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.

Allele frequencies and segregation of human polymorphic keratins K4 and K5.

Two electrophoretic variants for each of the human keratins K4 and K5 that are expressed in squamous nonkeratinizing epithelia lining the upper digestive tract could be distinguished on SDS-PAGE. Based on a sampling size of 1,299 unrelated individuals, calculation of allele frequencies showed the alleles to be in Hardy-Weinberg equilibrium. The genetic basis of this variation was confirmed by both quantitative gene dosage dependence and the transmission of the variants as Mendelian traits in two families. Thus the human keratin genes K4 and K5 are polymorphic, and each presents with two codominant alleles (a and b).

In situ hybridization demonstration of poly-adenylated RNA sequences in formalin-fixed paraffin sections using a biotinylated oligonucleotide poly d(T) probe.

An in situ hybridization technique has been developed for assessing poly(A)+ RNA preservation in routine pathology specimens. The method detects poly-adenylated RNA sequences in tissue sections using a biotinylated polydeoxythymidine (poly d(T)) probe. The probe was prepared from single-stranded 25-30 base oligo d(T) and was biotinylated using the enzyme terminal deoxynucleotide transferase with biotin-11-dUTP and dTTP in the ratio 1:4. The hybridization protocol uses varying concentrations of proteinase K to unmask mRNA sequences and the biotin-labelled hybrids are demonstrated after hybridization under standard conditions by the application of streptavidin and biotinylated alkaline phosphatase. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results have confirmed that the method is specific for poly(A)+ RNA and shows that poly(A)+ RNA can be demonstrated in routine formalin-fixed sections using non-radioactive techniques with retention of morphology. It also provides a means of optimizing the hybridization conditions for specific mRNA probes and produces a staining pattern demonstrating the relative level of poly(A)+ RNA per cell which may reveal new information about cell activity and tissue function.

Flow cytometric analysis of the Hermes homing-associated antigen on human lymphocyte subsets.

The homing of lymphocytes is controlled by interactions with high endothelial venules (HEV), specialized vessels that define sites of lymphocyte extravasation into lymph nodes and inflamed tissues. In humans, lymphocyte-HEV binding involves a lymphocyte surface glycoprotein (GP) of 85 to 95 kd (CD44, H-CAM), defined by monoclonal antibody (MoAb) Hermes-1. To define the expression of this homing-associated adhesion molecule during human lymphocyte development, we performed two-color immunofluorescence analyses of human bone marrow (BM), thymus, peripheral blood (PB), and tonsillar lymphocytes. The highest levels of Hermes-1 antigen are displayed by circulating B and T cells in the blood, which are uniformly positive and bear roughly twice the level of antigen present on mature lymphocytes within organized lymphoid tissues and BM. "Immature" (CD4+, CD8+) T cells in the thymus are Hermes-1lo to-, whereas thymocytes of mature phenotype (CD4+ or CD8+) are positive. The Hermes-1 antigen is present at high levels on the same population of thymocytes that bears high surface levels of CD3, a component of the T-cell antigen receptor complex, suggesting that levels of T-cell homing and antigen receptors characteristic of mature peripheral T cells appear coordinately during thymocyte maturation/selection. Essentially all T cells in the periphery are Hermes-1hi, including T blasts, and the homing-associated antigen is maintained at high levels on T cells stimulated in vitro by phytohemagglutinin (PHA) and on interleukin-2 (IL-2) maintained T-cell clones and lines. In contrast, although most resting IgD+ B cells are positive a significant fraction of B cells in tonsils are Hermes-1lo to-; these cells are predominantly PNAhi, IgD-, and CD20hi, a phenotype characteristic of sessile, activated B cells in germinal centers. In all lymphocyte populations examined, there is a linear correlation in staining for Hermes-1 and for Hermes-3, an antibody that defines a distinct functionally important epitope on this molecule. The results demonstrate a precise regulation of this homing-associated antigen during lymphocyte differentiation.

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.

Characterization of the human C1q receptor.

At the meeting new procedures were reported for the isolation of the receptor which binds to the collagen-stalks of C1q, a subcomponent to the macromolecular complex, C1. The C1q-receptor was isolated from human tonsil cells, by a two-step procedure which did not involve affinity chromatography on C1q-Sepharose. In solution the C1q-receptor from tonsil cells behaved as an elongated dimer of two approx. 60 kDa chains. A C1q-receptor preparation isolated from human phagocytes, using pepsin-digested C1q for affinity chromatography, was found to be predominantly a molecule of approx. 120 kDa as assessed by SDS-PAGE. The amino acid compositions of C1q-receptor preparations derived from Raji (B-lymphoblastoid) and U937 (monocytic) cell-lines were found to be very similar. These results suggest that the human C1q-receptor may be a one-chain molecule, and also raise the possibility that there is more than one type of C1q receptor on cell surfaces.

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.

Expression and functional role of CD23 on T cells.

We have found that approximately 10%-15% of tonsil, but not peripheral blood, T cells express the CD23 antigen following activation with 12-O-tetradecanoylphorbol 13-acetate (TPA), phytohemagglutinin (PHA) or recombinant interleukin 4. The proliferative response of tonsil T cells is significantly increased when CD23 monoclonal antibodies (mAb) are present in the cultures. In contrast, no such proliferative augmentation is seen when peripheral blood T cells are cultured in this way. Supernatant (SN) of Epstein-Barr Virus-transformed B lymphoblastoid cell lines (EBVLCL), is found to have a similar co-stimulatory effect on the proliferation of tonsil T cells to that seen with CD23 mAb. This effect is greatly diminished by preclearing SN with CD23 mAb. Similarly, SN from a CD23+ L cell transfectant augments the proliferative response of tonsil T cells to both TPA and PHA. The CD23 molecule expressed by TPA-driven T cell blasts appears identical in size to the 45-kDa glycoprotein present on EBVLCL and activated B cells. In contrast, a 42-kDa molecule is observed when CD23 is precipitated from T cells activated with PHA. The results presented here demonstrate that CD23 is expressed on activated tonsil, but not peripheral blood T cells and plays a role, via the binding of CD23 mAb and CD23+ material, present in EBVLCL and CD23+ transfectant SN, in the regulation of T cell proliferation in response to mitogens such as PHA and TPA.

Immunohistochemical investigations of the human palatine tonsil from surgical specimens using polyclonal and monoclonal antikeratin antibodies.

The keratinization process of tonsillary epithelium in patients with different age, who underwent surgery because of chronic inflammatory processes, has been studied by employing low (40 KD), medium (52-56-58 KD), high (56-64 and 68 KD) as well as broad spectrum antikeratin antibodies. The findings thus obtained outline the various keratin patterns of the tonsil in the outer covering and, even more, in the crypt labyrinth wall. These findings have been compared with those obtained by histochemical reactions for acid phosphatase and non-specific esterase activities (Favrz et al. 1986 II). In crypt epithelium keratinization, 56-64 KD keratins are involved in cells where also high enzymic activities are taking place, namely 1. in those elements delimiting the lumen as well as in those belonging to the reticulum mesh which are nearest to it and host lymphoid elements active in immunity reactions; 2. in elements at the bottom of small and large crypts, where excavation continues into the lymphoid tissue and interfollicular spaces. 68 KD keratin, although present in lower amounts, is synthesized almost everywhere, but only irregularly. It marks surface epithelium surrounding some dermal papillae, some meshes of the crypt reticulum, the "gallery" bottom as well as elements of interfollicular proliferation developing in the lymphoid tissue like arborescence. The path of congested blood vessels in the crypt wall towards the surface is marked by keratinized epitheliocytes. 56-64 KD keratin is present in the most superficial elements and is generally accompanied by the wall growing thinner, its blistering and breaking. The most frequent pathologic alterations of the epithelium (like cellular hypertrophy) involve variations in cytoplasm keratin pattern, in still un-flattened polyhedral elements of the intermediate layers.

Transfer factor therapy of thoracic sarcoidosis.

The authors repeatedly treated 59 patients with thoracic sarcoidosis with transfer factor (TF) since 1976. They utilized this therapy with TF from human tonsil lymphocytes (TFh) on account of the ineffectiveness of the corticosteroid treatment, because of the side effects of the corticosteroids, and as primary TF therapy, and to test an animal TF preparation from pig tonsil lymphocytes (TFp). In their observations only fraction II of the dialysable leukocyte extract was sufficient. Differences in the effectiveness between TFh and TFp do not exist on the whole. Our conclusion is that TF can stimulate the immunosystem of the patients, and can be an important mode of treatment. The mode of action is not clear.

Determination of 2'-acetyl erythromycin and erythromycin in human tonsil tissue by HPLC with coulometric detection.

A simple and sensitive high-performance liquid chromatographic method was developed for the determination of 2'-acetyl erythromycin and erythromycin in human tonsil tissue. Methyl tert-butyl ether was used as the extraction solvent after alkalization of tissue homogenates. Separation was achieved on a reverse phase C-18 column. The mobile phase consisted of acetonitrile-methanol-tetrahydrofuran-sodium acetate buffer, pH 4.5. Eluted compounds were monitored by a coulometric detector in the oxidative screen mode. The quantitation limits of 2'-acetyl erythromycin and erythromycin were 0.20 and 0.30 mg/kg of tissue, respectively. The method was linear over the concentration range of 0.60-10.0 mg/kg of tissue for 2'-acetyl erythromycin and erythromycin.

[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.

Antigenic phenotype and functional characterization of human tonsil B cells.

Human tonsil cells were labeled with anti-leu-16, a monoclonal antibody (MoAb) that recognizes the CD20 antigen and that is specific for B cells. Two populations of B cells were identified by flow cytometry on the basis of antigen density. One labeled brightly with anti-Leu-16, and the other labeled at a level comparable to blood B cells. These two populations were characterized with a panel of MoAbs in two- and three-color flow cytometric studies and appeared to correspond to germinal-center and mantle-zone B cells. The pattern of staining of anti-Leu-16 on sections of frozen tonsil supported this characterization. Anti-Leu-16 labeled germinal center cells more intensely than mantle zone cells and stained a few scattered B cells in the interfollicular zone. The ability of each Leu-16+ population to secrete IgG and IgM in response to mitogens was measured in a particle immunofluorescence assay. Dim Leu-16+ B cells (small, resting B cells and a subpopulation of preactivated cells) secreted IgG and IgM in response to pokeweed mitogen (PWM) but only IgG in response to B cell growth factor (BCGF). Bright Leu-16+ B cells (small to large activated cells and possibly memory cells) did not respond to PWM but secreted IgG in response to BCGF. The functional responses of dim Leu-16+ and bright Leu-16+ B cells were consistent with their identification as mantle-zone and germinal-center B cells. Phenotypic identification and functional studies of mantle-zone and germinal-center B cells may help clarify the differentiation pathway within the germinal center.

Southern blot analysis of DNA extracted from formal-saline fixed and paraffin wax embedded tissue.

Model experiments were designed to assess whether DNA could be recovered from formol-saline fixed peripheral blood lymphocytes and tonsil tissue for use in Southern blot gene analysis. Lymphocytes were fixed for 30 min and tonsil for 6 and 24 h, then paraffin embedded. High molecular weight DNA was extracted by prolonged digestion (2-7 days) with proteinase K or protease XXIV in the presence of 1 per cent sodium dodecyl sulphate. Restriction, transfer and hydridization were possible without modification of standard procedures. Multiple copy sequences were demonstrated using Mspl and Bst Nl restriction and hybridization for the Y chromosome (pHY 2.1 probe), single copy genes using EcoRI and BamHl restriction for the T-cell receptor beta chain (T beta probe), and Bgl II and Hind III for the immunoglobulin heavy chain (JH probe). Identical banding to unfixed tissue was achieved except when 24 h fixed extracts were used. With these, demonstration of the 24 KB Bam Hl/T beta and 9.2 KB Hind III/JH bands was not obtained. These findings suggest that as the fixation time is extended, alterations to DNA will limit the available range of restriction enzyme/probe combinations. However, with careful choice of these the extraction of DNA from formalin fixed and paraffin embedded pathological tissue for Southern blotting should be profitable.

Concentrations of phenoxymethylpenicillin and cefadroxil in tonsillar tissue and tonsillar surface fluid.

Thirty patients who underwent elective tonsillectomy were given phenoxymethylpenicillin (0.8 g) or cefadroxil (1 g) at different times before operation. The concentrations of the antibiotics were analysed in serum, tonsillar tissue, fluid from the surface of the tonsils, and mixed saliva. The concentrations in tonsillar tissue for both drugs were much lower than the corresponding serum concentrations. This apparently low tissue accessibility could be ascribed to the limited intracellular penetration of beta-lactam antibiotics. For both antibiotics the concentrations in the tonsillar surface fluid were higher than the levels in the tissue and well above the minimal inhibitory concentrations for streptococci. This was not due to antibiotics in saliva but probably a result of leakage from the interstitial fluid. Inability to reach active concentrations of phenoxymethylpenicillin or cefadroxil at the site of infection does not therefore seem to be a probable cause for relapse after treatment of streptococcal tonsillitis.

Phenotypic expression of cells of stationary elements in human lymphoid tissues. A histochemical and immunohistochemical study.

The stationary elements of lymphoid tissues are composed of four types of cells: histiocytes, interdigitating reticulum cells, follicular dendritic cells, and fibroblastic reticulum cells. The phenotypes of these cells were determined with a large panel of monocyte/histiocyte, C3 receptor monoclonal antibodies, and others. Based on the monocyte-marker expression, histiocytes can be separated into two groups: (1) free histiocytes (monocyte-marker positive) and (2) fixed histiocytes (monocyte-marker negative). The former are characterized by the expression of monocyte markers, such as OK M1, Co Mo2, BRL Mol/Mo2, and Leu M3, whereas the latter are not. Interdigitating reticulum cells are localized in the T-cell zone. These cells are characterized by the expression of 1E9, 2H9, and Leu M1. Interdigitating reticulum cells and fixed histiocytes are similar in terms of marker expression and enzyme histochemistry. However, in interdigitating reticulum cells, the Leu M1 antigen is localized on the membrane, in contrast to histiocytes, in which it has a Golgi distribution. Follicular dendritic cells are present in germinal centers and mantle zones. These cells express complement (C3) receptors and several monocyte markers (including OK M1 and Co Mo2). Follicular dendritic cells are capable of trapping antigens onto their membranes. This unique property makes us reluctant to conclude that follicular dendritic cells are related to monocytes. Fibroblastic reticulum cells express BA-1 and alkaline phosphatase, and they form a dendritic network, especially in the T-cell zone. The results of this study demonstrate that immunoperoxidase staining with monoclonal antibodies can reveal the distribution of histiocytes and dendritic/reticulum cells in lymphoid tissues.

Cytoskeletal components of lymphoid organs. I. Synthesis of cytokeratins 8 and 18 and desmin in subpopulations of extrafollicular reticulum cells of human lymph nodes, tonsils, and spleen.

Using light and electron microscopic immunolocalization with antibodies to cytoskeletal proteins, we have characterized the nonlymphoid cells of various human lymphoid organs (lymph nodes, tonsils, spleen). In all these tissues, the lymphoid follicles contain a three-dimensional meshwork of "dendritic reticulum cells" which are characterized by the presence of desmosomal junctions, as demonstrated by positive punctate staining with antibodies to the desmosome-specific proteins desmoplakin I and desmoglein, and by intermediate-sized filaments (IFs) of the vimentin type only. In contrast, the extrafollicular regions are characterized by an extended meshwork of other types of reticulum cells, which also contain vimentin IFs but lack desmosomal proteins. In addition, a considerable, although variable proportion of these extrafollicular reticulum cells forms IFs containing cytokeratins 8 and 18 and/or desmin-containing IFs. The occurrence of cytokeratins 8 and 18 in lymph nodes has also been shown by gel electrophoresis and immunoblotting. Results of double-label immunolocalization indicate that some of the extrafollicular reticulum cells coexpress all three kinds of IF protein. A large proportion of these cells also synthesizes another marker of myogenic differentiation, i.e., the isoform of alpha-actin specific for smooth muscle. This proportion includes some cells that are negative for desmin. Comparison of the distribution of cells expressing cytokeratins and/or desmin with that of reticulum cells showing strong alkaline phosphatase activity (as a marker for the so-called "fiber-associated (fibroblastic) reticulum cells") suggests that the former represent a subset of the latter. The biological meaning of these different patterns of expression in reticulum cells and of the resulting cell-type heterogeneity as well as possible implications of these observations for tumor diagnosis, notably of lymph-node metastases and lymphomas, are discussed.

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.