Fc epsilon receptor, a specific differentiation marker transiently expressed on mature B cells before isotype switching.

The expression of Fc epsilon R on human lymphocytes was studied with the anti-Fc epsilon R mAbs. Fc epsilon R was expressed on most mu+,delta+ circulating B cells, whereas T cells did not express Fc epsilon R even in patients with hyper-IgE syndrome. B cells with gamma, alpha, or epsilon phenotype did not express Fc epsilon R, moreover its expression could not be induced, suggesting that the Fc epsilon R expression was correlated with isotype switching. mu+delta+ B cells in bone marrow did not express Fc epsilon R, but PHA-sup (supernatant from PHA-stimulated cell cultures) could induce its expression, and the addition of IgE augmented this induction. Recombinant IL-2, IL-1, IFN-gamma or -beta, or purified B cell differentiation factor (BSF-2 B cell-stimulatory factor 2) could not induce Fc epsilon R expression in bone marrow B cells. IFN-gamma inhibited the Fc epsilon R expression induced by PHA-sup, suggesting that the human counterpart of BSF-1 may be responsible for Fc epsilon R expression in bone marrow B cells. B cells from patients with common variable immunodeficiency and ataxia telangiectasia did not express Fc epsilon R, but PHA-sup could induce its expression, indicating that circulating B cells of these patients are at a differentiation stage similar to B cells in bone marrow. The study showed that Fc epsilon R is a B cell-specific differentiation marker, the expression of which is restricted to a defined stage of B cell differentiation.

Determination of UTP and ATP pool sizes in human tonsillar lymphocytes by using Escherichia coli RNA polymerase.

The present paper describes a rapid, specific and sensitive method for quantitating ribonucleoside triphosphates (ATP and UTP) in cell extracts. The principle of the method is based on the synthesis of a ribonucleotide polymer in the presence of UTP, ATP and poly(dA-dT) as template. A method for calculation is also described, making the determination of UTP and ATP pool sizes in the cells possible under the same experimental conditions. The calculation takes into account the isotope dilution effect caused by the intracellular ATP. Our experiments show that the neutralized perchloric acid soluble fraction of human tonsillar lymphocytes contains no inhibitors for the RNA polymerase test. According to our results, this cell extract contains 80 pmol of UTP and 340 pmol of ATP per mug RNA.

Expression of three forms of thyroid hormone receptor in human tissues.

At least two thyroid hormone receptor (hTR) genes are present in humans, but the significance of this multiplicity is unknown. These receptors could have differences in tissue distribution or possess different functions. We studied the distribution and abundance of three hTR mRNAs (hTR beta, hTR alpha 1, and hTR alpha 2) by Northern blot analysis. Three mRNAs were expressed in all tissues examined. hTR beta was strongly expressed in brain and prostate predominantly as a 10.0-kilobase (kb) mRNA. This mRNA was also expressed in thyroid and was much less abundant in liver, kidney, placenta, tonsil, and spleen. hTR alpha 1 is represented by two mRNAs with sizes of 6.0 and 3.2 kb. The 6.0-kb mRNA was constantly less abundant than the 3.2-kb mRNA. hTR alpha 2 was detected as a single mRNA with a size of 3.2 kb, using a probe unique for this mRNA. Both hTR alpha 1 and hTR alpha 2 were strongly expressed in brain, prostate, and thyroid and much less in other tissues. The relative amounts of the three hTR mRNAs were roughly parallel in each tissue. It is of interest that none of these hTRs was abundant in liver, which is the major thyroid hormone-responsive organ. Another hTR may be present in liver.

The localisation of intracellular immunoglobulin and alpha-1-antitrypsin by immunoelectron staining of post-osmicated, resin-embedded tissue.

An indirect immunoperoxidase method is described to demonstrate intracellular immunoglobulins and alpha-1-antitrypsin in semithin and ultrathin sections from human tissue. The tissue was primarily fixed in glutaraldehyde, post-osmicated and resin-embedded.

Sensitivity and specificity of immunohistological methods on freeze-dried paraffin sections.

The application of freeze-dried paraffin sections for immunohistology eliminates many of the problems associated with the cryostat technique: the sections are thin sufficiently, the cell morphology is improved, there are no diffusion artifacts and the intensity of immunohistological reactions is superior to that obtained on cryostat sections. The preservation of antogenicity in freeze-dried paraffin sections is sufficient for the demonstration of light chain monotypia in lymphomas of B-cell origin. Indirect immunofluorescence is the method of choice for demonstrating surface Ig in freeze-dried paraffin sections.

Histochemical studies on lectin binding in reactive lymphoid tissues.

Using the avidin-biotin-labeled peroxidase complex (ABC) method, the staining reaction of a panel of 12 biotin-labeled lectins was studied in formalin-fixed, paraffin-embedded reactive lymph nodes and tonsils. Varying degrees of lectin binding were observed in lymphoid cells and macrophage-histiocytes with Concanavalin ensiformis (Con A), Lens culinaris (LCA), Phaseolus vulgaris (PHA), Pisum sativum (PSA), Ricinus communis (RCA), and Triticum vulgaris (WGA) agglutinins, but no evidence of binding was observed with Dolichos biflorus (DBA), Bandieraea simplicifolia (BSA), Arachis Hypogaea (PNA), Glycine soja (SBA), Sophora japonica (SJA), and Ulex europaeus (UEA) agglutinins. Three major patterns of binding were seen: the reaction products occurred along the plasma membranes (membranous), were confined to one pole of the cell membrane (cap-like), or were present diffusely in cytoplasm (cytoplasmic). The cells showing membranous and cap-like staining patterns corresponded to the lymphoid cells, as did the cytoplasmic to plasma cell and macrophage-histiocytes. Cap-like staining was observed on the lymphocytes at B and T cell areas with all six lectins. Thus, the presence of cap-like staining may not be useful for discrimination between B and T cells. Membranous staining, in contrast, was limited to lymphocytes of follicles (B cells) with PSA and LCA, and to germinal center cells with PHA, WGA, Con A, and RCA also reacted with the membrane of T-cell. The cytoplasmic staining reaction of macrophage-histiocytes varied markedly from one lectin to the other. Our study indicates that the carbohydrate moiety of the cells retains their binding sites for lectins through routine processing, providing a means of valid retrospective studies. Furthermore, these observations suggest that each lectin, despite its identical inhibitory sugar, should be tested for its unique reaction pattern, which is not predictable from the data derived from cell suspension studies.

The labeled antigen method of immunoenzymatic staining.

A two stage immunohistological technique (the "labeled antigen" procedure) has been assessed for the detection of a variety of human and animal cytoplasmic constituents in tissue sections. In this method specific antiserum is followed by antigen complexed to horseradish peroxidase or to alkaline phosphatase. The primary antibody acts bivalently, linking the labeled antigen to antigen in the tissue section. The major advantage of this technique is that nonantigen specific antibody in the primary antiserum cannot cause nonspecific staining since it has no affinity for the antigen:enzyme complex. Consequently the specificity of the reaction is assured, background staining is minimized and the total staining time (from wax section to mounted slide) can be reduced to as little as 30 min. Further advantages include the possibility of labeling Ig allotypes and the high efficiency of enzyme utilization. Covalent human IgG:horseradish peroxidase complexes can also be used in a triple sandwich in conjunction with human anti-viral or autoimmune antibodies.

Anatomical distribution of call antigen expressing cells in normal lymphatic tissue and in lymphomas.

Anatomical distribution of common acute lymphoblastic leukemia antigen (CALLA) was studied in lymphomas as well as in normal lymphatic organs using the monoclonal antibody VIL-A1. Twelve lymphomas were labelled by VIL-A1. Three of the 12 tumours also had T-cell marker, six lymphomas also showed immunoglobulin staining and only three tumours were pure CALLA lymphomas. Tonsils showed a distinct CALLA labelling of many germinal centre cells and of singular cells in interfollicular T-cell regions. Children's thymuses showed rare distinctly labelled cells in the cortex and medulla and slightly more cortical cells stained faintly by VIL-A1. Foetal thymuses of about the twelfth week of gestation contained many heavily labelled cells. The findings are discussed as evidence for the presence of CALLA on immature B as well as T lymphocytes. They favour the idea of CALLA as a common lymphocyte differentiation antigen although other possibilities of interpretation are also discussed.

A new approach to enzyme histochemical analysis of biopsy specimens.

A novel technique combining the freeze drying and embedding in glycol methacrylate at low temperature of tissue permitted the histochemical demonstration of a variety of enzymes, showing maintenance of enzyme activity, accurate enzyme localisation without apparent diffusion, and excellent morphological detail. The results obtained with this new approach were superior to standard techniques used for both enzyme histochemical and morphological studies. Moreover, blocks of the embedded tissue were stored for at least one year at room temperature without loss of enzyme activity. This method should find a wide range of applications in histopathology.

Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted.

[Assessment of the intensity of lymphocyte blast transformation in the crypts of the palatal tonsils by using DNA cytophotometry]. / Otsenka intensivnosti blastnoi transformatsii limfotsitov v kriptakh nebnykh mindalin s pomoshch'iu tsitofotometrii DNK.

Cell fractions isolated from lacunae of palatine tonsils with different functional activities were taken from 40 children 6-15 years of age. The cell fractions were examined using vital phase-contrast microscopy, light microscopy of Pappenheim-stained smears and the Feulgen cytophotometry. Lymphocytes with signs of immunological involvement were shown to constitute 95 per cent of the whole cell lacunar population of the active functional tonsils. 35 per cent of lymphocytes, being in different stages of blast transformation, were found. The most intense blast transformation was revealed in patients with tonsil inflammation. 5-13 per cent of lacunar nuclei with DNA content above the diploid level were found in these patients. Some multicellular islets accumulating small lymphocytes, lymphocytes at different stages of blast transformation, and macrophages were revealed to imitate the cooperation of immunocompetent cells. The data obtained can be helpful in the practical surgery of palatine tonsils.