Exosomal miR-147-3p regulates the growth of mouse bronchial epithelial cells and asthma progression via targeting RUNX3: a pilot study

Interferon-γ Inhibits the Growth of Human Bronchial Epithelial Cells Independently of Transforming Growth Factor-β-1 and Nitric Oxide (NO)

The colostrum of many species will stimulate the growth of cells in vitro and colostrum ingestion may play an important rôle in directly promoting the growth of the gastrointestinal tract in the newborn animal (Berseth, Lichtenberger and Morriss, 1984). Most studies have suggested however, that growth-promoting activity declines to zero, or to negligible levels in post-colostrum milk (Shing and Klagsbrun, 1984; Cera, Mahan and Simmen, 1987). We have investigated the ability of sow's milk, collected up to 21 days of lactation, to stimulate the growth of epithelial cells in vitro. For these studies we use cells derived from the mucosa of the small intestine: RIE-1 cells (Blay and Brown, 1984) and a lung-derived cell line: CHV79 cells.Milk was collected from three sows at each of the following times: 0 (colostrum), 7, 14, and 21 days post partum. The samples were defatted by centrifugation (30 000 g for 40 min), filter sterilized and diluted in phosphate buffered saline (PBS, pH 6.8). RIE-1 cells (a generous gift from K. Brown, Brabaham) or CHV79 cells were grown to confluence in Dulbecco's modified Eagle's medium (DME) containing foetal calf serum (FCS, 10%). The cultures were quiesced in a medium containing 0.5% FCS for 24 h and milk samples added in serum-free PBS for a further 24 h. [3H]-thymidine was added for the final 4 h before cells were harvested. Growth-promoting activity of milk samples are expressed as a stimulation index (SI) i.e. in relation to the amount of label incorporated after incubation with an equivalent volume of PBS. Fractionation of the growth-promoting activity of a pooled post-colostrum milk sample was performed on sephacryl S-200sf and S-300sf columns.

Measles virus (MeV) first infects immune cells in the respiratory tract of a human host, spreads to lymphoid organs throughout the body, and finally enters and grows in respiratory epithelial cells before being released and transmitted to the next host. Thus, efficient growth in respiratory epithelial cells is important for the person-to-person transmission of MeV. Upon viral entry, host cells detect viral nucleic acids and produce interferons (IFNs) to control viral growth. Type I (IFN-α/β) and type III (IFN-λ) IFNs have largely common induction and signaling mechanisms and stimulate expression of similar target genes but utilize distinct receptors. To determine the relative contributions of type I and type III IFNs to the control of MeV growth in epithelial cells, we examined the growth of MeV and that of its mutants lacking either type I or type III IFN receptor in the human lung epithelial cell line H358. Our results revealed that both type I and type III IFNs are required to restrict MeV growth in H358 cells and that the induction of type III as well as type I IFNs was increased in the absence of the MeV nonstructural V protein.

Until recently, oxalate was considered merely as a major component of calcium oxalate stones, forming crystals in the lumen of the renal tubules. However, new evidence suggests that oxalate is not only a major constituent of calcium oxalate stones but also has effect on renal tubular epithelial cells, affecting the pathogenesis of nephrolithiasis. We tried to elucidate the effect of oxalate on the growth of renal tubular epithelial cells of different species and locations and also to interpret the possible role of the oxalate in the pathogenesis of urolithiasis. Porcine proximal renal tubular epithelial cells (LLC-PK1) and canine distal renal tubular epithelial cells (MDCK) were incubated with different concentrations of oxalate, and the effect of oxalate on the growth of the cells was assessed by methylthiazoletetrazolium assay. Growth of the renal tubular epithelial cells was inhibited with increasing concentrations of oxalate in both proximal and distal lines. Oxalate may cause stone formation by affecting the growth of renal tubular epithelial cells as well as by providing a constituent of calcium oxalate stones.

The molecular mechanisms that regulate the synthesis of salivary proteins are unknown. The paucity of homogeneous cell populations of parotid acinar cells has become a limiting factor for such a study. Therefore, the establishment of immortalized clones of acinar cells is essential. This study has established primary cultures of rat and human parotid epithelial cells that are suitable for transfection with plasmid vectors, pSV2, pSV3, and pSV5 to generate immortalized cells in vitro. Among various techniques used, the rat and human parotid tissue or cellular clumps when restrained in chicken plasma clot allowed the outgrowth of epithelial cells that maintained epithelial cell morphology for over 4 wk. However, the initial growth requirements for rat and human parotid cells were different. The presence of 10% heat inactivated fetal bovine serum in supplemented MCDB-LB medium was essential for the outgrowth of rat parotid epithelial cells, but this was not needed for the outgrowth of human parotid epithelial cells. The growth of both human and rat parotid epithelial cells can be maintained in serum-free supplemented MCDB-LB. These primary cultures contained amylase-producing cells as demonstrated by immunofluorescent technique, and they were transfected with pSV2, pSV3, and pSV5 using primarily the calcium phosphate-DNA co-precipitation technique. After initial extensive cell death, many cells with epithelial cell morphology survived.

Explants of adult human bronchus were cultured in CMRL-1066 medium supplemented with heat-inactivated fetal bovine serum, hormones, antibiotics, and either 0.0, 0.1, 1, or 10 mM putrescine. The outgrowth of bronchial epithelial cells was stimulated in medium containing 1 mM putrescine, a concentration that partially inhibited the outgrowth of fibroblasts. In medium containing 10 mM putrescine, the outgrowth of epithelial cells was similar to that observed in the control medium, but the outgrowth of fibroblasts was completely inhibited for periods of at least 4 weeks. When 10 mM putrescine was added to cultures of bronchial fibroblasts, the fibroblasts were not killed. These results suggest that human bronchial epithelial cells have a higher requirement for putrescine for growth than fibroblasts, and the molecular basis for this observation is under investigation.

The capacity of mineralized cementum to support epithelial cell migration and growth and the effect that fibronectin and partial demineralization of cementum have on these processes were assessed in vitro. Dog gingival explants, 1 X 2 mm, were cultured on the cementum surfaces of pig root pieces in a defined medium consisting of DMEM and F12 (1V/1V), transferrin, insulin, epidermal growth factor, cortisone, selenium, and high-density lipoprotein. Sixty root pieces were divided into four equal groups according to the treatment: (1) untreated mineralized cementum; (2) treated with 5 micrograms of fibronectin; (3) partially demineralized in 18% EDTA for 30 min; and (4) both partially demineralized and fibronectin-treated as above. Epithelial cell migration and growth on each of the four differently treated cementum surfaces were assessed histomorphometrically by means of scanning electron microscopy. The defined culture medium supported the selective migration and growth of epithelial cells from the gingival explants onto the mineralized cementum. This was confirmed by the positive immunostaining of these cells with antikeratin antibodies. Partial demineralization of cementum inhibited epithelial migration and growth by 83% and 91%, respectively. Fibronectin treatment did not affect epithelial cell migration and growth on mineralized cementum, but it decreased the degree of epithelial cell migration and growth inhibition on partially demineralized cementum to 57% and 43%, respectively. The results indicate that: (i) mineralized cementum may consist of components that are recognized by gingival epithelial cells and support their migration and growth in vitro; (ii) these components can be removed by demineralization; and (iii) fibronectin partially restores epithelial cell migration and growth on partial demineralized cementum in vitro.

Growth and differentiation of fetal rat small intestinal epithelium in tissue culture: Relationship to fetal age

We have developed a co-culture system to study paracrine effects of fetal mesenchyme cells on the growth of primary mammary epithelial cells of the mouse. The method should be adaptable for study of interaction of a variety of cell populations. The procedure is simple and inexpensive. The culture consists of four layers: a monolayer of mesenchyme cells on a plastic culture dish, a soft agarose layer overlaying the cell monolayer, epithelial cells suspended in collagen gel placed atop the agarose, and culture medium above the collagen gel. The agarose layer prevents direct contact of two different cell populations but allows soluble molecules produced by either cell population to diffuse through the system, so as to contact and interact with the other cell population.

Epithelial cells in the thymic medulla are conspicuous in normal adult mice, but sparse in the early fetal thymus and the thymus of adult T cell-deficient SCID mice. To examine whether growth of medullary epithelial cells (MEC) depends upon local contact with mature T cells, we used the finding that the SCID thymus is unusually permeable to mature T cells entering from the bloodstream. When SCID mice received multiple injections of mature lymph node T cells from birth, the thymus accumulated large numbers of mature TCR+ T cells of resting phenotype, but contained virtually no immature (CD4+8+) cells. The injected T cells localized in the medullary region of the thymus and led to marked regeneration of MEC. These and other data suggest that the growth of MEC is under the control of mature T cells. Placing MEC under T cell control might be a device for regulating the size and integrity of the medulla, especially during the phase of rapid thymic growth. Maintaining the cellular components of the medulla in proper balance could be critical for ensuring efficient self tolerance induction.

The goal of this study was to evaluate the experimental effectiveness of Regenerative Factor Rich Plasma (RFRP) of human blood versus Fetal Bovim Serum (FBS) and neuropeptide Substance P (SP) on corneal epithelium cell proliferation. Rabbit corneal epithelium cell (CCL-60) growth was compared between different RFRP fractions, FBS and with the neuropeptide Substance P. The ability of the RFRP fractions and SP to revert the inhibitory effect of the CsA was also evaluated. All groups showed an increase (p<0.001) in corneal epithelial cell growth compared with the control group. The maximum capacity of cell growth was obtained with dilutions of 50% in the FBS, RFRP-I, RFRP -II, RFRP-III groups and with 100nM of SP. The highest growth was observed with 50% FBS, RFRP-I and RFRP-II. The group with SP and RFRP-III had significantly lower growth (p<0.001). When the NK1 receptor antagonist CsA was added at a dose of IC50, we found a significant decrease in cell growth (p<0.001) in all culture conditions, including the control group. The decrease was similar in all groups, but was especially pronounced in RFRP-II. RFRP I, II and III promoted growth more than SFB 10%. The RFRP of human blood promotes the growth of corneal epithelial cells in a significantly more efficient manner than FBS and SP. RFRP can be effective both in cell cultures and stem cell cultures.

Background: It is known that growth hormones such as insulin-like growth factor-I (IGF-I) and several kinds of cytokines are involved in the regeneration process of injured epithelial cells in chronic respiratory inflammatory diseases such as bronchial asthma. Repetitive degeneration/regeneration processes of the airway epithelial layer is supposed to be responsible for the remodeling and irreversible organic changes of the airway in bronchial asthma. The purpose of this study is to establish a simple and reliable in vitro method for studying airway epithelial cell growth and proliferation using IGF-I. Method: By altering the number of cultured epithelial cells (strain NCI-H₂₉₂), culture duration before stimulation with IGF-I, concentration of IGF-I, and duration of IGF-I stimulation, the optimum conditions for epithelial cell growth was determined. The epithelial cell growth was evaluated using [methyl-³H]thymidine uptake. Result: Among various culture conditions, the epithelial cells cultured at 1 × 10³ cells/well for 24 h followed by 24 h of stimulation by 10^–8 M of IGF-I showed the highest growth. Conclusion: The method for the evaluation of epithelial cell growth established in this study requires a small number of cells and has no complicated procedure. This simple model enables us to investigate the effect of various substances on bronchial epithelial cell growth in the presence of IGF-I.

Impact of capsaicin, an active component of chili pepper, on pathogenic chlamydial growth ( Chlamydia trachomatis and Chlamydia pneumoniae ) in immortal human epithelial HeLa cells

Rationale: CC16 is a protein mainly produced by nonciliated bronchial epithelial cells (BECs) that participates in host defense. Reduced CC16 protein concentrations in BAL and serum are associated with asthma susceptibility. Objectives: Few studies have investigated the relationship between CC16 and asthma progression, and none has focused on BECs. In this study, we sought to determine if CC16 mRNA expression levels in BECs are associated with asthma severity. Methods: Association analyses between CC16 mRNA expression levels in BECs (242 asthmatics and 69 control subjects) and asthma-related phenotypes in Severe Asthma Research Program were performed using a generalized linear model. Measurements and Main Results: Low CC16 mRNA expression levels in BECs were significantly associated with asthma susceptibility and asthma severity, high systemic corticosteroids use, high retrospective and prospective asthma exacerbations, and low pulmonary function. Low CC16 mRNA expression levels were significantly associated with high T2 inflammation biomarkers (fractional exhaled nitric oxide and sputum eosinophils). CC16 mRNA expression levels were negatively correlated with expression levels of Th2 genes (IL1RL1, POSTN, SERPINB2, CLCA1, NOS2, and MUC5AC) and positively correlated with expression levels of Th1 and inflammation genes (IL12A and MUC5B). A combination of two nontraditional T2 biomarkers (CC16 and IL-6) revealed four asthma endotypes with different characteristics of T2 inflammation, obesity, and asthma severity. Conclusions: Our findings indicate that low CC16 mRNA expression levels in BECs are associated with asthma susceptibility, severity, and exacerbations, partially through immunomodulation of T2 inflammation. CC16 is a potential nontraditional T2 biomarker for asthma development and progression.

Although estrogens can stimulate the growth of uterine epithelial cells in vivo, there is no clear effect of estrogens on the in vitro growth of epithelial cells from reproductive tract tissues; thus, we have established a defined culture system for mouse uterine epithelial cells. Pieces of uteri from immature CD-1 mice (21-23 days of age) were treated with trypsin, and the epithelial fragments were separated, enriched by Percoll gradient centrifugation, and seeded on collagen gels prepared from rat tail tendon. Initially, the cells were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's Medium supplemented with epidermal growth factor (EGF; 10 ng/ml), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), hydrocortisone (0.1 micrograms/ml), and vitamin A (10 ng/ml). The cells formed a monolayer on the collagen gel within 1-2 days, but with time, cells began to detach from the gel. Further studies revealed that the attachment and growth of these cells on collagen were markedly influenced by the calcium concentration. It was found that lowering the calcium concentration from 1.05 to 0.05-0.1 mM dramatically suppressed cell detachment; the number of cells doubled after 7 days of culture. Proliferation of uterine epithelial cells was enhanced by EGF, but not by fibroblast growth factor, platelet-derived growth factor, nerve growth factor, multiplication-stimulating activity, or somatomedin-C. The uterine epithelial cells exhibited a single class of high affinity binding sites for [125I]iodo-EGF (Kd, approximately 1.8 nM), with approximately 5 X 10(4) receptors/cell; binding was inhibited by EGF but not by the other polypeptides. This cell culture system will aid in our investigations on hormonal effects on the growth and differentiation of estrogen target cells.