Effects ofStaphylococcus aureusproducts on growth and function of bovine mammary myoepithelial cellsin vitro

1995 ◽  
Vol 62 (4) ◽  
pp. 577-586 ◽  
Author(s):  
Boris Zavizion ◽  
A. John Bramley ◽  
Ioannis Politis

SUMMARYThe effects of culture supernatants conditioned by the growth ofStaphylococcus aureusM60 onin vitrogrowth and functional properties of bovine mammary myoepithelial cells were examined. Myoepithelial cell proliferation was reduced byStaph, aurexisM60 culture supernatants. Exposure of myoepithelial cells to culture supernatants of isogeneic mutants ofStaph, aureusM60 that produced either α or β toxins reduced proliferation, but to a lesser extent than supernatants from the wild type strain. Thus, α and β toxins may play some role in affecting myoepithelial cell proliferation. Of the cells tested, 42% contracted following addition of oxytocin (10–7M) in the culture medium. Treatment of myoepithelial cells for 15 min withStaph, aureusM60 supernatants, prior to addition of oxytocin in the culture medium, increased the number of cells that contracted to 92%. Exposure of cells for 3 h to the same supernatant, prior to addition of oxytocin in the culture medium, abolished oxytocin responsiveness, had no effect on immunolocalization of actin and vimentin, but affected the localization of α-actinin within myoepithelial cells. Treatment of myoepithelial cells for 3 h with a combination of purified staphylococcal proteinases XVI and XVII-B abolished oxytocin responsiveness and mimicked the effect of theStaph, aureusculture supernatant. We conclude thatStaph, aureusM60 culture supernatant affected proliferation and functional properties of myoepithelial cells.

1993 ◽  
Vol 73 (2) ◽  
pp. 449-452
Author(s):  
B. Zavizion ◽  
I. Politis ◽  
R. C. Gorewit

The effects of mammary-derived growth inhibitor (MDGI) on proliferation of bovine mammary myoepithelial cells was examined. MDGI (10 ng L−1) inhibited myoepithelial proliferation. The specificity of the MDGI effect was tested with anti-MDGI rabbit serum which was shown to neutralize MDGI and abolish the effect of MDGI on myoepithelial cell proliferation. Key words: Bovine, MDGI, myoepithelial cells


2012 ◽  
Vol 94 (2) ◽  
pp. 123-131 ◽  
Author(s):  
Céline Miroux ◽  
Olivier Morales ◽  
Khaldoun Ghazal ◽  
Samia Ben Othman ◽  
Yvan de Launoit ◽  
...  

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 333-340 ◽  
Author(s):  
JW Jr Shands

Abstract The properties of mouse macrophage procoagulant induced by endotoxin in vitro were studied by the acceleration of clotting and by chromogenic assays using as substrates human plasma and bovine components, which are not activated by mouse tissue factor. Maximal macrophage procoagulant activity occurred when activated cells were lysed in culture supernatant fluids, suggesting the interaction of cellular and supernatant factors. This procoagulant was clearly able to activate bovine factor X. The procoagulant also appeared to have prothrombinase activity. However, additional experiments suggested that the bulk of this activity was due to the activation of factor X contaminating the prothrombin. The production of the procoagulant was inhibited by warfarin (5 microM). Its activity was inhibited by 1 mM diisopropylfluorophosphate and unaffected by iodoacetamide, indicating that the procoagulant is a serine protease. Macrophage culture supernatants contained factor-VII-like activity. Neither mouse tissue factor nor macrophage culture supernatants alone activated bovine factor X. The two combined served as an efficient factor-X activator. Active supernatant factor (factor-VII-like) was not produced by macrophages cultured in the presence of warfarin, while the production of the macrophage cellular factor was unaffected by the presence of warfarin. I conclude that exudate macrophages cultured in vitro make and secrete factor VII or a factor-VII-like substance into the culture supernatant. When activated macrophages are lysed in this supernatant, the interaction of a cellular factor (? tissue factor) and factor VII gives rise to a factor-X activator.


2020 ◽  
Vol 26 (1) ◽  
pp. 53-64
Author(s):  
Zahraa Alali ◽  
Amanda Graham ◽  
Kimberly Swan ◽  
Rebecca Flyckt ◽  
Tommaso Falcone ◽  
...  

Abstract Endometriosis is a female disease which is defined as the presence of ectopic endometrial tissue and is dependent on estrogen for its survival in these ectopic locations. Expression of the ribosomal protein large P1 (RPLP1) is associated with cell proliferation and invasion in several pathologies, but a role in the pathophysiology of endometriosis has not been explored. In this study, we aimed to evaluate the expression and function of RPLP1 with respect to endometriosis pathophysiology. RPLP1 protein was localised by immunohistochemistry (IHC) in eutopic and ectopic tissue from 28 subjects with confirmed endometriosis and from 20 women without signs or symptoms of the disease, while transcript levels were evaluated by qRT-PCR in 77 endometriotic lesions and 55 matched eutopic endometrial biopsies, and protein expression was evaluated using western blotting in 20 of these matched samples. To evaluate the mechanism for enhanced lesion expression of RPLP1, an experimental murine model of endometriosis was used and RPLP1 expression was localized using IHC. In vitro studies using an endometriosis cell line coupled with shRNA knockdown was used to demonstrate its role in cell survival. Expression of RPLP1 mRNA and protein were significantly higher in ectopic lesion tissue compared to paired eutopic endometrium and immunohistochemical localisation revealed predominant localisation to epithelial cells. This pattern of lesion RPLP1 was recapitulated in mice with experimentally induced endometriosis. Stable knockdown of RPLP1 protein resulted in a significant decrease in cell survival in vitro. These studies reveal that RPLP1 is associated with cell proliferation and/or survival and may play a role in the pathophysiology of endometriosis.


Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4899-4899
Author(s):  
Hisayuki Yokoyama ◽  
Maria Berg ◽  
Andreas Lundqvist ◽  
J. Philip McCoy ◽  
Shivani Srivastava ◽  
...  

Abstract The ability to expand NK cells in vitro has led to the recent initiation of protocols incorporating adoptive NK cell infusions after HCT. Calcineurin inhibitors such as CSA are commonly used to prevent graft versus host disease (GVHD) in HCT recipients. Recently, Hong et al found the phenotype and function of fresh NK cells cultured in vitro with CSA was altered, with CSA treated NK cell cultures having enhanced cytotoxicity against tumor targets. However, the impact of CSA on in vitro expanded NK cell function and phenotype has not been explored. We analyzed cell proliferation, IFN-gamma production, cell surface immunofluorescent staining and cytotoxicity against K562 and renal cell carcinoma cell lines by in vitro expanded vs freshly isolated NK cells cultured in physiological doses of CSA (40ng/ml, 200ng/ml, 1000ng/ml for 18hrs). Fresh NK cells were obtained from the PBMC of healthy donors using immunomagnetic beads to isolate CD56+/ CD3− cells. NK cells were expanded in vitro using irradiated EBV transformed B cells as feeder cells in media containing IL-2 [500U/ml] for 12–14 days. Comparing CSA containing cultures to controls, there was a significant reduction in IL-2 stimulated fresh NK cell proliferation (stimulation index 0.51± 0.1) and TRAIL expression (MFI 10.4 vs 3.01). Furthermore, an ELISA assay showed fresh NK cells treated with CSA had a significant reduction in IL-2 induced IFN-g production compared to controls (median 231 vs 57 pg/ml, p=0.025). In contrast, in vitro expanded NK cells cultured in CSA showed no significant reduction of proliferation or TRAIL expression. At the highest doses of CSA (1000ng/ml), minimal inhibition of K562 killing of freshly isolated NK cells was observed. In contrast, expanded NK cells cultured in CSA for 18 hours compared to controls had a significant reduction in the killing of K562 cells (E:T=10:1, median 66 vs 43% lysis, p=0.011) and RCC tumor cells (E:T=20:1, 14.8 vs 8.8%, p=0.043). Figure Figure These data confirm CSA alters the phenotype and function of CD3−/CD56 + NK cells. Importantly, CSA appears to have a deleterious effect on expanded NK cell tumor cytotoxicity that was not observed with fresh NK cells. These finding suggest the anti-tumor effects of in vitro expanded NK cells could be hindered when adoptively infused in HCT patients receiving CSA.


Reproduction ◽  
2020 ◽  
Vol 160 (3) ◽  
pp. 405-415
Author(s):  
Qiaoge Niu ◽  
Maosheng Cao ◽  
Chenfeng Yuan ◽  
Yuwen Huang ◽  
Zijiao Zhao ◽  
...  

Nerve growth factor (NGF) has been proved to play important roles in male reproductive physiology, but the molecular mechanisms of NGF action remain unclear. In this study, the effects of NGF on the growth of newborn bovine testicular Sertoli (NBS) cells and the related signaling pathways were investigated. The NBS cells were treated in vitro with NGF (100 ng/mL) for 18 h. The expression levels of cell proliferation related genes, INHBB, and cytoplasmic specialization related gene were determined using real-time PCR and Western blot. The roles of PI3K/AKT and MAPK/ERK pathways in NGF-induced cell proliferation were investigated. It was found that NGF regulates proliferation and function of NBS cells via its receptor NTRK1 by activating the PI3K/ATK and MAPK/ERK signaling pathways. The study will help to further understand the role of NGF in male reproduction and provide new therapeutic targets for reproductive dysfunctions in male animals.


Gut ◽  
2021 ◽  
pp. gutjnl-2020-323951
Author(s):  
Naoki Sugimura ◽  
Qing Li ◽  
Eagle Siu Hong Chu ◽  
Harry Cheuk Hay Lau ◽  
Winnie Fong ◽  
...  

ObjectiveUsing faecal shotgun metagenomic sequencing, we identified the depletion of Lactobacillus gallinarum in patients with colorectal cancer (CRC). We aimed to determine the potential antitumourigenic role of L. gallinarum in colorectal tumourigenesis.DesignThe tumor-suppressive effect of L. gallinarum was assessed in murine models of CRC. CRC cell lines and organoids derived from patients with CRC were cultured with L. gallinarum or Escherichia coli MG1655 culture-supernatant to evaluate cell proliferation, apoptosis and cell cycle distribution. Gut microbiota was assessed by 16S ribosomal DNA sequencing. Antitumour molecule produced from L. gallinarum was identified by liquid chromatography mass spectrometry (LC-MS/MS) and targeted mass spectrometry.ResultsL. gallinarum significantly reduced intestinal tumour number and size compared with E. coli MG1655 and phosphate-buffered saline in both male and female murine intestinal tumourigenesis models. Faecal microbial profiling revealed enrichment of probiotics and depletion of pathogenic bacteria in L. gallinarum-treated mice. Culturing CRC cells with L. gallinarum culture-supernatant (5%, 10% and 20%) concentration-dependently suppressed cell proliferation and colony formation. L. gallinarum culture-supernatant significantly promoted apoptosis in CRC cells and patient-derived CRC organoids, but not in normal colon epithelial cells. Only L. gallinarum culture-supernatant with fraction size <3 kDa suppressed proliferation in CRC cells. Using LC-MS/MS, enrichments of indole-3-lactic acid (ILA) was identified in both L. gallinarum culture-supernatant and the gut of L. gallinarum-treated mice. ILA displayed anti-CRC growth in vitro and inhibited intestinal tumourigenesis in vivo.ConclusionL. gallinarum protects against intestinal tumourigenesis by producing protective metabolites that can promote apoptosis of CRC cells.


Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 333-340
Author(s):  
JW Jr Shands

The properties of mouse macrophage procoagulant induced by endotoxin in vitro were studied by the acceleration of clotting and by chromogenic assays using as substrates human plasma and bovine components, which are not activated by mouse tissue factor. Maximal macrophage procoagulant activity occurred when activated cells were lysed in culture supernatant fluids, suggesting the interaction of cellular and supernatant factors. This procoagulant was clearly able to activate bovine factor X. The procoagulant also appeared to have prothrombinase activity. However, additional experiments suggested that the bulk of this activity was due to the activation of factor X contaminating the prothrombin. The production of the procoagulant was inhibited by warfarin (5 microM). Its activity was inhibited by 1 mM diisopropylfluorophosphate and unaffected by iodoacetamide, indicating that the procoagulant is a serine protease. Macrophage culture supernatants contained factor-VII-like activity. Neither mouse tissue factor nor macrophage culture supernatants alone activated bovine factor X. The two combined served as an efficient factor-X activator. Active supernatant factor (factor-VII-like) was not produced by macrophages cultured in the presence of warfarin, while the production of the macrophage cellular factor was unaffected by the presence of warfarin. I conclude that exudate macrophages cultured in vitro make and secrete factor VII or a factor-VII-like substance into the culture supernatant. When activated macrophages are lysed in this supernatant, the interaction of a cellular factor (? tissue factor) and factor VII gives rise to a factor-X activator.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4724-4724
Author(s):  
Taxiarchis V Kourelis ◽  
Akrivi D Manola ◽  
Despoina Adamidou ◽  
Lazaros Sakkas ◽  
Eyagelia Mperou ◽  
...  

Abstract Vasculogenesis is known to be defective in patients with scleroderma (SS) and psoriatic arthritis (PA) with vessel loss in the former and hypertrophic blood vessels in the latter in affected areas. We studied the number and function of peripheral blood endothelial progenitors (PBEP) in patients with SS and PA to elucidate the mechanism of EC dysfunction. Materials and Methods: Eleven patients with SS, 13 patients with PA and 7 healthy individuals were studied. We measured CD133+/CD146+ cells in peripheral blood (PB) by immunofluorescence. We performed cell cultures of isolated CD34+ cells in endocult medium and examined the endothelial colonies (EPC). We also performed cocultures of CD34+ and autologous bone marrow stromal cells (BMSC) in double chambers. We also performed cocultures of BMSC from patients and normal endothelial cells from cord blood. Results: The number of CD133+/CD146+ cell in PB was increased the 2 groups of patients compared to controls. The number of EC colonies in endocult did not differ in the 3 groups. The presence of autologous serum within the culture medium reduced the number of colonies in 3 patients with SS. The number and the size of EC colonies from SS patients in vitro were significantly reduced (p&lt;0.01) after co-cultures of autologous BMSC with CD34+ in culture plates with insert while they were increased (p&lt;0.01) from patients with PA. The same was true when cord blood CD34+ cells were cultured in endocult medium in the presence of BMSC of SS and PA patients. Conclusion: EC progenitors from patients with SS and PA are increased in PB and develop normal EC colonies in vitro. They developed decreased colonies in SS and increased colonies in PA when cultured together with with autologous BMSC. This means that possible cell-cell or humoral interactions between EC and some cellular component within BMSC affect the survival and differentiation of EC.


Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4298-4298
Author(s):  
Sara Trabanelli ◽  
Antonio Curti ◽  
Darina Očadlíková ◽  
Cecilia Evangelisti ◽  
Valentina Salvestrini ◽  
...  

Abstract Abstract 4298 Indoleamine 2,3-dioxygenase (IDO1) and indoleamine 2,3-dioxygenase-like (IDO2) are enzymes involved in the tryptophan catabolism along the kynurenine pathway. While it is established that IDO1-expressing dendritic cells (DCs) contribute to tolerance in a number of biological settings, little is known about the expression and function of IDO2 in DCs. Human DCs can be generated in vitro to obtain immunogenic antigen-presenting cells (APC), used as cellular vaccines. In the clinical setting, DCs are commonly matured with a cytokine cocktail (CC) which includes TNF-a, IL-1b, IL-6 and PGE2. In particular, PGE2 enhances APC function of DCs by increasing IL-12 production and facilitating DC migration to lymph nodes. However, PGE2 is also a strong IDO1 inducer, which by this route can also limit the anti-tumor activity of DC-based immunotherapies. Thus, understanding the roles of IDO1 and IDO2 in DCs may impact the development of vaccines or DC-based immunotherapies. In the present study, we fully characterized IDO1 and IDO2 expression and function in human monocyte-derived dendritic cells (Mo-DCs). Mo-DCs were generated from purified CD14+ monocytes after culture with GM-CSF and IL-4 and then matured with CD40L, LPS alone, LPS plus IFN-g and the CC. We observed that immature Mo-DCs had little if any expression of both IDO1 and IDO2, whereas mature Mo-DCs exhibited upregulation of both enzymes. Among the different maturation stimuli, CC was the most effective in upregulating IDO1 and IDO2, both at the message and protein levels. This effect was associated also with the highest kynurenine production. By means of IDO1 and IDO2 expression, mature Mo-DCs were inhibited in stimulating allogeneic T cell proliferation and generated a population of CD4+CD25+FOXP3+ Tregs which highly suppressed allogeneic and autologous T-cell proliferation. On the basis of evidence that IDO1 is preferentially inhibited by the L-isoform of 1 methyl-tryptophan (1-MT) and IDO2 by the D-isoform, we performed functional enzyme tests in presence of both isoforms. Notably, both isoforms exhibited inhibitory effects, although we observed a stronger effect of L-1-MT than with D-1-MT suggesting a greater contribution of IDO1 than IDO2. These results offer direct evidence that Mo-DCs express functional IDO1 and IDO2 proteins. During the maturation phase, Mo-DCs enhance their tolerogenic qualities, and in particular the capacity to induce Tregs, through the upregulation of both IDO1 and IDO2. Beside the critical role of IDO1 in enhancing the immunosuppressive capacity of DCs, we show, for the first time, that IDO2 is involved also. Our findings imply that, from a clinical standpoint, to improve the efficacy of DC-based vaccines mature DCs should be combined with molecules that can inhibit the activity of both IDO1 and IDO2. Disclosures: Metz: NewLink Genetics: Employment. Prendergast:New Link Genetics Corp: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.


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