Shiga toxin stimulates leukocyte adhesion in rat mesenteric microcirculation in vivo

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). N/OFQ causes hypotension and vasodilation, and we aimed to determine the role of histamine in inflammatory microvascular responses to N/OFQ. Male Wistar rats (220–300 g, n = 72) were anesthetized with thiopental (30 mg/kg bolus, 40–90 mg·kg−1·h−1 iv), and the mesentery was prepared for fluorescent intravital microscopy using fluorescein isothiocyanate-conjugated BSA (FITC-BSA, 0.25 ml/100 g iv) or 1 μm fluorescently labeled microspheres. N/OFQ (0.6–60 nmol/kg iv) caused hypotension (SAP, baseline: 154 ± 11 mmHg, 15 nmol/kg N/OFQ: 112 ± 10 mmHg, P = 0.009), vasodilation (venules: 23.9 ± 1.2 μm, 26.7 ± 1.2 μm, P = 0.006), macromolecular leak (interstitial gray level FITC-BSA: 103.7 ± 3.4, 123.5 ± 11.8, P = 0.009), and leukocyte adhesion (2.0 ± 0.9, 15.2 ± 0.9/100 μm, P = 0.036). Microsphere velocity also decreased (venules: 1,230 ± 370 μm/s, P = 0.037), but there were no significant changes in blood flow. Flow cytometry measured a concurrent increase in neutrophil expression of cd11b with N/OFQ vs. controls (Geo mean fluorescence: 4.19 ± 0.13 vs. 2.06 ± 0.38, P < 0.05). The NOP antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101; 60 and 150 nmol/kg iv), H1 and H2antagonists pyrilamine (mepyramine, 1 mg/kg iv) and ranitidine (1 mg/kg iv), and mast cell stabilizer cromolyn (1 mg·kg−1·min−1) also abolished vasodilation and macromolecular leak to N/OFQ in vivo ( P < 0.05), but did not affect hypotension. Isolated mesenteric arteries (∼200 μm, n = 25) preconstricted with U-46619 were also mounted on a pressure myograph (60 mmHg), and both intraluminally and extraluminally administered N/OFQ (10−5 M) caused dilation, inhibited by pyrilamine in the extraluminal but not the intraluminal (control: −6.9 ± 3.8%; N/OFQ: 32.6 ± 8.4%; pyrilamine: 31.5 ± 6.8%, n = 18, P < 0.05) experiments. We conclude that, in vivo, mesenteric microvascular dilation and macromolecular leak occur via N/OFQ-NOP-mediated release of histamine from mast cells. Therefore, N/OFQ-NOP has an important role in microvascular inflammation, and this may be targeted during disease, particularly as we have proven that UFP-101 is an effective antagonist of microvascular responses in vivo.

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Leukocyte Adhesion to the Coronary Microvasculature During Ischemia and Reperfusion in an In Vivo Canine Model

Circulation ◽

10.1161/01.cir.93.10.1784 ◽

1996 ◽

Vol 93 (10) ◽

pp. 1784-1787 ◽

Cited By ~ 55

Author(s):

Frank M. Sheridan ◽

Paul G. Cole ◽

David Ramage

Keyword(s):

Leukocyte Adhesion ◽

Canine Model ◽

Ischemia And Reperfusion

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Evaluation of Fab and F(ab′)2 Fragments and Isotype Variants of a Recombinant Human Monoclonal Antibody against Shiga Toxin 2

Infection and Immunity ◽

10.1128/iai.00867-09 ◽

2010 ◽

Vol 78 (3) ◽

pp. 1376-1382 ◽

Cited By ~ 17

Author(s):

Donna E. Akiyoshi ◽

Abhineet S. Sheoran ◽

Curtis M. Rich ◽

L. Richard ◽

Susan Chapman-Bonofiglio ◽

...

Keyword(s):

Monoclonal Antibody ◽

Shiga Toxin ◽

Chinese Hamster Ovary Cells ◽

Human Monoclonal Antibody ◽

Chinese Hamster ◽

The Body ◽

Neutralization Activity ◽

Shiga Toxin 2

ABSTRACT 5C12 HuMAb is a human monoclonal antibody against the A subunit of Shiga toxin 2 (Stx2). We have previously shown that 5C12 HuMAb effectively neutralizes the cytotoxic effects of this toxin by redirecting its transport within the cell and also by neutralizing the toxin's ability to inhibit protein synthesis. The 5C12 HuMAb and its recombinant IgG1 version protect mice at a dose of 0.6 μg against a lethal challenge of Stx2. The contribution of the Fc region to this observed neutralization activity of the 5C12 antibody against Stx2 was investigated in this study. Using recombinant DNA technology, 5C12 isotype variants (IgG1, IgG2, IgG3, and IgG4) and antibody fragments [Fab, F(ab′)2] were expressed in Chinese hamster ovary cells and evaluated in vitro and in vivo. All four 5C12 isotype variants showed protection in vitro, with the IgG3 and IgG4 variants showing the highest protection in vivo. The Fab and F(ab′)2 fragments also showed protection in vitro but no protection in the mouse toxicity model. Similar results were obtained for a second HuMAb (5H8) against the B subunit of Stx2. The data suggest the importance of the Fc region for neutralization activity, but it is not clear if this is related to the stability of the full-length antibody or if the Fc region is required for effective elimination of the toxin from the body.

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Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo

Cellular Microbiology ◽

10.1111/j.1462-5822.2005.00673.x ◽

2006 ◽

Vol 8 (5) ◽

pp. 869-879 ◽

Cited By ~ 65

Author(s):

Yukiko Miyamoto ◽

Mitsutoshi Iimura ◽

James B. Kaper ◽

Alfredo G. Torres ◽

Martin F. Kagnoff

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Human Colon ◽

Enterohaemorrhagic Escherichia Coli

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Peptides derived from phage display libraries as potential neutralizers of Shiga toxin-induced cytotoxicity in vitro and in vivo

Journal of Applied Microbiology ◽

10.1111/jam.12451 ◽

2014 ◽

Vol 116 (5) ◽

pp. 1322-1333 ◽

Cited By ~ 5

Author(s):

R.A. Bernedo-Navarro ◽

M.M. Miyachiro ◽

M.J. da Silva ◽

C.F. Reis ◽

R.A. Conceição ◽

...

Keyword(s):

Phage Display ◽

Shiga Toxin ◽

Phage Display Libraries

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Influence of ionic and non-ionic radiographic contrast media on leukocyte adhesion molecules

Mediators of Inflammation ◽

10.1080/09629350310001619690 ◽

2003 ◽

Vol 12 (5) ◽

pp. 269-275 ◽

Cited By ~ 1

Author(s):

Guy L. J. Vermeiren ◽

Roel Willems ◽

Marc J. Claeys ◽

Chris Vrints ◽

Herman Slegers ◽

...

Keyword(s):

Coronary Angiography ◽

Contrast Media ◽

Adhesion Molecules ◽

Leukocyte Adhesion ◽

Serum Samples ◽

Cd11b Expression ◽

Radiographic Contrast ◽

Radiographic Contrast Media

Background:Many papers have focused on the importance of granulocytes in the process of reperfusion and ischemia. Most of the clinical studies measured several parameters of this process during and after coronary angiography, without taking into account the effect of the radiographic contrast media (RCM) used during this procedure.Materials and methods:We performed a randomized patient study(n=37)to evaluate the effect of ionic and non-ionic RCM on granulocyte adhesion during coronary angiography. We also evaluated the influence of the ionicity and osmolarity of the different substances on granulocyte adhesion molecules inin vitroexperiments.Results:The osmolarity of patient serum samples increased from 302±1 to 309±1 mOsm/kg(P<0.05)after infusion of RCM. The CD11b expression in the samples of the non-ionic RCM treated group increased from 221±21 MFI to 377±30 MFI(P<0.05)measured as the absolute mean fluorescence intensity (MFI), yet did not alter significantly in the ionic RCM group. In contrast, thein vitroexperiments showed a reduction of the CD11b expression from 360±70 MFI to 149±30 MFI(P<0.05)in the ionic RCM group.Conclusions:The upregulation of adhesion molecules was significantly reducedin vivowith ionic RCM, while ionic substances caused opposite effectsin vitro. This effect should be taken into account when performing leukocyte functional analysis of samples taken during angiography.

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Human Recombinant Fab Fragment Neutralizes Shiga Toxin Type 2 Cytotoxic Effects in vitro and in vivo

Toxins ◽

10.3390/toxins10120508 ◽

2018 ◽

Vol 10 (12) ◽

pp. 508 ◽

Cited By ~ 4

Author(s):

Daniela Luz ◽

Maria Amaral ◽

Flavia Sacerdoti ◽

Alan Bernal ◽

Wagner Quintilio ◽

...

Keyword(s):

Shiga Toxin ◽

Lethal Dose ◽

Dependent Manner ◽

Fab Fragment ◽

Cell Viability Assay ◽

Cytotoxic Effects ◽

Morphological Alterations ◽

Viability Assay

Shiga toxin (Stx) producing Escherichia coli (STEC) is responsible for causing hemolytic uremic syndrome (HUS), a life-threatening thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure after bacterially induced hemorrhagic diarrhea. Until now, there has been neither an effective treatment nor method of prevention for the deleterious effects caused by Stx intoxication. Antibodies are well recognized as affinity components of therapeutic drugs; thus, a previously obtained recombinant human FabC11:Stx2 fragment was used to neutralize Stx2 in vitro in a Vero cell viability assay. Herein, we demonstrated that this fragment neutralized, in a dose-dependent manner, the cytotoxic effects of Stx2 on human glomerular endothelial cells, on human proximal tubular epithelial cells, and prevented the morphological alterations induced by Stx2. FabC11:Stx2 protected mice from a lethal dose of Stx2 by toxin-antibody pre-incubation. Altogether, our results show the ability of a new encouraging molecule to prevent Stx-intoxication symptoms during STEC infection.

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Effects of Shiga Toxin Type 2 on a Bioengineered Three-Dimensional Model of Human Renal Tissue

Infection and Immunity ◽

10.1128/iai.02143-14 ◽

2014 ◽

Vol 83 (1) ◽

pp. 28-38 ◽

Cited By ~ 10

Author(s):

Teresa M. DesRochers ◽

Erica Palma Kimmerling ◽

Dakshina M. Jandhyala ◽

Wassim El-Jouni ◽

Jing Zhou ◽

...

Keyword(s):

Cell Culture ◽

Renal Failure ◽

Shiga Toxin ◽

Three Dimensional ◽

Kidney Injury ◽

Renal Tissue ◽

Three Dimensional Model ◽

Drug Induced ◽

Tissue Model

Shiga toxins (Stx) are a family of cytotoxic proteins that can cause hemolytic-uremic syndrome (HUS), a thrombotic microangiopathy, following infections by Shiga toxin-producingEscherichia coli(STEC). Renal failure is a key feature of HUS and a major cause of childhood renal failure worldwide. There are currently no specific therapies for STEC-associated HUS, and the mechanism of Stx-induced renal injury is not well understood primarily due to a lack of fully representative animal models and an inability to monitor disease progression on a molecular or cellular level in humans at early stages. Three-dimensional (3D) tissue models have been shown to be morein vivo-like in their phenotype and physiology than 2D cultures for numerous disease models, including cancer and polycystic kidney disease. It is unknown whether exposure of a 3D renal tissue model to Stx will yield a morein vivo-like response than 2D cell culture. In this study, we characterized Stx2-mediated cytotoxicity in a bioengineered 3D human renal tissue model previously shown to be a predictor of drug-induced nephrotoxicity and compared its response to Stx2 exposure in 2D cell culture. Our results demonstrate that although many mechanistic aspects of cytotoxicity were similar between 3D and 2D, treatment of the 3D tissues with Stx resulted in an elevated secretion of the kidney injury marker 1 (Kim-1) and the cytokine interleukin-8 compared to the 2D cell cultures. This study represents the first application of 3D tissues for the study of Stx-mediated kidney injury.

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L-Selectin–Dependent Leukocyte Adhesion to Microvascular But Not to Macrovascular Endothelial Cells of the Human Coronary System

Blood ◽

10.1182/blood.v89.9.3228 ◽

1997 ◽

Vol 89 (9) ◽

pp. 3228-3235 ◽

Cited By ~ 45

Author(s):

A. Zakrzewicz ◽

M. Gräfe ◽

D. Terbeek ◽

M. Bongrazio ◽

W. Auch-Schwelk ◽

...

Keyword(s):

Endothelial Cells ◽

Time Course ◽

Leukocyte Adhesion ◽

Flow Chamber ◽

Shear Stresses ◽

Selectin Ligands ◽

Tnf Α ◽

Factor Α

Abstract To characterize L-selectin–dependent cell adhesion to human vascular endothelium, human cardiac microvascular endothelial cells (HCMEC) and human coronary endothelial cells (HCEC) were isolated from explanted human hearts. The adhesion behavior of human (NALM-6) and mouse (300.19) pre-B cells transfected with cDNA encoding for human L-selectin was compared with that of the respective nontransfected cells in a flow chamber in vitro. More than 80% of the adhesion to tumor necrosis factor-α (TNF-α)–stimulated HCMEC at shear stresses <2 dyne/cm2 was L-selectin dependent and could be equally well blocked by an anti–L-selectin antibody or a L-selectin-IgG-chimera. No L-selectin dependent adhesion to HCEC could be shown. The L-selectin dependent adhesion to HCMEC was insensitive to neuraminidase, but greatly inhibited by addition of NaClO3 , which inhibits posttranslational sulfation and remained elevated for at least 24 hours of stimulation. E-selectin dependent adhesion of HL60 cells to HCMEC was blocked by neuraminidase, but not by NaClO3 and returned to control levels within 18 hours of HCMEC stimulation. It is concluded that microvascular, but not macrovascular endothelial cells express TNF-α–inducible sulfated ligand(s) for L-selectin, which differ from known L-selectin ligands, because sialylation is not required. The prolonged time course of L-selectin dependent adhesion suggests a role in sustained leukocyte recruitment into inflammatory sites in vivo.

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