Immunohistochemical distribution of inter-alpha-trypsin inhibitor chains in normal and malignant human lung tissue.

The inter-alpha-trypsin inhibitor (ITI) family is a group of plasma proteins built up from heavy (HC1, HC2, HC3) and light (bikunin) chains synthesized in the liver. In this study we determined the distribution of ITI constitutive chains in normal and cancerous lung tissues using polyclonal antibodies. In normal lung tissue, H2, H3, and bikunin chains were found in polymorphonuclear cells, whereas H1 and bikunin proteins were found in mast cells. Bikunin was further observed in bronchoepithelial mucous cells. In lung carcinoma, similar findings were obtained on infiltrating polymorphonuclear and mast cells surrounding the tumor islets. Highly differentiated cancerous cells displayed strong intracytoplasmic staining with H1 and bikunin antiserum in both adenocarcinoma and squamous cell carcinoma. Moreover, weak but frequent H2 expression was observed in adenocarcinoma cells, whereas no H3-related protein could be detected in cancer cells. Local lung ITI expression was confirmed by RT-PCR. Although the respective role of inflammatory and tumor cells in ITI chain synthesis cannot be presently clarified, these results show that heavy chains as well as bikunin are involved in malignant transformation of lung tissue.(J Histochem Cytochem 47:1625-1632, 1999)     

Immunocytochemical localization and enzymatic distribution of rat ovarian aromatase

A rabbit antiserum directed against purified human placental aromatase was used for immunohistochemical localization of the enzyme in rat ovaries. Immunostaining was conducted on tissue from animals at various ages and in different reproductive states: immature; immature, eCG-treated; immature pseudopregnant; adult cycling; and adult pregnant. Various labeling protocols were employed (e.g. horseradish peroxidase-conjugated secondary antibody, peroxidase-antiperoxidase, and avidin-biotin-peroxidase on fresh frozen and Bouin's fixed paraffin-embedded sections), but the avidin-biotin-peroxidase method on paraffin sections proved to be superior to the others. In immature rats, most of the immunostaining, which was quite weak, was limited to the stroma. After stimulation with eCG, some of the granulosa cells of antral follicles exhibited immunostaining; in pseudopregnant rats, most staining occurred in the luteal cells. In mature animals, the corpora lutea of pregnant and cycling rats demonstrated the greatest degree of immunostaining. No significant immunoreactivity was detected in pre-antral follicles, but in early antral follicles and preovulatory follicles, both theca and granulosa cells exhibited immunostaining. Aromatase enzymatic activity was also determined on ovarian microsomal fractions of eCG-treated immature animals, pregnant animals at term, and cycling animals. Furthermore, enzyme activity and estradiol concentrations were examined after ovaries from proestrous rats were dissected into follicular, luteal, and residual components. Activity was found in all regions and correlated with immunostaining and estrogen production. These results argue against a model in which all the immunoreactive/enzymatically active protein is localized in granulosa cells of Graafian follicles and suggest that corpora lutea may be involved in estrogen synthesis during the rat estrous cycle as well as during pregnancy.     

Hysteresivity of the lung and tissue strip in the normal rat: effects of heterogeneities.

We measured lung impedance in rats in closed chest (CC), open chest (OC), and isolated lungs (IL) at four transpulmonary pressures with a optimal ventilator waveform. Data were analyzed with an homogeneous linear or an inhomogeneous linear model. Both models include tissue damping and elastance and airway inertance. The homogeneous linear model includes airway resistance (Raw), whereas the inhomogeneous linear model has a continuous distribution of Raw characterized by the mean Raw and the standard deviation of Raw (SDR). Lung mechanics were compared with tissue strip mechanics at frequencies and operating stresses comparable to those during lung impedance measurements. The hysteresivity (eta) was calculated as tissue damping/elastance. We found that 1) airway and tissue parameters were different in the IL than in the CC and OC conditions; 2) SDR was lowest in the IL; and 3) eta in IL at low transpulmonary pressure was similar to eta in the tissue strip. We conclude that eta is primarily determined by lung connective tissue, and its elevated estimates from impedance data in the CC and OC conditions are a consequence of compartment-like heterogeneity being greater in CC and OC conditions than in the IL.     

Localization and activity of iNOS in normal human lung tissue and lung cancer tissue

Inducible nitric oxide synthase (iNOS) is one of three enzymes generating nitric oxide (NO) from the amino acid L-arginine. iNOS-derived NO plays an important role in several physiological and pathophysiological conditions. NO is a free radical which produces many reactive intermediates that account for its bioactivity. In the human lung, the alveolar macrophage is an important producer of cytokines and this production may be modified by NO. Moreover, high concentrations of NO have been shown to increase nuclear factor kappaB (NF-kB) activation. Recent investigations of NO expression in tumor tissue indicated that, at least for certain tumors, NO may mediate one or more roles during the growth of human cancer. We have studied iNOS in two tissue groups: normal human lung tissue and human lung cancer tissue. We localized iNOS in these tissues by immunohistochemistry and tested the mRNA expression by RT-PCR, the protein level by Western blot, and the protein activity by radiometric analysis. The results demonstrate different expression, localization and activity of iNOS in normal versus tumor tissue. This is suggestive of a role for NO production from iNOS in human lung cancer because high concentrations of this short molecule may transform to highly reactive compounds such as peroxynitrite (ONOO-); moreover, through the upregulator NF-kB, they can induce a chronic inflammatory state representing an elevated risk for cell transformation to cancer.     

Immunocytochemical studies on the distribution pattern of daunomycin in rat gastrointestinal tract

The cancer drug daunomycin is used in treatment of leukemia but possesses severe side effects that involve the gastrointestinal tract. We therefore used a newly developed immunocytochemical procedure to determine the distribution of DM in the gastrointestinal tracts of rats after i.v. injection. Two hours after injection, DM was diffusely distributed in nuclei and most parts of the cytoplasm of intestinal epithelial cells. The cytoplasmic immunoreactivity for DM was most pronounced in small granules of the apical cytoplasm. Sixteen hours after injection, DM immunostaining was by and large absent in the villous epithelium but persisted in the intestinal crypts. In addition, staining was also detected in endothelial cells, scattered cells of the lamina propria and in smooth muscle cells. After 5 days, only little staining for DM remained. Similar findings were made in the colon. In the gastric mucosa, DM accumulation persisted at 16 h in some glandular cells but was lost from the surface epithelium. No staining was detected in saline-injected control rats. The distribution of DM accumulation correlated partially with the distribution of apoptotic cells as detected by the TUNEL procedure. Our results pinpoint that DM may exert prolonged effects on glandular and regenerative cells of the gastrointestinal tract—an observation that may explain the gastrointestinal toxicity of the drug. It seems possible that DM accumulation in surface epithelial cells is rapidly cleared through drug transporters.     

Subcellular distribution of the protein kinase activity in the normal and neoplastic thyroid tissue of the rat]

Thyroid tissue has been fractionnated by centrifugation (105 000 q) of its homogenate. Protein-kinase activity in presence of histone is distributed in nuclei (11.5%) mitochondira (22.8%), microsomes (9.8%) and soluble fraction (56%); it is activated by cyclic AMP and GMP, mostly in soluble and nuclear fractions. Protein-kinase activity of total homogenate of neoplasic thyroid (strain 1-5G Wollman) in presence of histone is 3 times higher than in normal tissue and more activated by cAMP. In absence of histone, protein-kinase activity is the more important in mitochondrial and microsomal fractions of normal thyroid and in soluble and nuclear fraction of neoplastic tissue.     

Immunocytochemical distribution of LHRH neurons and processes in the rat: Hypothalamic and extrahypothalamic locations

Summary The distribution of luteinizing hormone-releasing hormone (LHRH)-immunoreactive perikarya and processes was examined, in the untreated rat, with the unlabeled antibody enzyme method of immunocytochemistry on thick 50 m vibratome sections. LHRH neurons were primarily observed in the preoptico-anterior hypothalamic and septal areas. Projections from these cell bodies to the median eminence form three distinct pathways, one laterally along the course of the optic tracts, one medially through the periventricular stratum of the third ventricle, and one through the tractus infundibularis. In addition, some of these cell bodies project to the organum vasculosum of the lamina terminalis (OVLT) and the subfornical organ (SFO). LHRH immunoreactive neurons were also noted in the anterior olfactory regions; they project along the medial olfactory tract to the olfactory bulb.     

Immunocytochemical localization of substance P neurokinin-1 receptors in rat gingival tissue

The distributions of substance P (SP) and the neurokinin-1 receptor (NK1-R), the receptor preferentially activated by SP, were examined in rat gingiva by immunocytochemical methods with light and electron microscopy. SP-immunoreactive nerve fibers were located preferentially in the junctional epithelium (JE) but few in the other oral and oral sulcular epithelia. NK1-R immunoreactivity was found in the endothelial cells (capillaries and postcapillary venules underlying the JE). NK1-R-labeled and -unlabeled unmyelinated nerve fibers were located close to the blood vessels and partially or completely covered by a Schwann cell sheath. In the JE, labeled naked axons without Schwann cell sheaths were observed. Neutrophils and macrophages in the connective tissue underlying the JE and in the JE were also labeled with NK1-R. Furthermore, NK1-R was found in the JE cells. Basically, immunoreaction products for NK1-R were found throughout various cells (endothelial cells, neutrophils, and JE cells) at invaginations of the plasma membrane and in vesicular and granular structures that are probably endosomes and are found close to both the plasma membrane and the nucleus. This is a first report, demonstrating the presence of NK1-R in the gingival tissue in the normal nonstimulated condition. Furthermore, it is thought that SP may modulate the permeability of blood vessels beneath the JE, the production of antimicrobial agents in neutrophils, and the proliferation and endocytotic ability of JE cells through NK1-R.     

Abundance and tissue distribution of selenocysteine-containing proteins in the rat

The form and distribution of selenium (Se) in proteins from selected tissues of the rat were studied by measuring 75Se radioactivity in animals provided for 5 months with [75Se]selenite as the main dietary source of Se. Equilibration of the animals to a constant specific activity of 75Se allowed the measurement of 75Se to be used as a specific elemental assay for Se. Skeletal muscle, liver and blood accounted for 73% of the whole-body Se and 95% of the total Se-dependent glutathione peroxidase activity. Over 80% of the whole-body Se was in protein in the form of the selenoamino acid, selenocysteine. All other forms of Se that were measured accounted for less than 3% of the whole-body Se. The Se in protein was distributed in seven subunit sizes and nine chromatographic forms. The Se in glutathione peroxidase accounted for one-third of the whole-body Se. These results show that the main use of dietary Se, as selenite, in rats is for the synthesis of selenocysteine-containing proteins. Furthermore, the presence of two-thirds of the whole-body Se in nonglutathione peroxidase, selenocysteine-containing proteins suggests that there may be other important mammalian selenoenzymes besides glutathione peroxidase.     

Ribosome distribution in normal and infarcted rat hearts

Summary Distribution of ribosomes throughout the myocardium of normal and infarcted rat hearts was studied by immunofluorescence and laser confocal scanning microscopy. In addition, sections were labelled with peroxidase or immunogold particles for electron microscopic examination. Ligation of the proximal free left coronary artery produced severe myocardial ischaemia, and after 6 days of ligation most of the left ventricular wall was necrotic and partially replaced by granulation tissue. Immunofluorescence microscopy revealed the presence of ribosomes throughout the non-necrotic myocardium. Some cardiac muscle cells located in subendocardial areas and in the border areas surrounding the infarct were particularly intensely stained. Cells constituting the granulation tissue frequently exhibited strong ribosomal immunostaining. Within longitudinally sectioned cardiac muscle cells, ribosomes were organized in strands oriented along the long axis of the cell as well as in a cross-striated pattern. By double labelling of muscle cells with antibodies against ribosomes and Z-line-associated proteins (desmin or α-actinin), it was shown that the cross-striated bands of anti-ribosomal staining coincided with the I-bands along the myofibrils. Immunoelectron microscopy confirmed a wide distribution of ribosomes throughout the intermyofibrillar and subsarcolemmal sarcoplasm, and some labelling was also observed within the I-band. The present results indicate that ribosomes are distributed in a characteristic pattern throughout the sarcoplasm of cardiac muscle cells in association with the myofibrils. Furthermore, it is suggested that within viable cardiac muscle cells located adjacent to the infarct, protein synthesis is increased; this might be an important factor in regional development of compensatory hypertrophy of the surviving cardiac muscle cells.     

Chromium content of normal and pathologically altered lung tissue

Flameless atomic absorption spectrophotometry was used to determine the chromium (Cr) content of samples taken at autopsy from the lungs, bronchi, and regional hilar lymph nodes of 50 randomly selected patients from urban and rural areas; these patients were not known to have been excessively exposed to Cr. On the average, the Cr concentration in the lungs of patients younger than 40 yr of age was approximately 2 μg/g; for patients over 40, the average Cr values were between 5 and 15 μg/g dry wt. The highest values were found in samples from the apex of the lung. The Cr concentration in lung and lymph nodes increased in proportion to age and the degree of anthracosis. Chromium content in the bronchial wall was very low in all cases. Chromium values up to ten times greater as compared to age-matched average values were found in scarred lung tissue, probably caused by a postinflammatory lymph vessel blockade. Slightly elevated Cr values were found in smokers' lungs. Chromium values in tissue from primary lung carcinomas (n=9) were lower than those in neighboring lung tissue. Based on the results of this study the amount of Cr of lung and bronchial tissue does not appear to be associated with the induction of bronchial carcinoma.     

Simulation and measurement of microbeam dose distribution in lung tissue

Microbeam radiation therapy (MRT), a so far preclinical method in radiation oncology, modulates treatment doses on a micrometre scale. MRT uses treatment fields with a few ten micrometre wide high dose regions (peaks) separated by a few hundred micrometre wide low dose regions (valleys) and was shown to spare tissue much more effectively than conventional radiation therapy at similar tumour control rates. While preclinical research focused primarily on tumours of the central nervous system, recently also lung tumours have been suggested as a potential target for MRT.This study investigates the effect of the lung microstructure, comprising air cavities of a few hundred micrometre diameter, on the microbeam dose distribution in lung. In Monte Carlo simulations different models of heterogeneous lung tissue are compared with pure water and homogeneous air–water mixtures. Experimentally, microbeam dose distributions in porous foam material with cavity sizes similar to the size of lung alveoli were measured with film dosimetry at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France.Simulations and experiments show that the microstructure of the lung has a huge impact on the local doses in the microbeam fields. Locally, material inhomogeneities may change the dose by a factor of 1.7, and also average peak and valley doses substantially differ from those in homogeneous material.Our results imply that accurate dose prediction for MRT in lung requires adequate models of the lung microstructure. Even if only average peak and valley doses are of interest, the assumption of a simple homogeneous air–water mixture is not sufficient. Since anatomic information on a micrometre scale are unavailable for clinical treatment planning, alternative methods and models have to be developed.