Terminal deoxynucleotidyl transferase (TdT) enzyme in thymus and bone marrow: I. Age-associated decline of TdT in humans and mice

This study was aimed at characterizing terminal deoxynucleotidyl transferase (TdT) levels in populations of normal human and murine lymphocytes and toward correlating TdT enzyme levels with the biological process of aging. A newly developed method that utilizes a small number of cells was employed to determine TdT levels in bone marrow and thymus cells following cell fractionation at unit gravity sedimentation. By these methods, cell fractions with high TdT activity were found to comprise only 5–10% of the parent cell pools. In the human bone marrow, we show here that TdT-positive cell fractions are largely depleted of HTLA, E-rosette forming, and mitogen-responsive cells, whereas TdT-positive human thymocyte fractions contain a high percentage of HTLA and E-rosette-positive cells. Our observations in the murine model confirm the earlier observations that TdT activity decreases with age. We further show here that the age-associated decline of TdT in the bone marrow preceded that in the thymus. As is true for the mouse, TdT activity in human bone marrow and thymus was also found to decrease with advancing age. The decline in TdT was not associated with a change in cell distribution profiles after unit gravity sedimentation of bone marrow or thymus cells. From these data, the age-associated loss of TdT cannot be attributed to a loss of a particular subpopulation of cells.     

雌激素受体在小鼠睾丸表达的免疫组织化学研究

观察雌激素受体在小鼠睾丸的定位与分布。取Ａ／Ｊ系小鼠睾丸, 制备石蜡切片。用间接酶标免疫组织化学和高温处理抗原暴露技术显示雌激素受体的所在部位。睾丸所有Ｌｅｙｄｉｇ 细胞和约２０％ 的肌样细胞的胞核呈雌激素受体阳性反应。睾丸支持细胞和生精细胞为阴性。本研究首次用免疫组织化学技术证明了睾丸中雌激素受体的存在,并定位于Ｌｅｙｄｉｇ 细胞和部分肌样细胞的胞核。为研究雌激素对雄性生殖功能的调节提供了形态学依据。     

Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue

We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.     

Localization of eosinophils in the thymus by the peroxidase reaction

Summary Thymuses of human fetuses and infants and of young mice were investigated histochemically for peroxidase. Eosinophils were shown to be the only peroxidase-positive cells in the thymus.In human thymuses the eosinophilic cells were predominantly localized in medullar areas, with concentration of cell clusters at the cortico-medullar junction, around or inside Hassall's bodies and occasionally in high numbers in the intraseptal vessels of the cortex.In the normal mouse the eosinophils were evenly distributed throughout the medulla.Treatment with corticosteroids or X-rays produced a severe involution of the thymus with concommitant change in cellular pattern. The central areas of the thymus residue contained lymphocytes while the peripheral regions consisted of reticuloepithelia, macrophages and numerous eosinophils.Azathioprine did not change the morphology of the thymus. The numbers of eosinophils were slightly reduced, the distribution pattern remaining unchanged.     

Quantitative microphotometric studies of Feulgen-stained nuclei on sections of the human uterine cervix: optimization of preparation and measuring technique

The total extinction of apparently normal intermediate cell nuclei was microphotometrically measured in formalin-fixed Feulgen-stained paraffin sections of the uterine cervix. We investigated whether reproducible microphotometric results can be obtained from a histologic section expected to contain a certain amount of sectioned nuclei and stained by a standard technique. The results have shown that exact reproduction of microphotometrically measured mean total extinction values of intermediate cell nuclei can be achieved with a threshold value of 90% transmission in 10 microns sections.     

Histochemical Demonstration of DNA Double Strand Breaks by in Situ 3'-tailing Reaction in Apoptotic Endometrium

A new histochemical technique, called in situ 3'-tailing reaction (ISTR), to detect DNA double strand breaks (DSB) was developed and applied to tissue sections of apoptotic endometrium. To demonstrate DSB, biotin-labeled and unlabeled dATPs with terminal deoxynucleotidyl transferase (TdT) were added to the many 3-hydroxyl termini of DNA fragments generated in the apoptotic cells. For an efficient 3'-end labeling, it was necessary to treat the sections with λ-exonuclease (λEx) prior to the TdT reaction to generate 3'-protruding ends. The λEx-TdT reaction specifically labeled nuclear fragments in the apoptotic cells in paraformaldehyde fixed frozen sections. In paraffin sections, pretreatment with proteinase K was effective for 3'-tailing reaction. ISTR should be a useful tool for detecting dying cells in both physiological and pathological states.     

Comparison of monoclonal antibodies PC 10 and MIB 1 on microwave-processed paraffin sections

Abstract. Antibodies against the proliferation-associated nuclear antigen (PCNA) and against the Ki-67 protein are widely used as operational proliferation markers in human tumour diagnostics. The original Ki-67 antibody had the inherent drawback in that it could only be used when fresh-frozen material was available. The antibody PClO was supposed to offer the advantage that it could be applied on formalin-fixed and paraffin-embedded tissues. However, in cases in which the formalin fixation exceeded 4 h, PC10 staining proved to be inconsistent and often failed.
The aim of this study was to compare a recently prepared Ki-67 equivalent monoclonal antibody (MIB 1) and PC10 in routinely fixed histopathological material using antigen retrieval by microwave processing.
Antibody MIB 1 stained the nuclei of cells known to belong to the proliferative compartments in microwave-processing paraffin sections of formalin-fixed tissues. Quiescent cells were consistently negative for MIB 1 staining. In contrast, PC10 was positive in almost all nuclei of different tissues in microwave-treated paraffin sections. Thus, antigen retrieval by microwave processing is beneficial for the detection of the Ki-67 protein in paraffin sections, whereas it is not needed for the detection of the PCNA.     

MONONUCLEAR PHAGOCYTES (KUPFFER CELLS) AND ENDOTHELIAL CELLS : Identification of Two Functional Cell Types in Rat Liver Sinusoids by Endogenous Peroxidase Activity

The fine structural characteristics and phagocytic properties of peroxidase-positive and peroxidase-negative cells in rat hepatic sinusoids were investigated. Cells with a positive peroxidase reaction in the endoplasmic reticulum and the nuclear envelope make up approximately 40% of cells in rat hepatic sinusoids and have abundant cytoplasm containing numerous granules and vacuoles, and occasional tubular, vermiform invaginations. After intravenous injection of colloidal carbon, the luminal plasma membrane of these cells shows continuous sticking of carbon, and there is evidence of avid phagocytosis of colloidal carbon particles. Peroxidase-positive cells are the only cells in hepatic sinusoids which phagocytize large (0.8 µ in diameter) latex particles. In contrast, the peroxidase-negative endothelial cells, which make up 48% of cells, have scanty perinuclear cytoplasm and organelles, and their long cytoplasmic extensions that form the lining of the hepatic sinusoids have fenestrations; these cells ingest small amounts of colloidal carbon, principally by micropinocytosis, exhibit no sticking of carbon particles to their plasma membranes, and do not ingest the larger (latex) particles. The so-called fat-storing cells are peroxidase negative and totally nonphagocytic. The peroxidase reaction thus distinguishes the typical mononuclear phagocytes or Kupffer cells of rat liver from the endothelial-lining cells.     

Nuclear matrix bound terminal deoxynucleotidyl transferase in rat thymus nuclei. I. A possible site for TdT mediated function

Approximately 80% of the terminal deoxynucleotidyl transferase (TdT) in thymus glands from 3–4 week old rats was found to be localized in the nucleus and the remaining 20% in the cytosol. Following endogenous nuclease digestion of the thymus nuclei, 70–85% of the nuclear TdT could be removed by low salt and high salt extractions, whereas 15–30% of the enzyme remained tightly bound to the residual nuclear matrix. Low salt and high salt extracts of the nuclei contained a mixture of 58, 56, 45 and 44 kDa species of TdT whereas only 58 kDa species of the enzyme was found to be associated with the matrix. In addition to TdT, 20–25% of the nuclear DNA polymerase was also tightly bound to the isolated nuclear matrix. These observations lead us to propose that besides being the site of DNA replicationvia-matrix bound replicational complexes [Van der Velden H.M.W. & Wanka F., Molecular Biology Reports 12 (1987): 69], nuclear matrix may also be the site of TdT mediated function and that matrix bound TdT and free TdT could be the functional and nonfunctional forms of the enzyme, respectively, in the thymus gland.Abbreviations dNTP deoxyribonucleoside triphosphate - DTT dithiothreitol - Ig immunoglobulin - PMSF phenylmethylsulfonylfluoride - rNTP ribonucleoside triphosphate - SDS sodium dodecyl sulphate - TCR T cell receptor - TdT terminal deoxynucleotidyl transferase - VDJ variable, diversity and joining segments of Ig or TCR genes     

A rapid and simple staining method, using Toluidine Blue, for analysing mitotic figures in tissue sections

Summary Mitotic index is a clinically important parameter in cancer pathology. We developed a staining method using Toluidine Blue to detect efficiently and rapidly mitotic figures in sections of formalin-fixed paraffin-embedded human and rat tisues. Sections were stained at acid pH with a 0.01% Toluidine Blue solution after removal of RNA with hydrochloric acid or ribonuclease. The optimal pH of the TB staining solution was found to be 4.5 for rat tissues and 3.5 for human tissues. This procedure stained mitotic figures much more intensely than other (extra)cellular structures. A quantitative estimate of the total number of nuclei in the field where mitotic figures were counted, was obtained in an adjacent section hydrolysed in 5 N hydrochloric acid and stained by the Feulgen reaction with a Schiff-type reagent containing 0.01% Toluidine Blue. This method specifically stained interphase and mitotic nuclei and the field cellularity could be quantified by image cytometry. When these procedures were performed on two consecutive serial sections, a mitotic index could be determined accurately by relating the count of mitotic figures to the number of tumour cells.     

B lymphocyte-associated antigens on terminal deoxynucleotidyl transferase-positive cells and pre-B cells in bone marrow of the rat

To investigate early stages of B lymphocytopoiesis in rat bone marrow (BM) before the expression of surface IgM (s mu), the populations of cytoplasmic mu-chain-positive (c mu+) pre-B cells and terminal deoxynucleotidyl transferase-positive (TdT+) cells were studied by double immunofluorescence microscopy. B lymphocytes that were s mu+ constituted 5%, c mu+s mu- pre-B cells 23%, and TdT+ cells 4% of nucleated cells in the BM of juvenile rats. TdT+ and pre-B cells ranged between 7 and 17 microns in diameter. TdT+ cells were slightly larger, with a modal diameter of 10.5 microns against 9 microns for pre-B cells. mu-Chains were absent from nearly all TdT+ cells. Their surface antigenic phenotype was studied by using a panel of mouse monoclonal antibodies (MAb) to rat B lymphocyte-associated antigens (Ig, Ia, and others) and T lymphocyte-associated antigens. Both pre-B cells and TdT+ lacked surface Ig and Ia but carried most of the other B lymphocyte-associated antigens analyzed. TdT+ and pre-B cells lacked those antigens found only on the T lineage. By using MAb HIS24 (detecting a non-Ig/Ia B lymphocyte-associated antigen) and fluorescence-activated cell sorting, TdT+ and pre-B cells were highly enriched. The results show that most TdT+ cells in rat BM are mu- but demonstrate strong similarity with pre-B cells in surface antigenic phenotype. Therefore, as suggested for man, a major proportion of rat BM TdT+ cells may be B lineage-cells before mu heavy chain gene expression.     

APPEARANCE AND FUNCTION OF ENDOGENOUS PEROXIDASE IN FETAL RAT THYROID

Iodination within the thyroid follicle is intimately associated with a thyroid peroxidase. In order to locate the in vivo site of iodination, the initial cytochemical appearance of this enzyme has been determined in fetal rat thyroid and its presence correlated with the onset of iodinated thyroglobulin synthesis. Peroxidase first appears in follicular cells during the 18th day of gestation. It is seen first in the perinuclear cisternae, the cisternae of the endoplasmic reticulum, and within the inner few Golgi lamellae. These organelles presumably represent sites of peroxidase synthesis. During the 19th and 20th days of gestation, there is a tremendous increase in peroxidase activity. In addition to the stained sites described, there are now many peroxidase-positive apical vesicles in the follicular cells. Newly forming follicles stain most conspicuously for peroxidase, the reaction product being heavily concentrated at the external surfaces of apical microvilli and in the adjacent colloid. Iodinated thyroglobulin becomes biochemically detectable in thyroids during the 19th day of gestation and increases greatly during the 20th day. The parallel rise in peroxidase staining that just precedes, and overlaps, the rise in iodinated thyroglobulin, suggests that apical vesicles and the apical cell membrane are the major sites of iodination within the thyroid follicle.     

Immunohistochemical detection of strain-specific major histocompatibility complex class I antigens in paraffin-embedded rat osteochondral tissue

Summary An immunohistochemical method using formalin-fixed, paraffin wax-embedded sections is described for detecting strain-specific major histocompatibility complex class I antigens in knee-joint tissue from DA and Lewis strains of rat. The fixed osteochondral tissues were additionally decalcified in formic acid before processing for paraffin wax embedding. For immunohistochemistry, two monoclonal antibodies, one specific for DA class I allele RT1Aa and the other for Lewis class I allele RT1A1, were used together with the avidin-biotin immunoperoxidase procedure. It was necessary to use strain-specific normal rat serum as a diluent for the antibodies to suppress cross-strain recognition. DA-specific antibody stained positively only on DA rat sections, not on Lewis rat sections, and Lewis-specific antibody stained positively only on Lewis rat sections, and not on DA. Positive staining was localized in the bone marrow, osteochondral cells and endothelium. We propose that the use of a decalcification medium may have enhanced the immunoreactivity of the tissue. The method described can be used on sections of allografts from the two strains of rat to assess morphologically the extent of cellular replacement of the graft by the host's cells.     

Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis

Apoptotic cells in rat thymus were labeled in situ in paraffin-embedded and frozen tissue sections by ligation of double-stranded DNA fragments containing digoxigenin or Texas red. Two forms of double-stranded DNA fragments were prepared using the polymerase chain reaction: one was synthesized using Taq polymerase, which yields products with single- base 3' overhangs, and one using Pfu polymerase, which produces blunt- ended products. Both types of fragment could be ligated to apoptotic nuclei in thymus, indicating the presence in such nuclei of DNA double- strand breaks with single-base 3' overhangs as well as blunt ends. However, in nuclei with DNA damage resulting from a variety of nonapoptotic processes (necrosis, in vitro autolysis, peroxide damage, and heating) single-base 3' overhangs were either nondetectable or present at much lower concentrations than in apoptotic cells. Blunt DNA ends were present in such tissues, but at lower concentrations than in apoptotic cells. In contrast, in all of these forms of DNA damage, nuclei contained abundant 3'-hydroxyls accessible to labeling with terminal deoxynucleotidyl transferase. Thus, although single-base 3' overhangs and blunt ends are present in apoptotic nuclei, the specificity of the in situ ligation of 3'-overhang fragments to apoptotic nuclei indicates that apoptotic cells labeled in this way can readily be distinguished from cells with nonapoptotic DNA damage. These data are consistent with the involvement of an endonuclease similar to DNase I in apoptosis, which is predicted to leave short 3' overhangs as well as blunt ends in digestion of chromatin.     

Simultaneous immunohistochemical demonstration of antigen expression and 5-bromodeoxyuridine incorporation in plastic embedded sections

Summary In this study a double immunohistochemical staining procedure is described for the simultaneous demonstration of antigen expressing cells and replicating cells in rat thymus. As markers for cell surface antigen expression a monoclonal antibody against Ia-expressing cells (His 19) and a monoclonal antibody against cells of the monocyte-macrophage lineage (ED2) were used. Replicating cells were demonstrated by the incorporation of 5-bromodeoxyuridine (BrdUrd). Tissue pieces were fixed in a periodate-lysine-paraformaldehyde fixative and embedded in glycol methacrylate. To demonstrate Ia-expressing cells or ED2-positive macrophages in plastic embedded sections a digestion with trypsin is necessary. The staining procedure was applied sequentially and was performed with a peroxidase and an alkaline phosphatase labeled reagent yielding respectively a brown and a blue reaction product. Results with this staining procedure on plastic embedded sections of rat thymus, an organ with a high DNA synthesizing capacity, showed incorporation of BrdUrd predominantly in the cortex. ED2-positive macrophages were only found in the cortex. The la-positive epithelial reticular cells demonstrated extremely well their stellate form.     

Simultaneous immunohistochemical demonstration of antigen expression and 5-bromodeoxyuridine incorporation in plastic embedded sections

In this study a double immunohistochemical staining procedure is described for the simultaneous demonstration of antigen expressing cells and replicating cells in rat thymus. As markers for cell surface antigen expression a monoclonal antibody against Ia-expressing cells (His 19) and a monoclonal antibody against cells of the monocyte-macrophage lineage (ED2) were used. Replicating cells were demonstrated by the incorporation of 5-bromodeoxyuridine (BrdUrd). Tissue pieces were fixed in a periodate-lysine-paraformaldehyde fixative and embedded in glycol methacrylate. To demonstrate Ia-expressing cells or ED2-positive macrophages in plastic embedded sections a digestion with trypsin is necessary. The staining procedure was applied sequentially and was performed with a peroxidase and an alkaline phosphatase labeled reagent yielding respectively a brown and a blue reaction product. Results with this staining procedure on plastic embedded sections of rat thymus, an organ with a high DNA synthesizing capacity, showed incorporation of BrdUrd predominantly in the cortex. ED2-positive macrophages were only found in the cortex. The Ia-positive epithelial reticular cells demonstrated extremely well their stellate form.     

Cellular composition and ultrastructure of the thymus of the newborn mouse after immunization of pregnant females with homologous brain antigens

Immunization is performed with 20% of water-saline extract of medulla oblongata on the 6th, 8th, 10th days of pregnancy (1 g per 200 g body mass). In the thymus cortical substance of newborn rats no statistically significant difference in content of small lympocytes, lymphoblasts and large lymphocytes, mitotically deviding cells is revealed as compared to the normal. The part of middle lymphocytes decreases up to 5.7 +/- 0.7% (control--10.0 +/- 1.7%). The content of distroying cells and fagocytic macrophages is increasing. In cytoplasm of one macrophage several fagocyted degenerating cells with pyknotic nuclei and destructively altered organells are often present. In the interlobular connective tissue an increased amount of degenerating forms of mast cells is noted. In the thymus medullary substance small lymphocytes are growing in number. Certain changes in vessels of the microcirculatory bed are revealed.     

Human archival tissues provide a valuable source for the analysis of spatial genome organization

Sections from archival formalin-fixed, paraffin wax-embedded human tissues are a valuable source for the study of the nuclear architecture of specific tissue types in terms of the three-dimensional spatial positioning and architecture of chromosome territories and sub-chromosomal domains. Chromosome painting, centromeric, and locus-specific probes were hybridized to tissue microarrays prepared from formalin-fixed paraffin wax-embedded samples of pancreas and breast. The cell nuclei were analyzed using quantitative three-dimensional image microscopy. The results obtained from non-neoplastic pancreatic cells of randomly selected individuals indicated that the radial arrangement of the chromosome 8 territories as well as their shape (roundness) did not significantly differ between the individuals and were in accordance with assumptions of a probabilistic model for computer simulations. There were considerable differences between pancreatic tumor and non-neoplastic cells. In non-neoplastic ductal epithelium of the breast there was a larger, but insignificant, variability in the three-dimensional positioning of the centromere 17 and HER2 domains between individuals. In neoplastic epithelial breast cells, however, the distances between centromere and gene domains were, on average, smaller than in non-neoplastic cells. In conclusion, our results demonstrate the feasibility of studying the genome architecture in archival, formalin-fixed, paraffin wax-embedded human tissues, opening new directions in tumor research and cell classification.     

The localization of endogenous peroxidase in the lacrimal gland of the rat during postnatal development. Electron microscope cytochemical and biochemical studies

The distribution of endogenous peroxidase activity in the lacrimal gland of the rat during postnatal development was investigated by electron microscope cytochemistry Peroxidase activity is first found 6 hr after birth in only a few acinar cells At this stage, reaction product fills only localized segments of the scant rough endoplasmic reticulum and of the perinuclear cisternae. Peroxidase activity thus develops asynchronously in a given cell as well as in the secretory cell population as a whole 2 days after birth, all cisternae of the rough endoplasmic reticulum of a peroxidase-positive cell contain reaction product, but the majority of the acinar cells is still negative During the next days, the number of peroxidase-positive cells and the amount of the rough endoplasmic reticulum increase rapidly. By 15 days postparturition, all secretory cells are peroxidase-positive. Reaction product is then found in all cisternae of the rough endoplasmic reticulum including the perinuclear cisternae, in smooth surface vesicles located mainly between the rough endoplasmic reticulum and the Golgi stacks, in condensing vacuoles, and in all secretory granules The Golgi cisternae rarely contain reaction product In total homogenates and in fractions of glandular tissue of adult rats, peroxidatic and catalatic activities are demonstrable. The microsomal fractions and the postmicrosomal supernatants were used to separate peroxidase from catalase by precipitation with ammonium sulfate, and the following parameters were determined: substrate (H₂O_2-) optimum (∼ 2.0 x 10^-4 M), pH-optimum (pH 6 5), temperature-optimum (42°C), and the absorption maximum (415 nm before and 425 nm after addition of H₂O₂) The same parameters were obtained from lacrimal fluid peroxidase. Both peroxidase from lacrimal gland and that from lacrimal fluid are almost completely inhibited by 10^-3 M aminotriazole and are possibly identical enzymes. Peroxidase is secreted into lacrimal fluid, which does not contain catalase.     

Selective Demonstration of Histidine

Mounted paraffin sections of formalin-fixed tissue are treated for 24 hr at room temperature in an iodine solution (0.3% iodine, 0.6% potassium iodide) at pH 10 to block the aromatic nuclei of tyrosine and tryptophane. A coupled tetrazonium reaction using naphthanil diazo blue B (tetrazotized o-dianisidine) as a 0.1% solution at pH 9.2 for 15 min at 4°C, as the first coupling agent, and H acid (8-amino-1-naphthol-3, 6-dissulfonic acid), as a 2% solution at pH 9.2 for 15 min at 4°C, as the second coupling agent, stains sites of histidine a red-brown to red-purple color.