Bone marrow cells repair cigarette smoke-induced emphysema in rats

The therapeutic potential of stem cells in chronic obstructive pulmonary disease is not well known although stem cell therapy is effective in models of other pulmonary diseases. We tested the capacities of bone marrow cells (BMCs), mesenchymal stem cells (MSCs), and conditioned media of MSCs (MSC-CM) to repair cigarette smoke-induced emphysema. Inbred female Lewis rats were exposed to cigarette smoke for 6 mo and then received BMCs, MSCs, or MSC-CM from male Lewis rats. For 2 mo after injection, the BMC treatment gradually alleviated the cigarette smoke-induced emphysema and restored the increased mean linear intercept. The BMC treatment significantly increased cell proliferation and the number of small pulmonary vessels, reduced apoptotic cell death, attenuated the mean pulmonary arterial pressure, and inhibited muscularization in small pulmonary vessels. However, only a few male donor cells were detected from 1 day to 1 mo after BMC administration. The MSCs and cell-free MSC-CM also induced the repair of emphysema and increased the number of small pulmonary vessels. Our data show that BMC, MSCs, and MSC-CM treatment repaired cigarette smoke-induced emphysema. The repair activity of these treatments is consistent with a paracrine effect rather than stem cell engraftment because most of the donor cells disappeared and because cell-free MSC-CM also induced the repair.     

改变食物组成对大鼠胃肠道嗜铬细胞的影响

为研究食物组成对肠嗜铬（ＥＣ）细胞的影响，用免疫组织化学方法（ＰＡＰ法）对４组食物组成不同大鼠的胃肠道５－羟色胺免疫反应阳性（５－ＨＴＩＲ）细胞的数量和分布作了观察。发现食物改变会引起大鼠摄食量和胃肠形态的变化，但未见其生长发育异常。食物中米糠含量加倍后，大鼠５－ＨＴＩＲ细胞数量无显著增加，但分布型有明显的变化。食物中米糠含量或米糠和麦麸含量都减少到１０％后，大鼠５－ＨＴＩＲ细胞数量和分布都有明显     

Calcitonin-immunoreactive cells of the digestive tract of the amphioxus are distributed concentrically in a restricted region of the mid-gut

In amphioxus (Branchiostoma belcheri), calcitonin‐immunoreactive cells of the digestive tract were distributed concentrically in a 1‐mm region of the latter half of the mid‐gut. In the mid‐gut caecum these cells were also present, but were dispersed throughout the length. The total number of cells in both parts ranged from 280 to 1157, varying from individual to individual, although the number in the mid‐gut caecum was only 100–200. These cells were morphologically typical gut endocrine cells. Considering their morphology and the characteristics of their distribution pattern, these cells may be related to some phenomena of digestive processes.     

An immimohistochemical study of regulatory peptides in lungs of amphibians

Summary The lungs of five species of European Anura and one species of Urodela (Triturus alpestris) have been studied by immunohistochemical methods to determine the occurrence, localization and distribution of serotonin, neuron-specific enolase, and eight regulatory peptides reported in the mammalian respiratory tract.Single and groups of serotonin-immunoreactive cells, corresponding to neuroendocrine cells of the mammalian lung, were identified in lungs of all amphibian species studied. Immunoreactivity for neuron-specific enolase was localized mainly in pulmonary nerves, nerve cell bodies and neuroendocrine cells. The localization and distribution of regulatory peptides varied among species. Bombesin and gastrin-releasing peptide immunoreactivities (predominant peptides in human lung) were localized mostly in submucosal nerves. Single bombesin-immunoreactive cells were found only in lungs of Urodela, i.e., Triturus alpestris. Occasional single cells, immunoreactive for somatostatin and leu-enkephalin were identified in lungs of Bombina variegata and a few cholecystokinin-immunoreactive cells in Hyla arborea. In all anuran species, numerous substance P-immunoreactive nerves were identified in submucosa, pulmonary septa and around blood vessels. No immunoreactive cells or nerves were demonstrated with antibodies against calcitonin and vasoactive intestinal peptide.The term pulmonary neuroendocrine (NE) cells (used here) does not imply neural origin or classical endocrine function for these cells, but rather indicates their potential involvement in neurohormonal regulation of pulmonary function (Cutz 1982)Supported by grant to E.C. from Medical Research Council of Canada (MT-7641)     

Expression of nitric oxide synthase isoforms (NOS II and NOS III) in adult rat lung in hyperoxic pulmonary hypertension

Breathing air with a high oxygen tension induces an inflammatory response and injures the microvessels of the lung. The resulting development of smooth muscle cells in these segments contributes to changes in vasoreactivity and increased pulmonary artery pressure. This in vivo study determines the temporal and spatial expression of endogenous endothelial nitric oxide synthase (NOS III) and inducible NOS (NOS II), important enzymes regulating vasoreactivity and inflammation, in the adult rat lung during the development of experimental pulmonary hypertension induced by oxidant injury. We analyzed the cellular distribution of these NOS isoforms, using specific antibodies, and assessed enzyme activity at baseline and after 1-28 days of hyperoxia (FIO₂ 0.87). The number of NOS III-immuno-positive endothelial cells increased early in hyperoxia and then remained high. By day 28, the relative number of these cells had increased from 40% in proximal vessels and 13-16% in distal alveolar vessels of the normal lung to 73-86% and 40-59%, respectively, in hyperoxia. Pulmonary alveolar macrophages (PAMs), normally few in number and only weakly immunopositive for NOS II or III in the normal lung, increased in number in hyperoxia and were strongly immunopositive for each isoform. These morphological data were supported by a temporal increase in total and calcium-independent NOS activity. Thus NOS expression and activity significantly increased in hyperoxia as pulmonary hypertension developed, and NOS III expression increased selectively in vascular endothelial cells, while both NOS isoforms were expressed by the PAM population. We conclude that this increase in expression of a potent vasodilator, an antiproliferative agent for smooth muscle cells, and an antioxidant molecule represents an adaptive response to protect the lung from oxidant-induced vascular and epithelial injury.     

Ultrastructural and immunocytochemical study of skin fibroblasts from normal and sialidosis patients

The objectives of this study were to analyze morphologically, morphometrically and immunocytochemically the lysosomal compartment of normal fibroblasts and of fibroblasts with neuraminidase deficiency. The immunocytochemical analyses consisted of quantifying the distribution of saposins and -galactosidase in the lysosomes of these cells to test the hypothesis that neuraminisdase deficiency is associated with an impairment in the transport of these proteins to the lysosomal compartment. To test this idea, cultured skin fibroblasts of patients with or without sialidosis were prepared for electron microscopy and probed with antibodies against lysosomal -galactosidase and lysosomal saposins. The lysosomes of the affected cells had an abnormal accumulation of incompletely digested membranes which was associated with a significant lowering in the density of antigenic sites per lysosome. However, due to a significant increase in the number of lysosomes per affected cell, the total number of antigenic sites in control and neuraminidase deficient cells was similar. This presumably compensatory effect indicates that although the rate of production of -galactosidase and saposins remains unchanged, the transport of these molecules to the lysosomes is somehow affected. Our data also indicate that in the fibroblasts, lysosomes require a normal concentration of the three enzymes to maintain neuraminidase activity and sphingolipid degradation.     

Post-irradiation thymocyte regeneration after bone marrow transplantation. II. Physical characteristics of thymocyte progenitor cells

Bone marrow cells were separated according to buoyant density, velocity sedimentation and cell surface charge. Fractionated (C3H x AKR)F1 bone marrow cells were transplanted into lethally-irradiated C3H recipients. In all fractions, the CFUs content and the capacity to restore the thymus cell population were determined. For all the physical parameters tested, the thymocyte progenitor cells show the same distribution as CFUs. The relationship between number of thymocyte progenitor cells and number of CFUs is dependent on density. Bone marrow progenitors of PHA responsive cells are of low buoyant density and show a distribution which resembles the distribution of the progenitors of Thy 1 positive cells. After transplantation of large numbers of bone marrow cells into irradiated mice, no significant change in the CFUs content of the thymus was observed.     

Distribution of atrial natriuretic polypeptide (ANP)-containing cells in the rat heart and pulmonary vein Immunohistochemical study and radioimmunoassay

Summary The distribution of atrial natriuretic polypeptide (ANP) was immunohistochemically surveyed in the rat heart and lung using an antiserum raised against -human ANP. The ANP-immunoreactive cells were seen to be distributed in the atrial walls and proximal portions of the pulmonary vein and venae cavae, but were absent from the aorta, pulmonary arteries, trachea, bronchus, and alveolar cells. The immunoreactive cells were present in a narrow region just beneath the endothelium of the pulmonary vein and vena cavae, and, ultrastructurally and immunocytochemically, were seen to be striated muscle cells with ANP-containing specific granules similar to those seen in atrial cardiocytes. A radioimmunoassay for ANP revealed a content of 604±51 pg/mg wet weight in the pulmonary vein, and 3343±1620 pg/mg wet weight in the venae cavae. In addition to the atrial wall, the proximal portion of both the pulmonary vein and venae cavae are suggested to be constituents of an ANP-producing organ.     

V gamma 4+ gamma delta T cells regulate airway hyperreactivity to methacholine in ovalbumin-sensitized and challenged mice

The Vgamma4(+) pulmonary subset of gammadelta T cells regulates innate airway responsiveness in the absence of alphabeta T cells. We now have examined the same subset in a model of allergic airway disease, OVA-sensitized and challenged mice that exhibit Th2 responses, pulmonary inflammation, and airway hyperreactivity (AHR). In sensitized mice, Vgamma4(+) cells preferentially increased in number following airway challenge. Depletion of Vgamma4(+) cells before the challenge substantially increased AHR in these mice, but had no effect on airway responsiveness in normal, nonchallenged mice. Depletion of Vgamma1(+) cells had no effect on AHR, and depletion of all TCR-delta(+) cells was no more effective than depletion of Vgamma4(+) cells alone. Adoptively transferred pulmonary lymphocytes containing Vgamma4(+) cells inhibited AHR, but lost this ability when Vgamma4(+) cells were depleted, indicating that these cells actively suppress AHR. Eosinophilic infiltration of the lung and airways, or goblet cell hyperplasia, was not affected by depletion of Vgamma4(+) cells, although cytokine-producing alphabeta T cells in the lung increased. These findings establish Vgamma4(+) gammadelta T cells as negative regulators of AHR and show that their regulatory effect bypasses much of the allergic inflammatory response coincident with AHR.     

Expression of calponin in rabbit and human aortic smooth muscle cells

Summary Polyclonal antibodies to chicken gizzard calponin were used to localize calponin and determine calponin expression in rabbit and human aortic smooth muscle cells in culture. Calponin was localized on the microfilament bundles of cultured smooth muscle cells. Early in primary culture,ccalponin staining was accumulated preferentially in the central part of the cell body. With time in culture, the number of calponin-negative smooth muscle cells increased while the distribution of calponin in calponin-positive cells became more even along the stress fibers. Calponin content and the calponin/actin ratio decreased about 5-fold in rabbit aortic smooth muscle cells during the first week in primary culture and remained low in proliferating cells. The same tendency in calponin expression was observed when human vascular smooth muscle was studied. On cryostat sections of human umbilical cord, calponin antibodies mainly stained vessel walls of both the arteries and veins, although less intensive labelling was also observed in non-vascular tissue. When primary isolates of human aortic intimal and medial smooth muscle cells were compared with corresponding passaged cultures, it was found that calponin content was reduced about 9-fold in these cells in culture and was similar to the amount of calponin in endothelial cells and fibroblasts. Thus, high calponin expression may be used as an additional marker of vascular smooth muscle cell contractile phenotype.     

Active tuberculosis in Indochinese refugees in British Columbia.

The incidence of active tuberculosis in 8692 Indochinese refugees admitted to British Columbia between 1979 and 1981 was reviewed. In the first 3 months after entry into the province the rate was extremely high--estimated at 1890/100 000 (126 times the provincial average). A large proportion of these cases were of primary or minimal pulmonary tuberculosis. However, although the proportion of cases of minimal pulmonary tuberculosis was twice the provincial average, the proportion of these cases that were confirmed by culture was only one third the provincial average; this suggests some overdiagnosis in this period. In the subsequent 21 months of residence the incidence of active tuberculosis was also high, at 353/100 000, which was more than 20 times the provincial average. The distribution of cases by severity was closer to the provincial distribution in this period, but advanced disease accounted for a far smaller proportion of cases in both periods than it did in 1980 in the entire province.     

Total cellular RNA content: correlation between flow cytometry and ultraviolet spectroscopy

Total RNA content in Chinese hamster ovary and HeLa-S3 cells determined by ultraviolet spectroscopy is compared with the red fluorescence distribution of acridine orange-stained cells observed by flow cytometry. A correlation coefficient of 0.93 is obtained when these methods of estimating RNA content are compared after various RNAse treatments. These data suggest that acridine orange staining effectively quantitates total cellular RNA content when analyzed by flow cytometry, although DNA is also shown to contribute a low but significant background of red fluorescence.     

GLAND DISTRIBUTION AND CANNABINOID CONTENT IN CLONES OF CANNABIS SATIVA L.

The relationship between glandular trichomes and cannabinoid content in Cannabis sativa L. was investigated. Three strains of Cannabis, which are annuals, were selected for either a drug, a non-drug, or a fiber trait and then cloned to provide genetically uniform material for analyses over several years. The distribution of the number and type of glands was determined for several organs of different ages including the bract and its subtending monoleaflet leaf and the compound leaf on pistillate plants. Quantitation of glands on these structures was integrated with gas chromatographic analyses of organ cannabinoid profiles. A negative correlation was found between cannabinoid content and gland number for each of the three clones. Isolated heads of the capitate-stalked glands also were analyzed for cannabinoid content and found to vary in relation to clone and gland age. These studies indicate that cannabinoids may occur in plant cells other than glandular trichomes. The results of these studies emphasize the need for stringent sampling procedures in micromorphological studies on trichome distribution and analytical determinations of cannabinoid content in Cannabis.     

HISTAMINE AND MAST CELLS IN DEVELOPING RAT BRAIN

The number and distribution of mast cells in rat brain were determined at different postnatal ages. The number of brain mast cells was found to change during ontogenic development following the same pattern as brain histamine (HA) levels. The calculated HA content of brain mast cells was close to the HA content of the crude nuclear fraction at every age studied. Since most of the brain HA in the newborn sediments with the crude nuclear fraction, these results suggest that the developmental pattern of brain HA reflects changes in the number of brain mast cells, that is, in the size of the mast cell HA pool. The HA content of the supernatant of the crude nuclear fraction corrected for mast cell HA contamination, on the other hand, follows a developmental pattern similar to that of other known neurotransmitters.     

Post-Irradiation Thymocyte Regeneration After Bone Marrow Transplantation Ii. Physical Characteristics of Thymocyte Progenitor Cells

Bone marrow cells were separated according to buoyant density, velocity sedimentation and cell surface charge. Fractionated (C3H × AKR)F₁ bone marrow cells were transplanted into lethally-irradiated C3H recipients. In all fractions, the CFUs content and the capacity to restore the thymus cell population were determined. For all the physical parameters tested, thymocyte progenitor cells show the same distribution as CFUs. the relationship between number of thymocyte progenitor cells and number of CFUs is dependent on density. Bone marrow progenitors of PHA responsive cells are of low buoyant density and show a distribution which resembles the distribution of the progenitors of Thy 1 positive cells. After transplantation of large numbers of bone marrow cells into irradiated mice, no significant change in the CFUs content of the thymus was observed.     

The radiosensitivity of the chromosomes of the cells of human squamous cell carcinoma cell lines.

Measurement of the radiation sensitivity of chromosomes was used to address the influence of cell cycle distribution and of DNA content and ploidy on radiation responses in seven human squamous cell carcinoma cell lines. The cell lines varied about twofold in DNA content and chromosome number, and the X-ray sensitivities (D0) of the lines ranged from 1.1 to 2.7 Gy. The more resistant cell lines (D0 greater than 1.8 Gy) had faster growth rates and larger proportions of cells in S phase in asynchronous cultures. Aberration frequencies were measured in cells irradiated in G1 and G2 phase. The more resistant lines had fewer induced aberrations in both phases than did sensitive lines, implying that they were more resistant to radiation in both of these cell cycle phases. Therefore, while the larger S-phase population seen in the resistant cell lines probably contributes to the resistant phenotype, it cannot explain all of the intrinsic differences in radiation sensitivity. There was no relationship between DNA content and radiation sensitivity as measured by the cell survival assay or the induction of chromosome aberrations, although cells with larger DNA contents tended to have more chromosome damage per cell at equitoxic doses.     

Plastid‐targeted forms of restriction endonucleases enhance the plastid genome rearrangement rate and trigger the reorganization of its genomic architecture

Plant cells have acquired chloroplasts (plastids) with a unique genome (ptDNA), which developed during the evolution of endosymbiosis. The gene content and genome structure of ptDNAs in land plants are considerably stable, although those of algal ptDNAs are highly varied. Plant cells seem, therefore, to be intolerant of any structural or organizational changes in the ptDNA. Genome rearrangement functions as a driver of genomic evolutionary divergence. Here, we aimed to create various types of rearrangements in the ptDNA of Arabidopsis genomes using plastid‐targeted forms of restriction endonucleases (pREs). Arabidopsis plants expressing each of the three specific pREs, i.e., pTaqI, pHinP1I, and pMseI, were generated; they showed the leaf variegation phenotypes associated with impaired chloroplast development. We confirmed that these pREs caused double‐stranded breaks (DSB) at their recognition sites in ptDNAs. Genome‐wide analysis of ptDNAs revealed that the transgenic lines exhibited a large number of rearrangements such as inversions and deletions/duplications, which were dominantly repaired by microhomology‐mediated recombination and microhomology‐mediated end‐joining, and less by non‐homologous end‐joining. Notably, pHinP1I, which recognized a small number of sites in ptDNA, induced drastic structural changes, including regional copy number variations throughout ptDNAs. In contrast, the transient expression of either pTaqI or pMseI, whose recognition site numbers were relatively larger, resulted in small‐scale changes at the whole genome level. These results indicated that DSB frequencies and their distribution are major determinants in shaping ptDNAs.     

Pulmonary capillaries are recruited during pulsatile flow.

Capillaries recruit when pulmonary arterial pressure rises. The duration of increased pressure imposed in such experiments is usually on the order of minutes, although recent work shows that the recruitment response can occur in <4 s. In the present study, we investigate whether the brief pressure rise during cardiac systole can also cause recruitment and whether the recruitment is maintained during diastole. To study these basic aspects of pulmonary capillary hemodynamics, isolated dog lungs were pump perfused alternately by steady flow and pulsatile flow with the mean arterial and left atrial pressures held constant. Several direct measurements of capillary recruitment were made with videomicroscopy. The total number and total length of perfused capillaries increased significantly during pulsatile flow by 94 and 105%, respectively. Of the newly recruited capillaries, 92% were perfused by red blood cells throughout the pulsatile cycle. These data provide the first direct account of how the pulmonary capillaries respond to pulsatile flow by showing that capillaries are recruited during the systolic pulse and that, once open, the capillaries remain open throughout the pulsatile cycle.     

Combined treatment with <Emphasis Type="SmallCaps">l</Emphasis>-carnitine and a pan-caspase inhibitor effectively reverses amiodarone-induced injury in cultured human lung epithelial cells

Amiodarone is an effective class III antiarrhythmic drug, however, the pulmonary toxicity is one of the most life-threatening complications of its use. The present study was designed to determine the mechanisms underlying pulmonary toxicity of amiodarone. In cultured human lung epithelial cells A549, amiodarone caused cell injury characterized by mitochondrial membrane depolarization, ATP depletion, enhanced propidium iodide (PI) uptake and increase in the number of Annexin-V positive cells, although the population of PI-stained cells appeared earlier and was not identical to that of Annexin-V stained cells, suggesting that the apoptosis and necrosis appeared in different cells. The apoptosis was accompanied with the activation of caspase-2, -3 and -8 but not caspase-9, and reversed by these caspase inhibitors. However, the caspase inhibitors had no influence on mitochondrial membrane potential or PI uptake after exposure of A549 cells to amiodarone. In contrast, mitochondrial cofactors such as L-carnitine and acetyl-l-carnitine attenuated mitochondrial membrane depolarization, abrogated cellular ATP depletion and reversed PI uptake without affecting Annexin-V positive cells. These finding suggest that different intracellular events operate to cause apoptosis and necrosis after exposure of pulmonary epithelial cells to amiodarone.