Increased Circulating Proplatelets and Elevated Plasma TPO Levels: A Novel Pathological Mechanism of Cerebral Infarction

Background: Platelets are produced by megakaryocytes and are primarily regulated by thrombopoietin (TPO). In the conditions of inflammation or stress, increased 5-HT and other inflammatory cytokines may affect liver and bone marrow to increase TPO and platelet production (Yang et al., Stem Cells, 2007; 2014). Mature megakaryocytes are fragmented in pulmonary circulation to generate more platelet intermediate proplatelets, then entered the circulation. We hypothesized that in patients with cerebral infarction, the secretion of inflammatory cytokines increases under stress or inflammatory conditions, increasing circulating TPO levels, followed by an increase in platelet production, and an increase in the number of platelet intermediate proplatelets. At the same time, platelets also have varying degrees of activation. Therefore, the increase in the number of proplatelets in the circulation and the increase in TPO levels may be the new pathological mechanism of cerebral infarction. Methods: The plasma levels of TPO were detected by ELISA. The in vivo proplatelets from patients were stained with CD41-FITC, which was further confirmed under a fluorescence microscope and flow cytometry. The in vitro proplatelets were observed in a culture system by CD41-FITC staining under a fluorescence microscope and flow cytometry detection. Results: Our clinical data demonstrated that patients with acute inflammation states, plasma TPO levels were significantly higher compared with healthy subjects (181.1 ± 35.38 vs 96.1 ± 9.7 pg/ml, p<0.001, n=65), which may act as an acute response protein to protect the body. We also detected 20 patients with cerebral infarction and 20 normal controls and found that the plasma TPO levels of cerebral infarction patients (186.2±12.5 pg/ml) was significantly higher than that of the control group (122.3±10.2 pg/ml). The number of platelets in patients with cerebral infarction (222.11±8.55 ×109/L, n=20) was slightly higher than that in the control group (206.55±8.83 ×109/L, n=20). More importantly, we found more large platelets or proplatelets in the circulating blood of patients with cerebral infarction, which was significantly different from the control group (n=8). Furthermore, in vitro study confirmed that in 200 megakaryocytes (MKs), (18±2.5)% of MKs producing proplatelets in the TPO group (100 ng/ml), and in 5-HT group (100 nM), (15±3.2)% of MKs producing proplatelets, while the control group was only (7±3.2)% (n=5). At the same time, the effects of TPO and 5-HT were interfered by the corresponding receptor blockers, confirming that their effects were mediated by its receptors. The study further confirmed that both TPO and 5-HT affect the cytoskeleton by activating p-ERK1/2, reorganizing F-actin to generate proplatelets, and its role was blocked by PD98059. Conclusions: The study demonstrated that increased numbers of proplatelets in circulation and elevated plasma TPO levels may be novel pathological mechanisms of cerebral infarction Disclosures No relevant conflicts of interest to declare.

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Abstract 15499: The Increase of Proplatelets and Tpo in Circulation May be a New Pathological Mechanism of Thrombosis

Circulation ◽

10.1161/circ.142.suppl_3.15499 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Mo Yang ◽

Liang Li ◽

hua zhang ◽

kuan zeng ◽

Yanqi Yang

Keyword(s):

Flow Cytometry ◽

Cerebral Infarction ◽

Acute Inflammation ◽

Production Capacity ◽

Fluorescence Microscope ◽

Control Group ◽

Pathological Mechanism ◽

Receptor Blockers ◽

Flow Cytometry Detection

Introduction: Platelets are produced from megakaryocytes, which mainly regulated by thrombopoietin (TPO) and 5-HT. We hypothesized that the secretion of inflammatory cytokines increased in patients with thrombosis under stress or inflammation, leading to increased TPO levels, which in turn increased production capacity of platelet, and increased the number of platelet intermediates proplatelets into the circulation. Therefore, the increase in the number of proplatelets in the circulation, and the increase in plasma TPO levels may be a new pathological mechanism of thrombosis. Methods: TPO was detected by ELISA. Proplatelets from patients were stained with CD41-FITC and further confirmed by a fluorescence microscope and flow cytometry. The in vitro proplatelets were tested by CD41-FITC staining under a fluorescence microscope and flow cytometry detection. Results: Our clinical data demonstrated that patients' plasma TPO levels were significantly higher with acute inflammation state compared with healthy subjects (181.1 ± 35.38 vs 96.1 ± 9.7 pg/ml, p<0.001, n=65). The plasma TPO levels of cerebral infarction patients (186.2±12.5 pg/ml, n=20) were significantly higher than that of the control group (122.3±10.2 pg/ml, n=20). The number of platelets in patients with cerebral infarction (222.11±8.55 х10 9 /L, n=20) was slightly higher than that in the control group (206.55±8.83 х10 9 /L, n=20). More large platelets or proplatelets were found in the circulating blood of patients with cerebral infarction, which was significantly different from the control group (n=8, p<0.05). In vitro study confirmed that in 200 megakaryocytes (MKs), more proplatelets were produced in the TPO group (100 ng/ml, 18±2.5%) and in the 5-HT group (100 nM, 15±3.2%), while the control group was only (7±3.2) % (n=5). The effects of TPO/ 5-HT were interfered with by its receptor blockers, confirming that their effects were mediated by its receptors. Both TPO and 5-HT affect the cytoskeleton by activating p-ERK1/2, reorganizing F-actin to generate proplatelets, and its role was blocked by PD98059. Conclusions: The studies showed that the increase of proplatelets in circulation and TPO levels in plasma may be a new pathological mechanism of cerebral infarction or thrombosis.

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Evaluating the Effect of Cinnarizine on Promastigotes and Amastigotes forms of Leishmania major

Infectious Disorders - Drug Targets ◽

10.2174/1871526518666181113114820 ◽

2020 ◽

Vol 20 (4) ◽

pp. 550-555 ◽

Cited By ~ 1

Author(s):

Lima Asgharpour Sarouey ◽

Parvaneh Rahimi-Moghaddam ◽

Fatemeh Tabatabaie ◽

Khadijeh Khanaliha

Keyword(s):

Flow Cytometry ◽

Cutaneous Leishmaniasis ◽

Leishmania Major ◽

Inhibitory Effect ◽

Control Group ◽

Secondary Infections ◽

Ic50 Values ◽

J774 Cells ◽

Mtt Assays

: As an important global disease, cutaneous leishmaniasis is associated with complications such as secondary infections and atrophic scars. The first line treatment with antimonials is expensive and reported to have serious side effects and enhance resistance development. The main objective of this study was to evaluate the effect of Cinnarizine on standard strains of Leishmania major because of paucity of information on this subject. Methods: In this experimental study, four concentrations of the drug (5, 10, 15 and 20 μg/ml) were added to Leishmania major cultures at 24, 48 and 72 hours intervals. MTT assays were performed to determine parasite viability and drug toxicity. Leishmania major promastigotes were augmented to the in vitro cultured macrophages (J774 cells) and then incubated for 72 hours. Half maximal inhibitory concentration (IC50) was ascertained by counting parasites. The inhibitory effect of the drug was compared with that of Glucantime. Flow-cytometry was performed to investigate apoptosis. Each test was repeated thrice. Results: The IC50 values of Cinnarizine after 72 hours were calculated to be 34.76 μg/ml and 23.73 μg/ml for promastigotes and amastigotes, respectively. The results of MTT assays showed 48 % promastigote viability after 72 hour-exposure to Cinnarizine at 20 μg/ml concentration. Programmed cell death in promastigote- and amastigote-infected macrophages was quantified to be 13.66 % and 98.7 %, respectively. Flow- cytometry analysis indicated that Cinnarizine induced early and late apoptosis in parasites. All treatments produced results which differed significantly from control group (P<0.05). Conclusion: Cinnarizine showed low toxicity with anti-leishmanial and apoptosis effects on both promastigote and intracellular amastigote forms. Therefore, we may suggest further assessment on animal models of this drug as candidates for cutaneous leishmaniasis therapy.

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Allogenic Pure Platelet-Rich Plasma Therapy for Adhesive Capsulitis: A Bed-to-Bench Study With Propensity Score Matching Using a Corticosteroid Control Group

The American Journal of Sports Medicine ◽

10.1177/03635465211018636 ◽

2021 ◽

pp. 036354652110186

Author(s):

Min Ji Lee ◽

Kang Sup Yoon ◽

Sohee Oh ◽

Sue Shin ◽

Chris Hyunchul Jo

Keyword(s):

Muscle Strength ◽

Propensity Score ◽

Inflammatory Cytokines ◽

Platelet Rich Plasma ◽

Corticosteroid Injection ◽

Adhesive Capsulitis ◽

Shoulder Function ◽

Control Group ◽

Inflammatory Condition

Background: While platelet-rich plasma (PRP) has been widely studied for musculoskeletal disorders, few studies to date have reported its use for adhesive capsulitis (AC). Fully characterized and standardized allogenic PRP may provide clues to solve the underlying mechanism of PRP with respect to synovial inflammation and thus may clarify its clinical indications. Purpose: To clinically evaluate the safety and efficacy of a fully characterized pure PRP injection in patients with AC and to assess the effects of pure PRP on synoviocytes with or without inflammation in vitro. Study Design: Controlled laboratory study and cohort study; Level of evidence, 3. Methods: For the clinical analysis, a total of 15 patients with AC received an ultrasonography-guided intra-articular PRP injection and were observed for 6 months. Pain, range of motion (ROM), muscle strength, shoulder function, and overall satisfaction in the patients were evaluated using questionnaires at 1 week as well as at 1, 3, and 6 months after the PRP injection and results were compared with the results of a propensity score−matched control group that received a corticosteroid injection (40 mg triamcinolone acetonide). For the in vitro analysis, synoviocytes were cultured with or without interleukin-1β (IL-1β) and PRP. The gene expression of proinflammatory and anti-inflammatory cytokines as well as matrix enzymes and their inhibitors was evaluated. Results: At 6-month follow-up, pure PRP significantly decreased pain and improved ROM, muscle strength, and shoulder function to levels comparable with those after a corticosteroid injection. All pain values, strength measurements, and functional scores significantly improved up to 6 months in the PRP group, but these measures improved up to 3 months and then were decreased at 6 months in the corticosteroid group. ROM was significantly improved in the 2 groups at 6 months compared with baseline. Allogenic PRP did not cause adverse events. For the in vitro findings, PRP induced inflammation but significantly improved the IL 1β−induced synovial inflammatory condition by decreasing proinflammatory cytokines such as IL-1β, tumor necrosis factor−α, IL-6, cyclooxygenase-2, and microsomal prostaglandin E synthase−1 and decreased matrix enzymes (matrix metalloproteinase−1, −3, and −13 as well as a disintegrin and metalloproteinase with thrombospondin motifs−4 and −5) and further increasing anti-inflammatory cytokines such as vasoactive intestinal peptide. Conclusion: This study showed that PRP decreased pain and improved shoulder ROM and function to an extent comparable with that of a corticosteroid in patients with AC. Allogenic pure PRP acted in a pleiotropic manner and decreased proinflammatory cytokines only in the inflammatory condition. Clinical Relevance: Allogenic PRP could be a treatment option for the inflammatory stage of AC.

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Silica/OCP Affects the Viability of Osteoblasts through ROS-Induced Autophagy

Journal of Nanomaterials ◽

10.1155/2021/3159848 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Jianghao Gong ◽

Shangjun Fu ◽

Zhenghao Zhou

Keyword(s):

Flow Cytometry ◽

Cell Viability ◽

Protein Level ◽

Reactive Oxygen ◽

Octacalcium Phosphate ◽

Control Group ◽

Substrate Protein ◽

Autophagy Inhibitor ◽

Osteoblast Cell Line

Objective. To explore the effects of silicone gel nanoparticles modified with octacalcium phosphate on the surface (silica/OCP) polymer drugs on the proliferation of osteoblasts and autophagy. Method. Silica/OCP was prepared in vitro, and the quality of the sample preparation was tested through characterization experiments. The osteoblast cell line (hFOB1.19) was treated with silica/OCP, autophagy inhibitor (3-methyladenine (3-MA)), and silica/OCP+3-MA, respectively. The proliferation of hFOB1.19 cells was detected through the methylthiazolyldiphenyl-tetrazolium bromide (MTT) kit. Flow cytometry was used to detect the cell apoptosis. The change in protein beclin1 and P62 expression in hFOB1.19 cells was observed in Western blot. An ROS detection kit was used to detect the content of reactive oxygen species in hFOB1.19 cells. Results. Silica/OCP was a sphere with a particle size of 50 nm to 130 nm and had an OCP phase in electron projection microscopy and X-ray diffraction techniques. The results indicated that OCP successfully modified silica and the material was successfully prepared. An MTT kit and flow cytometry test showed that the cell viability of the cells treated with silica/OCP increased significantly ( P < 0.05 ), and the intracellular apoptosis phenomenon was significantly decreased ( P < 0.05 ) compared to the control group. Moreover, the inhibition of cell viability and promotion of apoptosis caused by the autophagy inhibitor 3-MA can be rescued. Western blotting demonstrated that the protein level of beclin1 in osteoblasts reached the highest after six hours of treatment with silica/OCP, and the protein level of p62, the substrate protein of autophagy, reached the lowest. At the same time, treatment of cells with the autophagy inhibitor 3-MA and silica/OCP+3-MA found that the protein levels of beclin1 and p62 in the silica/OCP+3-MA group were adjusted back compared to the 3-MA group. After adding the autophagy inhibitor, the reactive oxygen content in the cell was significantly increased ( P < 0.05 ) in the silica/OCP group. In the presence of intracellular reactive oxygen inhibitors catalase and silica/OCP, the cell viability of osteoblasts was significantly lower than that of the silica/OCP group but significantly higher than that of the silica/OCP+3-MA group. The apoptosis level of the silica/OCP+catalase group was also significantly lower than that of the silica/OCP+3-MA group ( P < 0.05 ) but was significantly higher than that of the silica/OCP group ( P < 0.05 ). Conclusion. Silica/OCP nanoparticles can upregulate the level of autophagy in osteoblasts and promote the proliferation of osteoblasts.

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Contributions of TRAIL-mediated megakaryocyte apoptosis to impaired megakaryocyte and platelet production in immune thrombocytopenia

Blood ◽

10.1182/blood-2010-02-267435 ◽

2010 ◽

Vol 116 (20) ◽

pp. 4307-4316 ◽

Cited By ~ 39

Author(s):

Lei Yang ◽

Lin Wang ◽

Chun-hong Zhao ◽

Xiao-juan Zhu ◽

Yu Hou ◽

...

Keyword(s):

Immune Thrombocytopenia ◽

Mononuclear Cells ◽

Lower Percentage ◽

Control Group ◽

Platelet Production ◽

Cd34 Cells ◽

Culture Supernatants ◽

Impaired Quality ◽

Necrosis Factor

Abstract Recent in vitro studies provide evidence for autoantibody-induced suppression of megakaryocytopoiesis and show a reduction in megakaryocyte production and maturation in the presence of immune thrombocytopenia (ITP) plasma. Here, we present CD34+ cells from healthy umbilical cord blood mononuclear cells cultured in medium containing thrombopoietin, stem cell factor, interleukin-3, and 10% plasma from either ITP patients or healthy subjects. The quantity, quality, and apoptosis of megakaryocytes were measured. We observed that most ITP plasma boosted megakaryocyte quantity but impaired quality, resulting in significantly less polyploidy cells (N ≥ 4) and platelet release. In these megakaryocytes, we found a lower percentage of cell apoptosis, a lower expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and a higher expression of Bcl-xL. Furthermore, there was a decrease of sTRAIL in ITP plasma and in cell culture supernatants of this group compared with the control group. Our findings suggest that decreased apoptosis of megakaryocytes also contributes to in vitro dysmegakaryocytopoiesis and reduced platelet production. The abnormal expression of sTRAIL in plasma and TRAIL and Bcl-xL in megakaryocytes may play a role in the pathogenesis of impaired megakaryocyte apoptosis in ITP.

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Octreotide ameliorates hypoxia/reoxygenation-induced cerebral infarction by inhibiting oxidative stress, inflammation and apoptosis, and via inhibition of TLR4/MyD88/NF-κB signaling pathway

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v20i11.4 ◽

2021 ◽

Vol 20 (11) ◽

pp. 2261-2266

Author(s):

Yanbin Hou ◽

Zhongze Lou ◽

Yunxin Ji ◽

Liemin Ruan ◽

He Gao

Keyword(s):

Oxidative Stress ◽

Cerebral Infarction ◽

Signaling Pathway ◽

Control Group ◽

N2a Cells ◽

Tnf Α ◽

Octreotide Treatment ◽

Vitro Model ◽

Hypoxia Reoxygenation

Purpose: To explore the effects of octreotide (OCT) on oxidative stress, inflammation and apoptosis in hypoxia/reoxygenation (H/R)-induced cerebral infarction.Methods: The in vitro model of cerebral infarction was established by treating N2A cells with hypoxia for 4 h and reoxygenation for 24 h. The viability of N2A cells was determined by CCK-8 assay. The cells were divided into 3 groups: control group, H/R group, and H/R+OCT group. The cells in H/R+OCT group were pretreated with OCT (60 ng/mL) before H/R treatment. The oxidative stress of N2A cells were assessed by determining the levels of superoxide dismutase (SOD), glutathione peroxidase (GSHPx), catalase (CAT), reactive oxygen species (ROS) and malondialdehyde (MDA). Inflammation of N2A cells was evaluated by evaluating the levels of TNF-α, IL-1β, IL-6, and IL-8, while the apoptosis of N2A cells was assessed by flow cytometry. Western blot analysis was used to determine the expression of Bcl-2, Bax, TLR4, MyD88, and NF-κB.Results: Octreotide treatment significantly reduced the level of oxidative stress. The inflammation of N2A cells caused by hypoxia/reoxygenation was inhibited by treatment with octreotide. Apoptosis of N2A cells was also inhibited by octreotide treatment. Hypoxia/reoxygenation activated TLR4/MyD88/NF-κB signaling pathway, while octreotide inhibits the activation of this pathway.Conclusion: The results reveal that octreotide inhibits hypoxia/reoxygenation-induced oxidative stress,as well as the inflammation, and apoptosis of N2A cells by inhibiting TLR4/MyD88/NF-κB signaling pathway. Thus, these findings may provide new insights into the treatment of cerebral infarction.

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Evaluation of Morphine with Imiquimod as Opioid Growth Factor Receptor or Nalmefene as Opioid Blocking Drug on Leishmaniasis Caused by Leishmania major in Vitro

Iranian Journal of Parasitology ◽

10.18502/ijpa.v14i3.1478 ◽

2019 ◽

Cited By ~ 1

Author(s):

Javad JABARI ◽

Fatemeh GHAFFARIFAR ◽

John HORTON ◽

Abdolhosein DALIMI ◽

Zohreh SHARIFI

Keyword(s):

Flow Cytometry ◽

Growth Factor ◽

Leishmania Major ◽

Growth Factor Receptor ◽

Adjunctive Treatment ◽

Control Group ◽

High Dose ◽

Macrophage Infection ◽

Opioid Growth Factor

Background: In this research, the effect of morphine on promastigotes and amastigotes of Leishmania major has been investigated in the presence of nalmefene as a blocking opioid drug and imiquimod as an opioid growth factor receptor. Methods: This study was conducted at Tarbiat Modares University, Tehran, Iran in 2015-2018. Morphine with different concentration (0.1, 1, 10 and 100 1µg/ml) alone and with imiquimod (0.01, 0.1 and 1µg/ml) and nalmefene (0.1, 1 and 10 µg/ml) on promastigotes and amastigotes in macrophages and also the percentage of infected macrophages was investigated. For evaluation of the apoptosis, we used flow cytometry method. The effect of imiquimod and nalmefene on glucantime and amphotericin B as current drugs for treatment of leishmaniasis was evaluated too. Results: The effect of morphine on promastigotes and amastigotes has a reverse relationship with its concentration. The results of flow cytometry for drug-treated promastigotes revealed that apoptosis and necrosis did not increase markedly relative to the control group. A combination of morphine and imiquimod in concentrations of 0.05, 5 and 5 µg/ml had a pronounced effect on reduction and prevention of macrophage infection with amastigotes. Morphine at a concentration of 0.1 µg/ml plays the role of adjunctive treatment. In amastigote assay we found the better results in group that get glucantime 25 µg/ml+ imiquimod 0.5 µg/ml. Conclusion: This effect is strengthened with imiquimod and weakened with nalmefene. Using high dose morphine and nalmefene had reverse effects. They suppress immune system and had no controlling effect in macrophages amastigote infection and reduction of promastigotes.

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β2M Signals Monocytes Through Non-Canonical TGFβ Receptor Signal Transduction

Circulation Research ◽

10.1161/circresaha.120.317119 ◽

2021 ◽

Author(s):

Zachary T Hilt ◽

Preeti Maurya ◽

Laura Tesoro ◽

Daphne N Pariser ◽

Sara K Ture ◽

...

Keyword(s):

Signal Transduction ◽

Flow Cytometry ◽

Nuclear Localization ◽

Ubiquitin Ligase ◽

Dependent Manner ◽

Elevated Plasma ◽

Imaging Flow Cytometry ◽

Tgfβ Receptor

Rationale: Circulating monocytes can have pro-inflammatory or pro-reparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet derived beta-2 microglobulin (β2M) and transforming growth factor beta (TGFβ) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes respectively, but each bind and signal through the same receptor. We now define the signaling pathways involved. Objective: To determine the molecular mechanisms and signal transduction pathways by which β2M and TGFβ regulate monocyte responses both in vitro and in vivo. Methods and Results: Wild-type (WT) and platelet specific β2M knockout (Plt-β2M -/- ) mice were treated intravenously with either β2M or TGFβ to increase plasma concentrations to those in cardiovascular diseases. Elevated plasma β2M increased pro-inflammatory monocytes, while increased plasma TGFβ increased pro-reparative monocytes. TGFβ receptor (TGFβR) inhibition blunted monocyte responses to both β2M and TGFβ in vivo. Using imaging flow cytometry, we found that β2M decreased monocyte SMAD2/3 nuclear localization, while TGFβ promoted SMAD nuclear translocation, but decreased non-canonical/inflammatory (JNK and NFκB nuclear localization). This was confirmed in vitro using both imaging flow cytometry and immunoblots. β2M, but not TGFβ, promoted ubiquitination of SMAD3 and SMAD4, that inhibited their nuclear trafficking. Inhibition of ubiquitin ligase activity blocked non-canonical SMAD-independent monocyte signaling and skewed monocytes towards a pro-reparative monocyte response. Conclusions: Our findings indicate that elevated plasma β2M and TGFβ dichotomously polarize monocytes. Furthermore, these immune molecules share a common receptor, but induce SMAD-dependent canonical signaling (TGFβ) versus non-canonical SMAD-independent signaling (β2M) in a ubiquitin ligase dependent manner. This work has broad implications as β2M is increased in several inflammatory conditions, while TGFβ is increased in fibrotic diseases.

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VIABILITAS BAKTERI PROBIOTIK IN-VITRO DAN PENGARUH PEMBERIAN AIR OKSIGEN TERHADAP PERTUMBUHAN BAKTERI PROBIOTIK SECARA IN-VIVO

Jurnal Gizi dan Pangan ◽

10.25182/jgp.2007.2.1.22-28 ◽

2007 ◽

Vol 2 (1) ◽

pp. 22

Author(s):

Enok Sobariah ◽

Ali Khomsan ◽

Ingrid S. Surono

Keyword(s):

Body Weight ◽

Lactic Acid ◽

Lactic Acid Bacteria ◽

Day Treatment ◽

The Body ◽

Probiotic Bacteria ◽

Control Group ◽

Oxygenated Water

The aim of this study were to identify the in-vitro tolerance of pro-biotic bacteria to acid and bile salt condition; and to prove a hypothesis that the supplementation of oxygenated water has a positive effect on the body weight of rat and on viability of pro-biotic bacteria. The first study was carried out at PAU Laboratory of Bogor Agricultural University, while the second study was conducted at Department of Community Nutrition of Bogor Agricultural University and Microbiology Laboratory of Indonesia Institute of Technology. Forty five rats aged 6 weeks were divided into three groups, i.e., control group without probiotic (a0), Lactobacillus casei Shirota (a1), and Lactobacillus IS-7257 (a2). Each group (consisting of 5 rats each) has three different treatments, namely, control without oxygenated water (b0), 50 ppm oxygenated water (b2), and 80 ppm oxygenated water (b2). Oxygenated water was administered to the rats twice a day in the morning (3.25 ml) and afternoon (3.00 ml). Observation was carried out on the body weight of the rats, fecal lactic acid bacteria, coliform, and anaerob bacteria by plate counting, for 4 periods, i.e, prior to the treatment (C0), after three-day treatment (C1), after seven-day treatment (C2), and on the 10th day treatment or three days after washed out period. The results indicated that probiotic bacteria are resistant to acid and bile acid condition. Oxygen concentration in water has a significant positive influence on the body weight of rats towards viability of probiotic bacteria (p-level < 0.05). The supplementation of oxygenated water 50 ppm significantly increase the population of viable fecal lactic acid bacteria in L. casei Shirota and Lactobacillus IS-7257 groups after 3 and 7 days of treatment. Lactobacillus IS-7257 gave better response than L. casei Shirota. The supplementation of oxygenated water 80 ppm significantly reduces the fecal coliform in-vivo in both L. casei Shirota and Lactobacillus IS-7257 groups (p-level < 0.05).

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In vitro observation: the GFP-E. coli adhering to porcine erythrocytes can be removed by porcine alveolar macrophages

PeerJ ◽

10.7717/peerj.6439 ◽

2019 ◽

Vol 7 ◽

pp. e6439 ◽

Cited By ~ 1

Author(s):

Wei Yin ◽

Chun Wang ◽

Kuohai Fan ◽

Na Sun ◽

Yaogui Sun ◽

...

Keyword(s):

Flow Cytometry ◽

Confocal Microscopy ◽

Fluorescence Intensity ◽

Alveolar Macrophages ◽

Control Group ◽

Laser Confocal Microscopy ◽

Functional Protein ◽

Blank Control Group ◽

E Coli

Although the activation of pathogen phagocytosis via complement system has been studied, erythrocyte-phagocyte interactions in pigs are not clearly understood. Therefore, we sought to investigate the ability of porcine erythrocytes to clear immune complexes (ICs) by using laser confocal microscopy and flow cytometry to observe the immune adhesion of porcine erythrocytes to fluorescent bacilli and the immune presentation process of transferring fluorescent bacilli to macrophages. Isolated porcine alveolar macrophages (PAMs) had uniform morphology and size, and a survival rate of 97.2%. The phagocytosis rate was 98.8%. After WT E. coli was labeled with Fluorescein Isothiocyanate (FITC), the bacteria showed a bright green fluorescence, and the labeling rate was 92.3%. When laser confocal microscopy was utilized to observe the co-incubation system of porcine erythrocytes, PAM, and fluorescent E. coli, the fluorescence intensity of bacilli decreased with increasing observation time and even disappeared. Flow Cytometry examination showed that the average fluorescence intensity of PAMs co-incubated with porcine erythrocytes adhered to WT-E. coli-FITC, was significantly higher than that of normal PAMs. Furthermore, when porcine erythrocytes adhered to WT E. coli were incubated with PAMs, the surface mean fluorescence intensity of porcine erythrocytes was significantly higher than that of the blank control group. This shows that PAMs can competitively bind to the oposinized E. coli adhered to the surface of porcine erythrocytes, and these oposinized pathogens can enter macrophages by the process of phagocytosis, which promoting the internalization of ICs or pathogens. During this process, the physical morphology of porcine erythrocytes was not damaged, but the levels of its main functional protein CR1-like were reduced.

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