture supernatants. The secretion of IgM was dependent on the addition of T lymphocytes. Moreover, the isolated B cells were functionally receptive to 'help' and 'suppression' by T cells with and without Fc receptors for IgG respectively. T cell subsets were fractionated on plastic culture dishes coated with heat aggregated rabbit or human IgG. the non-bound cells (enriched T(gamma-)) provided collaborative 'help' in the PWM induced IgM secretion response by human B lymphocytes. The bound cells (enriched T(gamma+)) eluted with 0.01 M EDTA in phosphate-buffered saline, suppressed IgM secretion. This method can be adapted to fractionate subsets of lymphocytes for which a fluoresceinated antibody is available. For routine functional studies, the isolation of cell types with conventional or monoclonal antibodies does not require the use of a fluorescence activated cell sorter, but only an antifluorescein labeled Petri dish. In conclusion, a rapid solid-phase technique enables us to prepare enriched populations of functionally active lymphocytes.

Animals↗
PubMed1981

Effect of levamisole on pokeweed mitogen stimulation of immunoglobulin production in vitro.

The effects of levamisole (LMS) on immunoglobulin (Ig) production were studied in vitro using peripheral blood lymphocytes from normal subjects stimulated with pokeweed mitogen (PWM). Cells were cultured for 9 days with varying concentrations of LMS and PWM, and immunoglobulin secretion in the supernatants was quantified by solid phase radioimmunoassay. The results showed that 1) the effect of LMS in vitro depends upon the degree of lymphocyte stimulation by PWM. When PWM stimulation is optimal, typical pharmacologic concentrations of LMS (0.5 micro/ml) decrease both IgM and IgG production by 50%. However, at lower suboptimal doses of PWM, LMS, at similar concentrations, enhances immunoglobulin production by 24% (p less than 0.01). Unstimulated lymphocytes are not affected by LMS. 2) The target cell upon which LMS acts is present among a T subpopulation that lacks the Fc receptor for aggregated rabbit IgG (T gamma -negative). We suggest that the diverse effects of LMS on autoimmune disease in vivo may depend upon both the size and degree of activation of the T gamma -negative lymphocyte pool.

B-Lymphocytes↗
PubMed1981

T lymphocyte-mediated cytotoxicity against autologous EBV-genome-bearing B cells.

We have stimulated human peripheral blood lymphocytes in vitro with autologous EBV-infected or noninfected B cells. A cytotoxic response was obtained only when virally infected cells were used. The activity of the effector cells was restricted by the major histocompatibility complex and was directed against EBV-genome-bearing targets. The highest cytolytic response was obtained when lymphocytes of individuals previously exposed to the virus (EBV-VCA positive) were used. Lymphocytes of noninfected donors (EBV-VCA negative) gave a low response; the relative frequency of their effector cells was at least 4-fold lower. Lymphocytes of newborns did not respond. The cytotoxic activity was mediated by T lymphocytes of the cytotoxic/suppressor subset, as determined by cytofluorographic analysis and antibody plus complement-mediated lysis, using monoclonal antibodies to human lymphocyte surface antigen.

Antibodies↗
PubMed1981

Age-associated changes in Epstein-Barr virus-induced human lymphocyte autoantibody responses.

In the present investigation we have studied the capacity of the Epstein-Barr virus (EBV), a ubiquitous human B lymphocyte polyclonal activator, to induce cultured peripheral blood lymphocytes from umbilical cords, young adults (20 to 40 yr old), and elderly adults (75 to 90 yr old) to form IgM antibodies to human IgG or human thyroglobulin. The EBV preparation used was shown to exert its B cell stimulatory effect independently of T cell-suppressor effects. The cultures were studied in limiting dilution analyses, and the data were taken to represent relative numbers of B cell precursors of autoantibody-forming cells in the 3 age groups. The results showed: 1) the EBV-inducible IgM anti-IgG and anti-thyroglobulin-producing cells increased in number from birth to old age; 2) the rise occurred at different times of life for the 2 autoantibodies, anti-IgG reactive B cells increasing between birth and young adulthood, and anti-thyroglobulin reactive B cells between young adulthood and old age; 3) the apparent relative avidity of the anti-IgG for antigen was higher in the elderly adults than in the younger adults. We believe these findings are determined by differences in the frequencies of the respective self-reactive B cells in the circulation. What physiologic or environmental factors determine the differential expansions of the human autoreactive B lymphocytes for IgG and thyroglobulin are not known. It seems possible that individual variations in the sizes of these pools may be a factor in determining susceptibility to autoimmune disease.

Adult↗
PubMed1980

Validation of conditions for efficient detection of HPRT and APRT mutations in suspension-cultured Chinese hamster ovary cells.

Conditions for reliable and efficient assay of mutations affecting the activity of HPRT (hypoxanthine phosphoribosyltransferase EC 2.4.2.8) and APRT (adenine phosphoribosyltransferase EC 2.4.2.7) have been determined for a strain of CHO (Chinese hamster ovary) cells that has been adapted for rapid growth both in suspension culture and in monolayer. To facilitate measurement of mutation at the aprt locus, clones were derived that are presumptively heterozygous at that locus. At a limiting concentration of 8 microgram/ml of azaadenine, 14/16 of the resistant clones picked and tested had approximately 1/2 of the APRT activity of the wild-type cells. One such clone, strain AA8, was chosen for further studies and found to be readily mutable to resistance to 80 microgram/ml azaadenine. Most of the highly resistant colonies isolated (21/24) had very low in vitro APRT activity. The optimal conditions for detection of TGr and AAr mutations were determined for two critical parameters, expression time and cell density. Cultures treated with mutagen either in monolayer or in suspension were allowed to express mutations in suspension. The expression of mutations induced by UV light, EMS, and ICR-191 was complete by 3 days for AAr and by 4-5 days for TGr. The time required to reach a maximal frequency of mutants was essentially independent of the type of mutagen and the level of survival after treatment. Induced mutation frequencies for both loci were notably stable during the time intervals examined. With respect to cell-density conditions, both markers were detected at frequencies that were independent of the cell inocula over the range of 1 x 10(5) to 1 x 10(6) cells per 100-mm petri dish (i.e. 1.6 x 10(3) to 1.6 x 10(4) cells/cm2) containing 20 ml of medium. These results were obtained with both mutagenized populations and with reconstructed mixtures obtained by adding drug-resistant cells to varying numbers of wild-type cells. The rapid expression of mutations for both markers, particularly AAr, combined with the advantage that large inocula can be plated for selection of mutants, make this CHO strain an attractive system for the simultaneous measurement of mutations at the autosomal aprt and X-linked hprt loci.

Adenine↗
PubMed1980

Inheritance of immunoglobulin M rheumatoid-factor idiotypes.

The idiotypic determinants on IgM rheumatoid factor (RF) from a single family have been analyzed. Rabbit Fab'2 antiidiotypic antibody was prepared against purified IgM-RF from a patient with rheumatoid arthritis. As measured by radioimmunoassay, the antiidiotype reacted with at least 90% of the patient's RF, but not with non-RF immunoglobulins from the same serum, nor with 10 of 11 polyclonal and monoclonal RF from unrelated individuals. Cross-reacting idiotypes were detected on RF in four of the patients' first degree relatives, spanning three generations, without apparent relation of HLA type or clinical rheumatoid arthritis. These results suggest that IgM-RF associated idiotypes were inherited in this family.

Adult↗
PubMed1979

Macrophage-T cell interaction mediated by immunogenic and non-immunogenic forms of a monofunctional antigen.

As an approach to the elucidation of the essential steps in the immune pathway, the uptake and retention of immunogenic and non-immunogenic analogs of a monofunctional antigen by guinea pig macrophages and the efficiency of macrophages pulsed with the compounds to present antigen to sensitized T lymphocytes were compared. L-Tyrosine-azobenzene-p-arsonate (RAT) and its non-immunogenic analog, 4-hydroxyphenyl-n-propane-3-azobenzene-p-arsonate (RAN), react similarly with antiarsonate antibody, but RAN, unlike RAT, is unable to induce cellular immunity in guinea pigs. The uptake and retention patterns of the two compounds by macrophages differed in that, at a given time, more RAN than RAT was retained and detectable on cell surfaces by anti-arsonate antibody. Equivalent numbers of T lymphocytes from guinea pigs sensitized to RAT formed antigen-dependent clusters with macrophages pulsed with either RAT or RAN after 24 hr in culture, but not with macrophages pulsed with an azobenzenoid compound of unrelated specificity. On the other hand, T lymphocytes from guinea pigs immunized with RAN showed no significant capacity to bind to macrophages which had been pulsed with any of the compounds. The number of lymphocytes from RAT-sensitized animals which bound to RAT-pulsed macrophages remained relatively stable over a 48 hr period, whereas clusters of the same lymphocytes with RAN-pulsed macrophages dissocitated to background levels within that time. Early cluster formation mediated by RAN, as well as its ability to induce transient specific T cell unresponsiveness to RAT in vivo, indicate that T cells are capable of recognizing (binding) the non-immunogen. However, such early, and perhaps weak, interaction with RAN-pulsed macrophages did not induce DNA synthesis by T cells. Anti-Ia serum completely blocked cluster formation mediated by either RAT or RAN. Thus, the only significant distinction disclosed by these studies between the immunogenic and non-immunogenic compounds was the stability of macrophage-T cell interaction as determined by the persistence of antigen mediated cell clusters in culture, suggesting that this may be a factor in immunogenic discrimination.

Animals↗
PubMed1979

113mIn-tripolyphosphate: a new radiopharmaceutical for bone scanning.

A compound was synthetized, which by X-Ray diffraction studies proved to be hexahydrated sodium tripolyphosphate. By means of in vitro procedures, it was demonstrated that this compound was able to complex trivalent cations, and that 113mIn could be completely incorporated to it. In vivo studies in rats, and scans performed with this new radiopharmaceutical in humans showed its highly selective accumulation in metastasic bone tumors.

Animals↗
PubMed1979

DNA crosslinking, sister-chromatid exchange and specific-locus mutations.

Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.

Adenine Phosphoribosyltransferase↗
PubMed1978

Spatial requirements between haptenic and carrier determinants for T-dependent antibody responses.

To gauge the proximity between cooperating T and B cells required for effective triggering of antibody production, guinea pigs were immunized with bifunctional antigens in which the haptenic and carrier determinants were separated by rigid spacers of varied dimension. These took the form 2,4-dinitrophenol-(proline)n-L-tyrosine-p-azobenzenearsonate, where n varied from 1 to 40 proline residues. Animals immunized with n = 10 and n = 22 compounds made strong anti-DNP antibody responses, whereas animals immunized with bifunctional compounds containing longer spacers did not make antibody detectable by precipitation. It can be calculated on the basis of very strong physicochemical evidence for the rigidity and axial translation of poly-L-proline chains in solution that the cut-off point for effective interaction between T and B cells lies between 69 and 97 A U.

Animals↗
PubMed1978

T-lymphocyte activation by immunogenic determinants.

Synthetic antigens have been of great value in elucidating the relationships between antigen structure and lymphocyte activation. The compound RAT behaves as a monofunctional antigen in guinea pigs and mice, inducing T-lymphocyte responses without appreciable circulating antibody, although the ABA-specific B cell population is expanded by immunization with the monovalent molecule. On the other hand, bifunctional antigens composed of one RAT moiety serving as a carrier and a second chemical group, either identical to or different from RAT, serving as a hapten, induced antibody responses. In such responses, T cell specificity was always directed against the RAT component. Using symmetrical bifunctional antigens with rigid or flexible spacers between the two determinants, marked differences in structural requirements for cell triggering, assessed by antigen-induced lymphocyte proliferation, and for cell cooperation, determined by antibody formation, were found. Rigidly spaced bifunctional antigens serve admirably for cooperation but poorly for T cell activation, underscoring the advantage of two-point binding for the latter.

Animals↗