Localization of surfactant-associated proteins SP-A and SP-B mRNA in rabbit fetal lung tissue by in situ hybridization.

Pulmonary surfactant is a lipoprotein substance, comprised of approximately 80% phospholipid and approximately 10% protein, that lowers surface tension at the air-alveolar aqueous interface. Surfactant is synthesized and secreted by alveolar type II epithelial cells where it is stored intracellularly in lamellar bodies. In the present study, we used the technique of in situ hybridization to localize the mRNA for two surfactant-associated proteins, SP-A and SP-B, in developing rabbit fetal lung tissue. We found that SP-A mRNA was first localized in rabbit fetal lung alveolar type II cells on day 26 of gestation, the time at which lamellar bodies are first observed within fetal lung type II cells. On day 28 of gestation, a very small amount of SP-A mRNA was also detectable in the epithelial cells of some bronchioles. In neonatal and adult rabbit lung tissue, SP-A mRNA was primarily restricted to alveolar type II cells; however, the epithelial cells of some bronchioles contained small amounts of SP-A mRNA. SP-B mRNA was first detected in cuboidal epithelial cells in the prealveolar region of the rabbit fetal lung tissue on day 24 of gestation, i.e., at least 2 days before the appearance of SP-A mRNA and lamellar bodies within differentiated alveolar type II cells. SP-B mRNA was detected in most bronchiolar epithelial cells of the rabbit fetal lung tissue at day 28 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucocorticoid regulation of surfactant-associated proteins in rabbit fetal lung in vivo

The effects of a maternally administered synthetic glucocorticoid, betamethasone, on the levels of mRNA for the surfactant proteins SP-A, SP-B, and SP-C and on the levels of SP-A protein were investigated in day 27 gestational age rabbit fetal lung tissue. Betamethasone administration to the pregnant rabbit caused approximately a twofold increase in the fetal lung level of SP-A protein and a threefold increase in fetal lung SP-A mRNA levels when compared to levels in fetuses obtained from saline-treated or uninjected animals. SP-B mRNA was increased fourfold in fetal lung tissue obtained from glucocorticoid-treated pregnant does when compared to levels in fetuses of uninjected pregnant does. However, SP-B mRNA levels in fetal lung tissue from saline-injected controls were also significantly elevated, ～twofold, when compared to fetal lung SP-B mRNA levels in the uninjected control condition. SP-C mRNA levels in lung tissue of fetuses from both saline-injected and betamethasone-injected pregnant does were increased similarly, ～twofold, over SP-C mRNA levels in fetal lung tissue obtained from uninjected control does. These data are suggestive that betamethasone treatment increases fetal lung SP-A and SP-B mRNA levels and that maternal stress alone can increase the expression of SP-B and SP-C mRNA in rabbit fetal lung tissue. Using in situ hybridization, SP-A mRNA was shown to be present primarily in alveolar type II cells in fetuses of control and saline-injected does. However, SP-A mRNA was easily detected in both alveolar type II cells and bronchiolar epithelial cells of rabbit fetal lung tissue following maternal betamethasone treatment. In contrast, SP-B and SP-C mRNA were present only in alveolar type II cells of lung tissue obtained from fetuses of control, saline, or betamethasone-treated does. Thus maternal administration of glucocorticoids increased SP-A protein as well as SP-A and SP-B mRNA levels in rabbit fetal lung tissue. SP-A mRNA was localized to both alveolar type II cells and in smaller amounts in bronchiolar epithelial cells of rabbit fetal lung tissue. However, SP-B and SP-C mRNA were detected only in alveolar type II cells. © 1993 Wiley-Liss, Inc.     

Localization of abutilon mosaic virus (AbMV) DNA within leaf tissue by in situ hybridization

Abutilon mosaic virus (AbMV) is a whitefly-transmitted geminivirus with a a bipartite genome. Using in situ hybridization AbMV DNA was detected exclusively in the phloem of infected Abutilon sellovianum leaves and intracellularly predominant in the nuclei. No AbMV DNA was found in cells from palisade and spongy parenchyma, the tissues which show the predominant cytopathological effects. A hypothesis is discussed to account for the finding that, while AbMV accumulates in the phloem, symptoms are observed in other tissues. Shoot tips were analyzed by in situ hybridization to determine the earliest stage of leaf development in which AbMV is detectable. We found the first hybridization signals in the sixth to seventh leaf of the shoot tip, whereas we could not detect any viral DNA in younger leaves and meristems. These results are discussed with reference to the relation of AbMV multiplication to the cell cycle.     

Localization of parvalbumin mRNA in rat brain by in situ hybridization histochemistry

Summary Parvalbumin mRNA was localized in rat brain by in situ hybridization using a ³⁵S labelled rat parvalbumin cDNA and a synthetic oligodeoxyribonucleotide (corresponding to base sequences 140 to 183 of rat parvalbumin cDNA). Strongest hybridization signals were detected in the Purkinje cells of the cerebellum and in neurones of the reticular nucleus of the thalamus. Signal was also detected in the cerebral cortex, hippocampus, basal ganglia and brain stem in agreement with the distribution of parvalbumin immunoreactivity.     

Localization of acidic fibroblast growth factor (aFGF) mRNA in mouse and bovine retina by in situ hybridization.

Acidic fibroblast growth factor (aFGF) mRNA has been detected in adult mouse or bovine retina by in situ hybridization with bovine aFGF cDNA clones. It is localized on ganglion cell layer, inner nuclear layer, photoreceptors and slightly on pigmented epithelium. This synthesis of aFGF in highly specialized retinal cell types is discussed in the framework on current views about the role of FGF in retinal cell biology.     

Localization of interleukin 6 mRNA in human tonsils by in situ hybridization

We have investigated which areas produce interleukin 6 (IL 6) in human tonsils. This growth factor is required for the terminal differentiation of B lymphocytes into plasmocytes. Using 35S-labeled IL 6 cDNA we demonstrated IL 6 gene expression over various areas of the tonsils, with consistent exception of the follicles, by in situ hybridization. It is, therefore, proposed that B cells are stimulated during their migration out of the follicles.     

Localization of IFN-gamma and IL-6 mRNA in murine intestine by in situ hybridization.

Previous studies have highlighted the importance of CD4+ T cells in regulation of IgA responses and have indicated a functional heterogeneity among these cells between inductive (Peyer's patch) and effector (lamina propria) sites in the intestine. To determine whether these functional differences could be accounted for by differences in cytokine profile of cells in each of these sites, the distribution of mRNA for interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) was investigated by in situ hybridization techniques using 35S-labelled riboprobes. Whereas message for IL-6 is abundant in all regions of the lamina propria from the base of the mucosa to the tips of the villi, very little is expressed in Peyer's patches or in the epithelium. In contrast, message for IFN-gamma is expressed predominantly by cells localized only in the base of the lamina propria and, as with IL-6, very little message was detected in Peyer's patches although occasional strongly positive IFN-gamma cells were observed in the epithelium. These results indicate that, at least in the absence of deliberate intestinal stimulation, functional expression of these cytokines is restricted to effector rather than induction sites in the intestine. This is consistent with our previous observations demonstrating a requirement for T-cell signals in promoting post-extravasation differentiation and proliferation of IgA-committed B cells in vivo and the implications of these findings to the role of the Th1 and Th2 subsets of CD4+ cells in mucosal immune responses is discussed.     

Ureaplasma in lung. 1. Localization by in situ hybridization in a mouse model

Ureaplasma urealyticum is a common inhabitant of mucosal surfaces but is also associated with a higher incidence of pneumonia and bronchopulmonary dysplasia in preterm infants. Culture and polymerase chain reaction demonstrate high isolation rates of ureaplasma in clinical specimens documenting their presence but do not associate the organism directly with the diseased tissue. In this study, lung tissue samples from newborn mice inoculated intranasally with U. urealyticum were used to develop an in situ hybridization (ISH) test for the organism. In situ hybridization allows the localization of gene expression for visualization within the context of tissue morphology. New techniques which use biotinyl-tyramide based signal amplification have been able to greatly enhance the sensitivity of ISH. Using the Dako GenPoint Catalyzed Signal Amplification system to detect a biotinylated DNA probe specific for an internal nucleotide sequence within the urease gene of U. urealyticum, the organism was detected within the infected murine lung tissues. Electron microscopy was used to verify the presence of the organisms in the positive ISH areas. The ISH procedure developed in this study can be used to analyze the presence of ureaplasma in human neonatal lung tissue with the corresponding histopathology.     

Localization of copper-zinc superoxide dismutase mRNA in human hippocampus by in situ hybridization

The cellular localization of copper-zinc superoxide dismutase (CuZn SOD) mRNA was determined in the human hippocampus by in situ hybridization with a 35S-labelled DNA probe complementary to human CuZn SOD mRNA. A positive hybridization signal was detected in pyramidal cell layers CA1-CA4 of Ammon's horn (CA), pyramidal cells of subiculum and in the granule cells of the dentate gyrus. The fact that CuZn SOD gene expression is important in neurones which are preferentially vulnerable in neurodegenerative processes such as Alzheimer's disease, suggests a role played by oxygen free radicals in the mechanism of nerve cell death.     

Localization of keratin mRNA in human tracheobronchial epithelium and bronchogenic carcinomas by in situ hybridization.

An in situ hybridization technique was applied to detect expression of keratin mRNAs in xenotransplanted human tracheobronchial epithelium and lung carcinomas. Tissues from eight tracheas repopulated with cells from five different noncancerous donors and 15 squamous cell carcinomas were used. Using a K6 (56 kd) human keratin cDNA (KA-1) and a K14 (50 kd) cDNA (KB-2) as probes, radiolabeled by nick-translation with 3H-dATP/TTP, the specificity and significant differences in the levels of silver grains on various epithelial lesions in formalin-fixed, paraffin-embedded tissue sections were demonstrated. In situ hybridization with either KA-1 or KB-2 probe showed similar localization of silver grains in all histologic types in consecutive tissue sections. In xenotransplanted tracheobronchial epithelia, very few grains were seen over cells of simple, pseudostratified, or stratified epithelia two to three cell layers thick. Nonkeratinizing stratified hyperplastic epithelia of more than three cell layers showed uniform localization of numerous grains throughout the lesions. In contrast, epidermoid metaplasias exhibited a dense and localized pattern of grains on the basal and parabasal cell layers with a decrease in grain density toward the surface layers. Carcinoma cells from bronchogenic squamous cell carcinomas showed a higher density and more uniform localization of grains. Well-differentiated carcinoma cells contained more keratin mRNAs than moderately to poorly differentiated carcinoma cells. This evidence obtained with the KA-1 and KB-2 probes demonstrates the different localization patterns of keratin mRNAs in different epithelial lesions. In addition, the levels of mRNA expressed show a positive correlation with the degree of squamous differentiation. It was of particular interest that an ordered program of keratin mRNA expression proportional to the level of cellular differentiation was observed in epidermoid metaplasias. Both of these probes serve as keratinization markers of human tracheobronchial epithelial lesions.     

Localization of immunoglobulin light chain mRNA expression in Hodgkin's disease by in situ hybridization

In situ hybridization techniques were used to detect immunoglobulin light chain messenger RNA (mRNA) in 28 formalin-fixed, paraffin-embedded samples of Hodgkin's disease. Cocktails of biotinylated oligonucleotide probes specific for the constant regions of kappa and lambda light chain mRNA were used. None of the Reed-Sternberg cells or their variants in any of the cases studied showed positive staining with either probe, in contrast to normal plasma cells which showed strong staining in the same sections. It was concluded, therefore, that the cytoplasmic immunoglobulin frequently detected within these cells by immunocytochemistry is present not as a result of synthesis, but as a result of some other mechanism.     

Localization of the 92 kd gelatinase mRNA in squamous cell and adenocarcinomas of the lung using in situ hybridization.

We have used in situ hybridization for RNA to localize cells containing mRNA for the 92 kd gelatinase in carcinomas of the lung. We used archival material to analyze sections from 12 cases of squamous cell carcinomas of the lung including six stage I and three stage II and from three cases of adenocarcinoma of the lung. Presence of mRNA in the tissue was verified by in situ hybridization for gamma actin. The 92 kd gelatinase mRNA was found in all 12 squamous cell carcinomas tumors and was highly expressed in the tumor cells themselves. In addition, it was found in host stromal cells surrounding the tumor, but not in normal lung fibroblasts. In contrast it was not found in the adenocarcinomas of the lung or in the stroma surrounding these tumors. The mRNA for the 92 kd gelatinase was present in normal pulmonary tissue, bronchial epithelium, basal cell hyperplasia of bronchial epithelium, alveolar macrophages, and focally in bronchial mucous glands. It was not present in normal alveoli, vascular cells, cartilage, or most lymphocytes. We corroborated the presence of the mRNA for the 92 kd gelatinase by ribonuclease protection assay. The levels of mRNA for the 92 kd gelatinase in two specimens of squamous cell carcinoma were 6- to 10-fold greater than in the nonneoplastic tissue and two adenocarcinoma specimens.     

Localization of dopamine D2 receptor mRNA with non-radioactive in situ hybridization histochemistry.

A digoxigenin-labeled antisense 42-mer oligonucleotide was used for the localization of the dopamine D2 receptor mRNA in the rat brain. The digoxigenin label was identified with alkaline phosphatase conjugated sheep-anti-digoxigenin. In good analogy with the known terminal fields of the dopaminergic system, various nuclei throughout the brain were labeled. Positive in situ hybridization signals were also found in dopamine cell groups of the substantia nigra and ventral tegmental area and in regions where a dopaminergic innervation is controversial, like the cerebellar cortex and the hippocampus. The non-radioactive in situ hybridization procedure described, shows the localization of the dopamine D2 receptor mRNA with a very high contrast and an optimal histological resolution.     

Detection of polyoma virus DNA in PML-brain tissue by (in situ) hybridization.

The human papova (JC) virus was extracted from brain of a patient with progressive multifocal leukoencephalopathy. A single band of virus was obtained at a density of 1.345 g/ml CsCl.JC virus DNA was purified and a highly specific cRNA was generated in vitro. In situ hybridization with JC virus cRNA and autoradiography on sections of the same brain revealed silver grains over oligodendrocytes, astrocytes and possible vascular endothelial cells, indicating the presence of JC virus DNA in these different cell classes.     

肺癌组织中EB病毒感染的检测

目的探讨原发性肺癌中EB病毒（Epstein—Barr virus，EBV）的存在情况及EBV与原发性肺癌的关系。方法唐山市人民医院和开滦医院病理科储存的2001--2006年肺癌手术切除石蜡包埋肺癌组织108份，癌旁组织22份，以EBV阳性鼻咽癌组织为阳性对照，用原位杂交法（ISH）检测肺癌患者石蜡包埋组织标本中EBV编码的小RNA（EBERl），并采用图像分析法进行形态学定量。结果癌组织及癌旁组织EBERl的阳性率分别为33．3％（36／108）和4．5％（1／22），二者间差异有统计学意义（P〈0．01）。鳞癌、腺癌、小细胞癌及大细胞癌中EBV感染率分别是35．9％（14／39）、31．6％（12／38）、31．0％（9／29）和1／2。EBV感染与患者年龄、性别和组织学类型无关，但与肺癌的部位、癌组织分化程度有关，右肺明显高于左肺，中低分化癌明显高于高中分化癌。结论唐山地区原发性肺癌组织中EBV感染率为33．3％，EBV感染可能是肺癌的潜在病因之一，在癌组织分化的不同阶段有不同的作用。     

Localization of type III procollagen mRNA in areas of liver fibrosis by in situ hybridization

Localization of type III procollagen mRNA in human liver was studied by in situ hybridization using human alpha 1(III) procollagen cDNA. Frozen and paraformaldehyde-fixed sections of biopsied human liver from patients with chronic hepatitis and cirrhosis were examined using the digoxigenin-labeled cDNA probe. Localization of the type III procollagen mRNA was demonstrated not only in the cytoplasm of mesenchymal cells but also in a large number of hepatocytes, in proportion to the extent of fibrosis. These results suggest that hepatocytes play an important role in fibrogenesis in the liver.     

Localization of an endoplasmic reticulum calcium ATPase mRNA in rat brain by in situ hybridization

SERCA-2 is an endoplasmic reticulum Ca2+ ATPase present in brain [Gunteski-Hamblin A.-M. et al. (1988) J. biol. Chem. 263, 15032-15040]. We sought to map the distribution of this pump in the rat brain and investigate its relationship to Ca2+ uptake by brain endoplasmic reticulum. Using in situ hybridization and Northern blots with antisense oligonucleotide probes, we found that SERCA-2 is concentrated most densely in the cerebellum, especially in Purkinje cells, and in the hippocampus, with heavy labeling also in cortex, thalamus, pontine nuclei and the mitral cell layer of the olfactory bulb. 45Ca2+ uptake displayed a similar pattern with heaviest accumulation in cerebellum, hippocampus, cortex, thalamus and olfactory bulb. In corpus striatum and substantia nigra, relative 45Ca2+ accumulation was greater than SERCA-2 mRNA. Thus, SERCA-2 appears to be involved in Ca2+ uptake into endoplasmic reticulum in brain for release by inositol 1,4,5-trisphosphate and other agents.     

Localization of plasminogen activator inhibitor-1 production in inflamed appendix by in situ mRNA hybridization

The peritoneum has been shown to possess fibrinolytic activity which is thought to play a role in the prevention of intra-abdominal adhesion formation. Recently inflamed peritoneal tissue has been shown to have reduced fibrinolytic activity secondary to increased levels of plasminogen activator inhibitor-1 (PAI-1). The aim of this study was to localize the production of PAI-1 in appendix tissue using in situ mRNA hybridization. Sections of normal and inflamed appendix were hybridized with a digoxigenin-labelled cDNA probe. PAI-1 production was localized to both mcsothelium and serosal blood vessel endothelium in all inflamed appendix samples. Cell identities were confirmed using immunohistochemistry directed against mesothelial and endothlial cell markers. Staining was not seen on sections of normal appendix or on negative control slides of inflamed appendix (hybridization with plasmid DNA, PAI-1 probe following ribonuclease digestion). The identification of the cells expressing the PAI-1 gene in peritoneum increases our understanding of the pathophysiological changes in fibrinolytic activity which occur in inflammation and may lead to adhesion formation.     

Localization of collagenase mRNA in rheumatoid arthritis synovium byin situ hybridization histochemistry

Collagenase has been implicated as playing an important role in the connective tissue destruction that is a major feature of rheumatoid arthritis. Numerous cell types in the hyperplastic rheumatoid synovium are capable of synthesizing collagenase. Past studies have used predominately synovial fibroblasts in culture as a model system for the regulation of collagenase production, but the major cellular source of the enzymein vivo has not been determined. Using the techniques ofin situ hybridization histochemistry and indirect immunofluorescence, we determined the cellular source of collagenase in frozen sections of human synovium. Collagenase mRNA production was localized to cells along the synovial lining layer in rheumatoid arthritis. These were identified as the macrophage-like Type A synovial lining cells by immunofluorescence with antibody LeuM3. Endothelial cells, fibroblasts, and T and B lymphocytes were devoid of detectable collagenase mRNA. Synovial tissue sections from patients with osteoarthritis and trauma did not contain detectable collagenase mRNA. These data identify the Type A macrophage-like synovial lining cell as the primary source of collagenase mRNAin vivo in the rheumatoid arthritis synovium and, potentially, as a major effector cell in the tissue destruction of the disease.