The response of human peripheral blood lymphocytes to phytohemagglutinin: determination of cell numbers.

Optimization and definition of conditions for studying lymphocyte function in vitro resulted in exponential proliferation of lymphocytes from day 2 to day 5 with an average doubling time of 20 hr. The number of cells in culture on day 5 was 5–10 times as great as the number initially planted and 10–20 times as great as the number surviving in culture on day 2. An improved pronase-cetrimide technique was used to determine the number of viable lymphocytes as a function of time after addition of PHA. The volume changes in nuclei, obtained after cetrimide treatment, were quantitated using a curve-fitting computer program.The response could be described in terms of an induction phase (0–2 days) characterized by a decrease in cellularity and an increase in nuclear volume, a proliferation phase (2–5 days) characterized by an exponential proliferation and a continued increase in the number of cells having a large nuclear volume, and a lysis phase (5–14 days) characterized by a decrease in cellularity and a decrease in nuclear volume. The results reported here suggest that the ratio of the number of cells cultured to the volume of culture medium was crucial for optimal transformation and proliferation, 10⁵ cells/ml producing far better responses than 10⁶ cells/ml.     

1H-NMR structural determination of the N-linked carbohydrate chains on glycopeptides obtained from the bean lectin phytohemagglutinin.

Phytohemagglutinin, the lectin of the common bean Phaseolus vulgaris, is a N-linked glycoprotein with one high-mannose-type and one xylose-containing oligosaccharide side chain per polypeptide. The high-mannose-type glycan is attached to Asn12 and the complex-type glycan to Asn60 [Sturm, A. & Chrispeels, M. J. (1986) Plant Physiol. 81, 320-322]. The structures of the oligosaccharides were elucidated from two glycopeptides obtained from the lectin by Pronase digestion, affinity chromatography on concanavalin-A--Sepharose and gel-filtration chromatography on a column of BioGel P-4. The N-linked glycan structures were investigated by 500-MHz 1H-NMR spectroscopy and were established to be: [formula; see text]     

Non-mitogenic binding of phytohemagglutinin to lymphocytes

³H-PHA is bound to lymphocytes both at 37°C and at 0°C. However, the lectin is rapidly shed from cells which have been pulsed at 0°C and no DNA-synthesis takes place even if the pulsed cells are kept at 37°C for 64 hours. Cells pulsed at 37°C and subsequently at 0°C do not shed the bound lectin. They are induced to synthesize DNA when subsequently incubated at 37°C for 64 hours.     

Immunological characteristics of the 2-stage method of smallpox vaccination]

As a result of observations carried out on children the authors present immunological characteristics of two-stage smallpox vaccination at different intervals (1 to 60 days) between the injection of inactivated and live vaccine. There proved to be acceleration and intensification of antibody formation after two-stage immunization in comparison with the rutine vaccination. A seven-day interval between the injection of the inactivated and live preparations was recommended on the basis of the data obtained.     

Secretion of phytohemagglutinin by monkey COS cells

The entire coding region of a gene, which encodes a polypeptide of phytohemagglutinin (PHA-L), obtained from a library of genomic DNA of the common bean Phaseolus vulgaris cv. Greensleeves, was introduced into the SV40 expression vector pJC119. Monkey COS1 cells were transfected with the recombinant clone and the synthesis, glycosylation, and transport of PHA-L studied and compared with the normal processes in bean cotyledons. In the bean, phytohemagglutinin is synthesized on the rough endoplasmic reticulum and transported via the Golgi complex to protein bodies, vacuole-like organelles. Phytohemagglutinin was synthesized and glycosylated at the ER and processed in the Golgi apparatus of the transfected COS1 cells. After passing the Golgi apparatus, PHA-L was slowly secreted into the culture medium (half-time of 3-6 h), a result indicating that the signals for targeting proteins beyond the Golgi apparatus in plant cells are different from those in animal cells. PHA, which is stored in protein bodies in the plant cells, is secreted by animal cells. Tunicamycin inhibited both glycosylation and secretion of PHA by the COS1 cells, a finding indicating an essential role of the oligosaccharides for transport of PHA in these cells in contrast to the situation found in bean cotyledons. PHA, secreted into the culture medium, was partially sensitive to endo H, a result indicating the presence of one high-mannose and one complex oligosaccharide chain, a situation identical to that in beans.     

Immunological determination of labeling index on human tumor tissue sections using monoclonal anti-BrdUrd antibody

The use of a monoclonal antibody against the thymidine analogue bromodeoxyuridine together with an in vitro labeling technique allowed rapid determination of the labeling index in human tumors. The labeling index estimated by these relatively simple immunofluorescence or immunoenzymatic staining methods was equivalent to that obtained by autoradiography. The interpretation of the preparations is easy since there is a minimum of background staining. This immunohistochemical technique combined with in vitro labeling provides a suitable alternative for determining the labeling index of human tumors.     

Two categories of lymphocyte unresponsiveness to phytohemagglutinin

Peripheral lymphocytes from healthy subjects, sarcoidosis and influenza patients were studied in vitro by measurement of the tritiated thymidine uptake of unstimulated and phytohemagglutinin. (PHA) stimulated cells. When the mitogen induced metabolic response is defined as the ratio between thymidine uptake by stimulated and unstimulated cells (stimulation index), PHA responsiveness was significantly decreased in both diseases and varied inversely with the level of isotope incorporated by unstimulated cells (p = 0.0002). The uptake of isotope by unstimulated cells from influenza patients was significantly increased (p = 0.0001). Isotope incorporation by mitogen stimulated cells from the same patients did not differ significantly from controls (p = 0.0925). In contrast, the impaired PHA responsiveness of lymphocytes from sarcoidosis patients was associated with levels of isotope incorporation in unstimulated cell cultures similar to those observed in healthy controls (p = 0.6444). These observations suggest that two different mechanisms may be responsible for low lymphocyte PHA stimulation indices associated with disease states. Methods are presented for minimizing variation of replicate observations and identification of both categories of lymphocyte unresponsiveness.     

Radiocompetitive method of H antigen determination

The authors describe a radiocompetitive method of H-d-monomere determination with the sensitivity of 2 ng/ml in double antibodies modification; this method was used for comparing the immunological affinity of the affiliated H-antigens. A difference between the immunological affinity to the antibodies in a monomere, polymere and the flagellum was shown.