The Role of Elastase in the Digestion of E. coli Proteins by Human Polymorphonuclear Leukocytes I. Experiments in Vitro

The adherence of human polymorphonuclear leukocytes (PMN) to nylon fibers is inhibited in a dose-dependent fashion by exposure in vitro of these cells to either colchicine or VM-26, both of which agents prevent microtubule assembly. Mean adherence of human PMN was 48% +/- 2%, following treatment with 10(-5) M colchicine or 10(-4) M VM-26 it was reduced to 31% +/- 2% and 7%, respectively. Peritoneal PMN obtained from mice and mink with Chediak-Higashi syndrome (CHS) thought to have a microtubule-membrane disorder affecting the PMN had a mean adherence of 29% +/- 3% and 40% +/- 8% compared to control values of 46% +/- 5% and 73% +/- 8%, respectively, from the mice and mink. Both ascorbic acid and bethanechol, shown previously to enhance microtubule assembly in humans with CHS, normalized granulocyte adherence in PMN obtained from mice with CHS. Cyclic nucleotide levels were not altered by treatment of human PMN with colchicine, nor did they differ between normal and CHS animals. Thus it appears that the state of microtubule assembly may directly affect the properties of the PMN plasma membrane without requiring alterations of cyclic nucleotides as an intermediary.

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Role of microtubules in granulocyte adherence

Blood ◽

10.1182/blood.v51.6.1045.1045 ◽

1978 ◽

Vol 51 (6) ◽

pp. 1045-1050 ◽

Cited By ~ 24

Author(s):

LA Boxer ◽

JM Allen ◽

AM Watanabe ◽

HR Jr Besch ◽

RL Baehner

Keyword(s):

Ascorbic Acid ◽

Cyclic Nucleotides ◽

Polymorphonuclear Leukocytes ◽

Microtubule Assembly ◽

Dose Dependent Fashion ◽

Chediak Higashi Syndrome ◽

Human Polymorphonuclear Leukocytes ◽

Dose Dependent

Abstract The adherence of human polymorphonuclear leukocytes (PMN) to nylon fibers is inhibited in a dose-dependent fashion by exposure in vitro of these cells to either colchicine or VM-26, both of which agents prevent microtubule assembly. Mean adherence of human PMN was 48% +/- 2%, following treatment with 10(-5) M colchicine or 10(-4) M VM-26 it was reduced to 31% +/- 2% and 7%, respectively. Peritoneal PMN obtained from mice and mink with Chediak-Higashi syndrome (CHS) thought to have a microtubule-membrane disorder affecting the PMN had a mean adherence of 29% +/- 3% and 40% +/- 8% compared to control values of 46% +/- 5% and 73% +/- 8%, respectively, from the mice and mink. Both ascorbic acid and bethanechol, shown previously to enhance microtubule assembly in humans with CHS, normalized granulocyte adherence in PMN obtained from mice with CHS. Cyclic nucleotide levels were not altered by treatment of human PMN with colchicine, nor did they differ between normal and CHS animals. Thus it appears that the state of microtubule assembly may directly affect the properties of the PMN plasma membrane without requiring alterations of cyclic nucleotides as an intermediary.

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Isolation, Characterization, cDNA Cloning, and Antimicrobial Properties of Two Distinct Subfamilies of α-Defensins from Rhesus Macaque Leukocytes

Infection and Immunity ◽

10.1128/iai.67.11.6139-6144.1999 ◽

1999 ◽

Vol 67 (11) ◽

pp. 6139-6144 ◽

Cited By ~ 41

Author(s):

Yi-Quan Tang ◽

Jun Yuan ◽

Christopher J. Miller ◽

Michael E. Selsted

Keyword(s):

Rhesus Macaque ◽

Host Defense ◽

Polymorphonuclear Leukocytes ◽

Nonhuman Primates ◽

Antimicrobial Properties ◽

Posttranslational Processing ◽

E Coli ◽

Gram Positive Bacteria

ABSTRACT Experiments to isolate and characterize rhesus macaque myeloid α-defensins (RMADs) were conducted. Seven RMAD peptides were isolated and sequenced, and the cDNAs encoding six of these peptides and one other α-defensin from bone marrow were also characterized. Four of the RMADs were found to be highly similar to human neutrophil α-defensins HNP-1 to HNP-3, while the remaining four peptides were much more similar to human enteric α-defensin HD-5. Two α-defensin pairs differed only by the presence or absence of an additional arginine at the amino termini of their mature peptides, indicative of alternate posttranslational processing. The primary translation products of RMAD-1 to -8 are 94- and 96-amino-acid prepropeptides that are highly similar to those of human α-defensins. Immunolocalization experiments revealed a granular cytoplasmic pattern in the cytoplasms of neutrophils, indistinguishable from the pattern observed after immunostaining of human myeloid α-defensins in polymorphonuclear leukocytes. Each of the purified peptides was tested for its in vitro activities against Staphylococcus aureus 502a,Listeria monocytogenes EGD, Escherichia coliML35, and Cryptococcus neoformans 271A. Several of the peptides were microbicidal for the gram-positive bacteria and C. neoformans at defensin concentrations in the range of 2 to 5 μM. All of the peptides were bacteriostatic against E. coli, but none were bactericidal for this organism. This study is the first to characterize the sequences and activities of α-defensins from nonhuman primates, data that should aid in delineating the role of these peptides in rhesus macaque host defense.

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Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

Applied Sciences ◽

10.3390/app11156865 ◽

2021 ◽

Vol 11 (15) ◽

pp. 6865

Author(s):

Eun Seon Lee ◽

Joung Hun Park ◽

Seong Dong Wi ◽

Ho Byoung Chae ◽

Seol Ki Paeng ◽

...

Keyword(s):

Cold Stress ◽

Wild Type ◽

Rna Chaperone ◽

E Coli ◽

Cold Sensitive ◽

Thioredoxin H ◽

Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

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The Use of the Vitality and Chemotaxis of Human Polymorphonuclear Leukocytes for the In Vitro Estimation of the Acute Toxicity of the First Ten Chemicals from the MEIC List

Alternatives to Laboratory Animals ◽

10.1177/026119299302100112 ◽

1993 ◽

Vol 21 (1) ◽

pp. 73-80

Author(s):

Matteo Valentino ◽

Francesca Monaco ◽

Maria Antonietta Pizzichini ◽

Mario Governa

Keyword(s):

Ethidium Bromide ◽

Polymorphonuclear Leukocytes ◽

Rank Correlation ◽

Fluorescein Diacetate ◽

Live Cells ◽

In Vitro Toxicity ◽

Ic50 Values ◽

Acute Cytotoxicity ◽

Human Polymorphonuclear Leukocytes

The acute cytotoxicity of the first ten MEIC chemicals has been estimated by others in various cell lines. In the present investigation, isolated human polymorphonuclear leukocytes (PMN) from ten healthy non-smoking laboratory personnel were used to assess in vitro toxicity of the same chemicals. The cells were treated with different concentrations of the respective chemicals for three hours and their vitality and chemotaxis were tested. Vitality was measured by fluorescence microscopy after the addition of fluorescein diacetate and ethidium bromide. Living cells which took up and hydrolysed fluorescein diacetate, and dead cells, stained by ethidium bromide, were counted and the percentage of live cells was calculated. Locomotion stimulated by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (F-MLP), was measured in blind-well Boyden chambers and a chemotactic index was calculated. The results were mathematically transformed to produce a linear curve, and then fitted by the linear least squares procedure, from which LC50 and IC50 values were obtained by interpolation. All the chemicals decreased the vitality and inhibited the chemotaxis of the PMN. Obviously the chemotactic test was more sensitive than the vitality one. A correlation (r = 0.933) was found between vitality and chemotaxis inhibition. Spearman rank correlation analysis revealed significant correlations between our results and those from in vitro experiments conducted in other laboratories, as well as with data concerning mouse, rat and human lethal doses.

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Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa315 ◽

2020 ◽

Author(s):

Lina Y Alkaissi ◽

Martin E Winberg ◽

Stéphanie DS Heil ◽

Staffan Haapaniemi ◽

Pär Myrelid ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Ex Vivo ◽

Ussing Chambers ◽

E Coli ◽

Follicle Associated Epithelium ◽

Vitro Model ◽

Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

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The role of adhesive interactions and extracellular matrix fibronectin from human polymorphonuclear leukocytes in the respiratory burst

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/0167-4889(91)90187-3 ◽

1991 ◽

Vol 1091 (3) ◽

pp. 257-260 ◽

Cited By ~ 10

Author(s):

G.F. Sud'ina ◽

A.V. Tatarintsev ◽

A.A. Koshkin ◽

S.V. Zaitsev ◽

N.A. Fedorov ◽

...

Keyword(s):

Extracellular Matrix ◽

Respiratory Burst ◽

Polymorphonuclear Leukocytes ◽

Adhesive Interactions ◽

Human Polymorphonuclear Leukocytes

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Influence of Gelatin-Thrombin Matrix Tissue Sealant on Bacterial Colony Formation and Risk of Pelvic Infection

Infectious Diseases in Obstetrics and Gynecology ◽

10.1155/2016/2649708 ◽

2016 ◽

Vol 2016 ◽

pp. 1-6

Author(s):

Michael J. Jarrett ◽

Andres Vázquez-Torres ◽

Daniel N. Frank ◽

Bruce D. McCollister ◽

Patrick K. Henthorn ◽

...

Keyword(s):

Colony Formation ◽

Rrna Gene ◽

Contributing Factors ◽

Vaginal Cuff ◽

Pelvic Infection ◽

Bacterial Colony ◽

E Coli ◽

Tissue Sealant

Objective. Gelatin-thrombin matrix (GTM) tissue sealant use was previously identified as an independent predictor of pelvic infection following hysterectomies. We aim to elucidate contributing factors by assessing influence of GTM on bacterial colony formation and characterizing bacteria present at the vaginal cuff.Methods.Escherichia coliwas incubated in phosphate-buffered saline (PBS) and pelvic washings with and without GTM to assess influence on colony formation. Pelvic washings of the vaginal cuff were collected from hysterectomies occurring from June through October 2015.In vitrotechniques, 16S rRNA gene qPCR, and 16S amplicon sequencing were performed with washings to characterize bacteria at the vaginal cuff.Results. Mean bacterial colony formation in PBS was greater forE. coliincubated in the presence of GTM (1.48 × 107 CFU/mL) versus without (9.95 × 105 CFU/mL) following 20-hour incubation (p=0.001). Out of 61 pelvic washings samples, 3 were culture positive (≥5000 CFU/mL) withEnterococcus faecalis.Conclusion.In vitroexperiments support a facilitating role of GTM on colony formation ofE. coliin PBS. However, given the negative results of surgical site washings following adequate disinfection, the role of GTM in promoting posthysterectomy pelvic infections may be limited. Analysis of pelvic washings revealed presence ofE. faecalis, but results were inconclusive. Further studies are recommended.

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