Patent Application
20250114511
The present disclosure relates to a blood perfusion device, and more specifically to a blood perfusion device which includes an anti-mitochondrial N-formyl peptide (mtFP) antibody to remove mtFP, thereby blocking the binding of mtFP to formyl peptide receptor 1 (FPR1) on the PMN membrane by removing the mtFP, and is capable of suppressing the occurrence of secondary infection in hospital by restoring the chemotaxis of polymorphonuclear leukocytes (PMN).
Immune paralysis increases susceptibility to secondary infections and worsens the clinical outcome of septic shock patients who survive the initial hyperinflammatory phase. The chemotaxis of polymorphonuclear leukocytes (PMN) to sites of secondary infection is important in inhibiting progression to secondary invasive infection in patients with septic shock. During septic shock, multiorgan damage occurs primarily due to systemic inflammation, and secondarily, tissue hypoperfusion causes mitochondrial N-formyl peptide (mtFP) to be released into the circulation system. Circulating mtFP binds to formyl peptide receptorl (FPR1) on PMN membranes. This binding internalizes FPR1 (homologous internalization) and other chemokine receptors (heterologous internalization) into the cytoplasm and blocks FPR1 from binding to newly synthesized bacterial peptides. Additionally, it blocks the binding of newly released chemokines to other chemokine receptors and secondarily inhibits PMN chemotaxis to the site of infection. It is known that after internalization of the FPR1-mtFP complex, mtFP is removed from the endosomal compartment, and FPR1 is rapidly recycled to the PMN membrane or degraded by lysosomes.
The inventors of the present disclosure have confirmed in previous studies that increased circulating mtFP levels are independently associated with the development of secondary infections and 90-day mortality in patients recovering from septic shock, and increased susceptibility to secondary infection is mainly due to the inhibition of FPR1-mediated PMN chemotaxis, and it was confirmed that circulating mtFP does not affect the bactericidal activity of PMN.
Furthermore, the inventors of the present disclosure confirmed that blocking FPR1 can preserve other chemokine receptors on PMN membranes by inhibiting heterologous internalization of non-FPR1 chemokine receptors. However, in septic shock patients, FPR1 blocking may not be an effective treatment to rescue PMN chemotaxis to secondary infection sites. In a previous study, in order to reduce secondary infections in hospitals that occur after the end of acute treatment for severe trauma patients, there were efforts to maintain the migration of neutrophils (PMN) to the site of secondary bacterial infection by inhibiting the intracellular movement of other chemotactic receptors instead of giving up the activity of FPR1 by administering an antagonist that blocks the mtFP binding site of FPR1. However, in sepsis patients, the concentration of chemokine in the systemic blood is relatively high, and the difference between the concentration of chemokine at the site of secondary bacterial infection and the concentration of systemic chemokine is not large, thereby reducing the effectiveness of using FPR1 antagonists. Actually, in a previous study, plasma chemokine levels were significantly higher at admission in patients with septic shock, as well as at the onset of secondary infection in patients recovering from septic shock, than in healthy volunteers. Unlike trauma patients, in patients recovering from septic shock, the chemokine gradient from the circulation system to the site of secondary infection may not be sufficient to induce PMN chemotaxis. In this situation, formyl peptide receptor 1 (FPR1)-mediated chemotaxis against newly synthesized bacterial peptides will be responsible for a significant portion of PMN migration to the sites of secondary infection.
Accordingly, the inventors of the present disclosure made diligent efforts to develop a technique to rescue PMN chemotaxis and inhibit the development of secondary infection in patients recovering from septic shock, and as a result, since FPR1, which can bind to newly synthesized bacterial peptides, must be preserved as much as possible in the PMN membrane, mtFP itself is removed from the blood through an antibody, thereby blocking the rebinding of mtFP to FPR1 re-expressed on the surface of neutrophils, and the FPR1-mediated chemotaxis of neutrophils to the sites of secondary bacterial infection is restored, thereby completing the present disclosure.
An object of the present disclosure is to provide a blood perfusion device for reducing secondary infection in hospital.
In order to achieve the above object, in one aspect, the present disclosure provides a blood perfusion device, including a blood inlet for allowing blood to flow into the device; an adsorption medium including an anti-mitochondrial N-formyl peptides antibody (anti-mtFP antibody); and a blood outlet for allowing blood to flow out of the device.
In the present disclosure, the blood perfusion device may suppress the occurrence of secondary infection in hospital.
In the present disclosure, a target subject of the blood perfusion device may be at least one selected from the group consisting of sepsis patients, septic shock patients, severe trauma patients, organ transplant patients, hemorrhagic shock patients, myocardial infarction patients, cardiogenic shock patients, stroke patients and patients surviving after cardiac arrest.
In the present disclosure, the mtFP may be at least one selected from the group consisting of mitochondrial NADH-ubiquinone oxidoreductase chain 6 (MT-ND6), MT-ND3, MT-ND4, MT-ND5 and mitochondrial cytochrome c oxidase 1 (MT-COX1).
In the present disclosure, the blood perfusion device may further include oxygen, a blood-anticoagulant and leukotriene B4 (LTB4), and the leukotriene B4 (LTB4) may be included at a concentration of 10 to 100 μg/mL.
In the present disclosure, the adsorption medium may include the anti-mtFP antibody at a concentration of 0.5 μg/mL to 20.0 μg/mL.
In the present disclosure, the adsorption medium may have an anti-mtFP antibody attached or coated on a surface thereof.
In the present disclosure, the adsorption medium may be at least one selected from the group consisting of fiber form, bead form, film form and hollow-fiber form.
The present disclosure can suppress the progression of systemic secondary infection through the sterilizing effect of neutrophils on secondary infected bacteria by restoring the chemotaxis of neutrophils (PMN) to the site of secondary infection, and this can reduce late-term mortality and improve long-term survival by improving the clinical course of sepsis patients who are recovering after the acute phase.
In addition, the present disclosure has the advantage of being applicable to all cases where secondary infections may occur in the hospital after the completion of acute treatment in diseases that have pathophysiology that allows mtFPs to be released from damaged tissues into the blood. In particular, it is expected to improve long-term survival by suppressing the occurrence of secondary infections in hospitals for patients who require long-term hospitalization in the intensive care unit or hospital room, such as severe trauma patients, organ transplant patients and patients surviving after cardiac arrest.
The above and other objects, features and advantages of the present disclosure will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the accompanying drawings, in which:
Hereinafter, the present disclosure will be described in detail.
Unless otherwise defined, all technical and scientific terms used in the present specification have the same meaning as commonly understood by a person skilled in the art to which the present disclosure pertains. In general, the nomenclature used herein is well known and commonly used in the art.
Mitochondrial N-formyl peptide (mtFP), which is released into the blood from damaged tissues in sepsis patients, binds to formyl peptide receptor 1 (FPR1) on the surface of neutrophils (PMN) to suppress the chemotaxis of neutrophils against secondary invading bacteria such that it causes the progression of secondary infection in hospital by the invading bacteria. The mechanism is due to a decrease in receptors on the surface of neutrophils (homologous internalization) due to the intracellular movement of FPR1 bound to mtFP and a decrease in chemotactic receptors on the surface of neutrophils due to the accompanying intracellular movement of other chemotactic receptors (heterologous internalization).
The inventors of the present disclosure have confirmed in previous studies that the increased levels of circulating mtFP are independently associated with the development of secondary infections and 90-day mortality in patients recovering from septic shock, and increased susceptibility to secondary infection is mainly due to FPR1-mediated inhibition of neutrophil (PMN) chemotaxis, and it was confirmed that circulating mtFP does not affect the bactericidal activity of PMNs.
Furthermore, the inventors of the present disclosure confirmed that blocking FPR1 can preserve other chemokine receptors on PMN membranes by inhibiting the heterologous internalization of non-FPR1 chemokine receptors. However, in septic shock patients, FPR1 blocking may not be an effective treatment to rescue PMN chemotaxis to secondary infection sites.
As such, the inventors of the present disclosure hypothesized that removing circulating mtFP by an mtFP-specific antibody before redistributing recycled FPR1 to the PMN membrane would allow a sufficient amount of recycled FPR1 to bind to newly synthesized bacterial peptides, and accordingly established a hypothesis that PMN chemotaxis rescue would occur in response to secondary bacterial infection even in septic shock patients who developed secondary infection. Therefore, the inventors of the present disclosure investigated whether removing circulating mtFP from plasma via mtFP-specific antibodies can rescue FPR1-mediated PMN chemotaxis against secondary bacterial infection in patients recovering from septic shock who developed secondary infection.
First of all, in order to determine the effect of antibody treatment specific for mitochondrial NADH-ubiquinone oxidoreductase chain 6 (MT-ND6), MT-ND3, MT-ND4, MT-ND5 and mitochondrial cytochrome c oxidase 1 (MT-COX1), plasma from sepsis patients was separated, and changes in the chemotaxis of neutrophils from normal people and sepsis patients exposed to the same were measured. There was no therapeutic effect when the existing anti-mtFP antibody was administered directly to plasma. In other words, previously, it was confirmed that anti-mtFP antibodies have no therapeutic effect when administered directly because they do not block FPR1 binding to the N-terminal of mtFP. As such, it was confirmed whether mtFP in the blood can be removed when beads and antibodies are combined and then removed by centrifugation. As a result, when beads and 4 anti-mtFP specific antibodies were combined and administered to the plasma of a sepsis patient, it was confirmed that mtFP can be effectively removed from the blood (
FPR1 bound to mtFP enters the cell, removes mtFP from the endosome, and is recycled back to the neutrophil surface for expression. Therefore, by blocking the recombination of mtFP in the blood to FPR1 expressed on the surface of neutrophils, it promotes the movement of neutrophils to the site of secondary bacterial infection, so as to eliminate the secondary invading bacteria and suppress the progression to systemic secondary infection (
As used herein, the term “perfusion” refers to the flow of fluid to organs or body tissues through the circulatory or lymphatic system, and especially refers to the delivery of blood to the capillaries within the tissues.
As used herein, the term “anti-mtFP antibody” (anti-mitochondrial N-formyl peptides antibody) is also referred to as a mitochondrial N-formyl peptide (mtFP) specific antibody, and it may remove mitochondrial N-formyl peptide (mtFP) included in blood through the mtFP-specific antibody. By removing the mtFP, the binding of the mtFP to the formyl peptide receptor 1 (FPR1) of the PMN membrane may be blocked, and it is possible to restore the chemotaxis of polymorphonuclear leukocytes (PMN). The mtFP may be characterized as being released into the blood from peripheral blood mononuclear cells (PBMCs) stimulated by damaged tissues and infectious agents.
As used herein, the term “adsorption medium” refers to a substance to which cells, organisms, viruses, toxins, pathogens, polypeptides, polynucleotides, chemical molecules, small molecules, biological molecules or fragments thereof can be attached to the surface thereof.
In one aspect, the present disclosure relates to a blood perfusion device, including a blood inlet for allowing blood to flow into the device; an adsorption medium including an anti-mitochondrial N-formyl peptides antibody (anti-mtFP antibody); and a blood outlet for allowing blood to flow out of the device.
In the present disclosure, the blood perfusion device may suppress the occurrence of secondary infection in hospital. Therefore, the blood perfusion device can be used for patients whose symptoms and clinical outcomes may worsen due to secondary infection in the hospital.
In the present disclosure, a target subject of the blood perfusion device may be characterized as at least one selected from the group consisting of sepsis patients, septic shock patients, severe trauma patients, organ transplant patients, hemorrhagic shock patients, myocardial infarction patients, cardiogenic shock patients, stroke patients and patients surviving after cardiac arrest, but the present disclosure is not limited thereto, and the blood perfusion device may be used for patients who require hospitalization for a considerable period of time even after acute treatment.
In the present disclosure, the mtFP may be characterized as at least one selected from the group consisting of mitochondrial NADH-ubiquinone oxidoreductase chain 6 (MT-ND6), MT-ND3, MT-ND4, MT-ND5 and mitochondrial cytochrome c oxidase 1 (MT-COX1), but the present disclosure is not limited thereto.
In the present disclosure, the blood perfusion device may remove blood from a subject and add desired substances such as, but not limited to, oxygen, blood-anticoagulant, anesthetic and the like to the blood before returning the same to the subject. Additionally, unwanted substances such as naturally occurring toxins or poisons may be removed from the blood.
In the present disclosure, the blood perfusion device may further include oxygen, a blood-anticoagulant and leukotriene B4 (LTB4), and the leukotriene B4 (LTB4) may be included at a concentration of 10 to 100 pg/mL.
In the present disclosure, the anti-mtFP antibody may be included at a concentration of 0.5 to 20.0 μg/mL, preferably, 1.0 to 15.0 μg/mL, more preferably, 2.0 to 10.0 μg/mL, and most preferably, 4.0 to 8.0 μg/mL, but the present disclosure is not limited thereto, but if the anti-mtFP antibody is included at a concentration of less than 0.5 μg/mL and/or more than 20.0 μg/mL, it may not effectively remove mtFP from the blood or may cause side effects.
In the present disclosure, the adsorption medium may have an anti-mtFP antibody attached or coated on the surface thereof. The adsorption medium may include fiber form, bead form, film form, hollow-fiber form and the like, but the present disclosure is not limited thereto, and it may also include various forms that attach or coat the anti-mtFP antibody to the blood contact site.
In the present disclosure, the blood perfusion device may be connected to a hemodialysis device, a continuous renal replacement therapy (CRRT) device, an extracorporeal membrane oxygenation (ECMO) device or other various extracorporeal circulation devices to perform blood perfusion, but the present disclosure is not limited thereto.
In another aspect, the present disclosure provides an in vitro method for reducing secondary infection in hospital, including the following steps:
In the present disclosure, the subject may be characterized as at least one selected from the group consisting of sepsis patients, septic shock patients, severe trauma patients, organ transplant patients, hemorrhagic shock patients, myocardial infarction patients, cardiogenic shock patients, stroke patients and patients surviving after cardiac arrest, but the present disclosure is not limited thereto, and the above method may be used for patients who require hospitalization for a considerable period of time even after acute treatment.
In the present disclosure, the mtFP may be characterized as at least one selected from the group consisting of mitochondrial NADH-ubiquinone oxidoreductase chain 6 (MT-ND6), MT-ND3, MT-ND4, MT-ND5 and mitochondrial cytochrome c oxidase 1 (MT-COX1), but the present disclosure is not limited thereto.
In the present disclosure, the anti-mtFP antibody may be included at a concentration of 0.5 to 20.0 μg/mL, preferably, 1.0 to 15.0 μg/mL, more preferably, 2.0 to 10.0 μg/mL, and most preferably, 4.0 to 8.0 μg/mL, but the present disclosure is not limited thereto, but if the anti-mtFP antibody is included at a concentration of less than 0.5 μg/mL and/or more than 20.0 μg/mL, it may not effectively remove mtFP from the blood or may cause side effects.
In the present disclosure, the adsorption medium may be characterized by having an anti-mtFP antibody attached or coated on the surface thereof. The adsorption medium may include fiber form, bead form, film form, hollow-fiber form and the like, but the present disclosure is not limited thereto, and it may also include various forms that attach or coat the anti-mtFP antibody to the blood contact site.
Hereinafter, the present disclosure will be described in more detail through examples. These examples are only for illustrating the present disclosure, and it is apparent to those skilled in the art that the scope of the present disclosure should not be construed as limited by these examples.
The collection of blood samples from healthy volunteers and septic shock patients was approved by the Institutional Review Board (IRB) of Seoul National University College of Medicine/Seoul National University Hospital (IRB number: 1605-044-760). The Clinical Data Repository Protocol for Patients with Septic Shock has also been approved by the IRB (IRB number: 1707-012-865) and registered at ClinicalTrials.gov (NCT01670383). Additionally, written consent was obtained from healthy volunteers, patients with septic shock or legally authorized representatives.
The inventors of the present disclosure selected sepsis patients who visited the emergency room based on a qSOFA (quick sequential organ failure assessment) score of 2 or higher. Afterwards, the inventors of the present disclosure calculated the SOFA score and injected 30 mL/kg crystalloid if the score was 2 or more. Patients were diagnosed with septic shock if the mean arterial pressure (MAP) was less than 65 mmHg and the serum lactate level was more than 2 mmol/L despite a crystalloid infusion of 30 mL/kg. Patients who were diagnosed with septic shock received a continuous intravenous infusion of vasopressors to maintain MAP above 65 mmHg, and after collecting blood samples for incubation studies, broad-spectrum antibiotics were administered intravenously within 1 hour, and the patients were admitted to the intensive care unit as soon as possible. Demographic and laboratory data were collected from patients who were admitted to the intensive care unit.
Human polymorphonuclear cells (PMN) were isolated according to the following method. Heparinized (10 U/mL) whole blood samples collected from healthy volunteers and patients with septic shock were centrifuged at 200 g for 10 minutes at 4° C. The plasma layer was obtained and centrifuged again at 20,000 g for 10 minutes at 4° C. to obtain a supernatant (cell-free plasma).
5 mL of buffy coat and red blood cells overlaid on 5 mL of 1-Step™ polymorph (AN221725, Accurate Chemical, Westbury, NY) were centrifuged at 550 g for 30 minutes at 24° C., and the PMN layer was collected separately. In order to restore the osmotic pressure, an equal volume of ice-cold 0.45% sodium chloride (NaCl) was mixed for 5 minutes. After washing with Roswell Park Memorial Institute (RPMI)-1640 medium (11875093, Thermo Fisher, Waltham, MA), the remaining red blood cells were lysed in 20 mL of ice-cold 0.2% NaCl for 20 s, and then, 20 mL of ice-cold 1.6% NaCl was administered. After centrifugation at 200 g for 10 minutes at 4° C., the supernatant was discarded.
The hexapeptide of ND6 (N-formyl-methionine-methionine-tyrosine-alanine-leucine-phenylalanine, N-formyl-MMYALF), ND3 (N-formyl-methionine-asparagine-phenylalanine-alanine-leucine-isoleucine, N-formyl-MNFALI), ND4 (N-formyl-methionine-leucine-lysine-leucine-isoleucine-valine, N-formyl-MLKLIV), and ND5 (N-formyl-methionine-threonine-methionine-histidine-threonine-threonine, N-formyl-MTMHTT) was synthesized by requesting at Peptron (Daejeon, Korea). N-formyl-methionine-leucine-phenylalanine (fMLF, standard bacterial FP) was purchased from Sigma-Aldrich (f506, St. Louis, MO).
Calcium mobilization within PMN cells was measured according to the following method. The separated PMN pellet was resuspended in pH 7.4 hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution. This HEPES buffer solution includes 20 mM HEPES, 140 mM NaCl, 5 mM potassium chloride (KCl), 1 mM magnesium chloride (MgCl2), 1 mM calcium chloride (CaCl2)), 10 mM glucose and 0.1% bovine serum albumin (BSA, A2058 Sigma-Aldrich). PMNs were loaded with fura2-AM (F1221, Thermo Fisher, Waltham, MA) for 45 minutes at 37° C.
Fluorescence was measured at 505 nm with 340/380 nm dual-wavelength excitation at 37° C. by using a spectrofluorometer (FluoroMax Plus-C, Horiba, Ltd., Kyoto, Japan). For the quantitative measurement of endoplasmic reticulum (ER) calcium depletion, PMNs were centrifuged at 200 g for 10 minutes at 4° C. PMN pellets were then resuspended in a calcium-free HEPES buffer solution including 0.3 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, E3889, Sigma-Aldrich, St. Louis, MO), and stimulated with ND6 and fMLF or with ND6 alone. 0.1% dimethyl sulfoxide (DMSO) was added to the medium without ND6 or fMLF. After endoplasmic reticulum calcium was depleted, 1.8 mM CaCl2) was added to induce cytoplasmic calcium influx. Endoplasmic reticulum calcium depletion and calcium influx were assessed by measuring the 340 nm/380 nm ratio and quantified as AUC over 150 seconds (AUC150) and AUC over 100 seconds (AUC100), respectively. The role of FPR1 in calcium mobilization was determined by pretreatment with 10 μM CsH 1 minute prior to the induction of endoplasmic reticulum calcium depletion. Three independent experiments were performed.
PMN chemotaxis was assessed by using multiScreen 96-well plates consisting of a 3-μm pore membrane (MAMIC3S10, Merck Millipore, Darmstadt, Germany). After preplanned treatment with ND6 or patient plasma, isolated human PMNs were centrifuged at 200 g for 10 minutes at 4° C. Afterwards, the PMN pellet was resuspended in Dulbecco's modified Eagle medium (DMEM, 11885-084, Thermo Fisher) containing 10% fetal bovine serum (FBS, F2442, Sigma-Aldrich). Before use, FBS was inactivated by heating at 56° C. for 1 hour. PMNs were placed in the upper chamber of the plate (0.1×106 PMNs at 75 μL per well), and medium containing 100 nM fMLF was placed in the lower chamber (150 μL per well). In medium without ND6 or fMLF, 0.1% DMSO was added.
Plates were incubated for 60 minutes in an incubator at 37° C. with 5% carbon dioxide (CO2). In order to determine the role of FPR1 in PMN chemotaxis, 10 μM CsH was added to one group of PMNs 15 minutes prior to incubation. After 60 minutes of incubation, the upper and lower chambers of the plate were separated, and the medium in the lower chamber was transferred to a 1.5 mL-Eppendorf (EP) tube. The EP tube was centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was aspirated. The pellet was resuspended in 200 μL of distilled water including a lysis buffer solution (1:20 dilution) and CyQuant GR dye (1:400 dilution, C7026, Thermo Fisher). PMNs were vortexed and dissolved for 15 minutes at room temperature in the dark. Afterwards, the contents were transferred to a 96-well plate, and fluorescence was measured at an excitation/emission wavelength of 480/520 nm with a cut-off of 515 nm. The number of migrated PMNs was calculated by using a standard curve derived from known PMN numbers. A 4-well replicate experiment was performed for each experimental group, and three independent experiments were performed.
The purity of isolated human PMNs was assessed by flow cytometry using a BD FACSCalibur (BD Biosciences, San Jose, CA). Human bone marrow cells were sorted by using fluorescein (FITC)-conjugated anti-human CD15 antibody (301903, BioLegend, San Diego, CA). Among these cells, CD49d negative and CD16 positive cells were counted as PMN. Phycoerythrin/cyanine 7 (PE/Cy7)-conjugated anti-human CD49d antibody (304313, BioLegend) and allophycocyanin (APC)-conjugated anti-human CD16 antibody (360705, BioLegend) were used.
FPR1 expression on PMN membranes and the amount of CaMK2 in the homogenized PMN pellet were also detected by flow cytometry. In order to detect FPR1 on PMN membranes, PMNs (1.0×106 in 200 μL) were incubated with FITC-conjugated anti-human FPR1 antibody (2 μL, FAB3744F, R&D Systems, Minneapolis, MN) for 30 minutes at room temperature in the dark. FITC-conjugated mouse IgG2a, κ isotype control antibody (400210, BioLegend, San Diego, CA) was used as a negative control. After washing twice, fluorescence was measured. APC-conjugated anti-CaMK2 antibody (2 μL, ab225178, Abcam, Cambridge, UK) was used to detect the amount of CaMK2 in 200 μL of homogenized PMN pellet obtained by homogenization of 1.0×106/l mL PMN for 30 seconds at 4° C. and centrifugation at 20,000 g for 10 minutes. FPR1 expression in PMN membranes and the amount of CaMK2 in homogenized PMN pellets were quantified as mean fluorescence intensity (MFI) by using FlowJo version 10.0 (FlowJo, LLC, Ashland, OR). At least three independent experiments were performed.
CaMK2 levels in the supernatants of homogenized PMNs obtained by homogenization of 1.0×106/l mL PMN for 30 seconds at 4° C. and centrifugation at 20,000 g for 10 minutes were measured by using the human calcium/calmodulin-dependent protein kinase II (CAMK2) ELISA kit (MBS005464, MyBioSource, San Diego, CA) according to the manufacturer's instructions.
Plasma ND6, ND3, ND4 and ND5 levels were respectively measured by using human NADH ubiquinone oxidoreductase chain 6(MT-ND6) ELISA Kit(MBS7246296, MyBioSource), human NADH ubiquinone oxidoreductase chain 3(MT-ND38) ELISA Kit (MBS7244982, MyBioSource), human NADH ubiquinone oxidoreductase chain 4(MT-ND4) ELISA Kit (MBS9715152, MyBioSource) and human NADH ubiquinone oxidoreductase chain 5(MT-ND5) ELISAKit (MBS7236655, MyBioSource).
Plasma tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10 levels were respectively measured by using the TNF alpha beta human ELISAKit (KHC3011, Thermo Fisher), IL-1 beta human ELISA Kit(KHCOO11, Thermo Fisher), IL-6 human ELISA Kit (KHCO061, Thermo Fisher) and IL-10 human ELISA Kit (KHCO101, Thermo Fisher). The plasma growth regulated oncogene (GRO)-α, IL-8 and leukotriene B4 (LT B4) levels were respectively measured by using the GRO alpha (CXCL1) human ELISA Kit (BMS2122, Thermo Fisher), IL-8 human ELISA Kit (KHCO081, Thermo Fisher) and LTB4 parameter analysis kit (KGE006B, R&D Systems, Minneapolis, MN). All experiments were performed in duplicate.
Isolated human PMNs obtained from healthy volunteers in DMEM containing 10% FBS (control medium) were treated with 100 nM of ND6 for 30 minutes on a 10-rpm rotator at 37° C. After centrifugation at 200 g for 10 minutes, the supernatant was discarded. PMN pellets were resuspended in 100 nM ND6 treatment medium (ND6-ND6 group) or control medium (ND6-DMSO group). 0.1% DMSO was added to the control medium without ND6. Next, PMNs were incubated at 37° C. for 90 minutes on a 10-rpm rotator. For calcium mobilization experiments, PMNs were extracted from the ND6-ND6 and ND6-DMSO groups at 30, 60 and 90 minutes, respectively. For chemotaxis experiments, PMNs were extracted at 30 and 90 minutes.
Commercial polyclonal anti-ND6 antibody (orb6548) was purchased from Biorbyt Ltd. (Cambridge, UK). Protein A Sepharose® was purchased from Abcam (ab193256). First of all, 0.125 μg/mL, 0.25 μg/mL, 0.5 μg/mL, 1.0 μg/mL or 2.0 μg/mL of the anti-ND6 antibody was directly administered to plasma obtained from a septic shock patient through an arterial catheter. After 2 hours of incubation on a 10-rpm rotator at 37° C., the plasma containing the anti-ND6 antibody was centrifuged at 20,000 g for 10 minutes at 4° C., and the supernatant was obtained. Afterwards, plasma ND6 levels were measured by ELISA.
The inventors of the present disclosure prepared a beads-anti-ND6 antibody complex. 80 μL of Protein A Sepharose® was washed twice with phosphate-buffered saline (PBS, pH 7.4, 10010023, Thermo Fisher) and co-incubated with the anti-ND6 antibody at 0.25 μg/mL, 0.5 μg/mL, 1.0 μg/mL, 2.0 μg/mL, 4.0 μg/mL or 8.0 μg/mL for 4 hours on a 10-rpm rotator at 4° C. The bead-anti-ND6 antibody complex was centrifuged at 3,000 g for 2 minutes at 4° C., and the supernatant was discarded. After washing twice with 3,000 g of PBS for 2 minutes at 4° C., the bead-anti-ND6 antibody complex was co-incubated with 500 μL plasma obtained from a septic shock patient via an arterial catheter for 2 hours on a 10-rpm rotator at 37° C. The bead-anti-ND6 antibody complex that could be attached by circulating ND6 was removed by centrifugation at 20,000 g for 10 minutes at 4° C., and the supernatant was obtained. Plasma ND6 levels before and after the bead-anti-ND6 antibody complex treatment were measured by ELISA. Three independent experiments were performed, and ELISA was performed in duplicate.
In addition to the anti-ND6 antibody, commercially available polyclonal anti-ND3 (orb185335), anti-ND4 (orb546348) and anti-ND5 (orb6547) antibodies were purchased from Biorbyt Ltd. Based on experimental results that estimated the required anti-ND6 antibody dose (5.0 μg/mL with 80 μL beads in 500 μL plasma), 5.0 μg/mL of each antibody specific for ND6, ND3, ND4, and ND5 was co-incubated with 320 μL beads for 4 hours on a 10-rpm rotator at 4° C. After washing twice with PBS, 500 μL plasma was added to the bead-anti-mtFP antibody complex (bead-anti-mtFP mixture) and co-incubated for 2 hours at 37° C. on a 10-rpm rotator. Next, plasma containing the bead-anti-mtFP mixture was centrifuged at 20,000 g for 10 minutes at 4° C., and the supernatant was obtained. Plasma ND6, ND3, ND4 and ND5 levels were measured by ELISA before and after the bead-anti-mtFP mixture treatment. Plasma TNF-α, IL-10, IL-6, IL-10, GRO-α, IL-8 and LTB4 levels were also measured. Plasma obtained from healthy volunteers was used as a negative control group.
The effect of bead-anti-mtFP mixture treatment on PMN chemotaxis rescue was analyzed. PMS was isolated from blood samples obtained from healthy volunteers and exposed to untreated plasma from septic shock patients for 30 minutes in a 10-rpm rotator at 37° C. After centrifugation at 200 g for 10 minutes at 4° C., the supernatant was discarded. PMN pellets were resuspended in untreated plasma or bead-anti-mtFP mixture-treated plasma and incubated for 90 minutes on a 10-rpm rotator at 37° C. Next, PMN chemotaxis to 100 nM fMLF was measured. In order to estimate the capacity of non-FPR1-mediated PMN chemotaxis, separate PMNs exposed to bead anti-mtFP mixture-treated plasma were pretreated with 10 μM CsH 15 minutes prior to the chemotaxis experiment. A 4-well replicate experiment was performed for each experimental group, and three independent experiments were performed.
Before the experiment, 320 μL of protein A Sepharose® was washed twice with PBS and co-incubated with 5.0 μg/mL of each antibody specific for ND6, ND3, ND4 and ND5 for 4 hours at 4° C. on a 10-rpm rotator. Blood samples were collected between days 10 and 14 of hospitalization through an arterial catheter from patients with septic shock who were aware of secondary infection or from patients with septic shock who did not develop secondary infection. Immediately after blood collection, plasma was separated, and cell-free plasma was prepared. Half of the plasma was divided into 500 μL per EP tube and incubated with the bead-anti-mtFP mixture for 2 hours on a 10-rpm rotator at 37° C. The other half of the plasma was incubated without the bead-anti-mtFP mixture. Plasma obtained from patients negative for secondary infection was also incubated without the bead-anti-mtFP mixture. The levels of ND6, ND3, ND4 and ND5 in the plasma of septic shock patients who developed secondary infection and in the plasma of patients negative for secondary infection were measured by ELISA before and after treatment with the bead anti-mtFP mixture. Plasma TNF-α, IL-1, IL-6, IL-10, GRO-α, IL-8 and LTB4 levels were also measured.
PMNs from the patients were isolated during a 2-hour plasma incubation period. After this preparation, PMNs were exposed to plasma untreated with the bead-anti-mtFP mixture for 30 minutes on a 10-rpm rotator at 37° C. After centrifugation at 220 g for 10 minutes at 4° C., the supernatant was discarded. PMN pellets were resuspended in the bead-anti-mtFP mixture untreated plasma or bead-anti-mtFP mixture treated plasma and incubated for 90 minutes at 37° C. on a 10-rpm rotator. Next, PMN chemotaxis to 100 nM fMLF was measured. In order to investigate the role of LTB4 in the recovery of FPR1-mediated PMN chemotaxis after the removal of circulating mtFP, at the start of the incubation for PMN migration, 50 μg/mL of recombinant LTB4 (L0517, Sigma Aldrich) was added to separate PMNs exposed to bead-anti-mtFP mixture-treated plasma. A 4-well replicate experiment was performed for each experimental group, and three independent experiments were performed.
Data were analyzed by using one-way analysis of variance with Tukey's post hoc test. At least three independent experiments were performed. A p-value of less than 0.05 was considered statistically significant (significance level: two-sided test). Statistical analyzes were performed by using IBM SPSS version 27.0 for Windows (SPSS, Chicago, IL).
In order to investigate which steps of intracellular calcium mobilization stimulated by FPR1 agonists directly correlate with PMN chemotaxis, the inventors of the present disclosure quantitatively measured the amounts of endoplasmic reticulum (ER) calcium depletion and subsequent calcium influx into the cytoplasm in isolated human PMNs obtained from healthy volunteers. The average purity of PMNs isolated from three independent experiments was 97.5% (
ND6 treatment induced ER calcium depletion, but dose-dependently inhibited fMLF-stimulated secondary ER calcium depletion and subsequent calcium influx (
After exposure to 100 nM ND6 for 30 minutes, PMNs were centrifuged at 220 g for 10 minutes at 4° C., and the supernatant was discarded. The PMN pellet was resuspended in medium containing 0.1% dimethyl sulfoxide (DMSO) (ND6-DMSO group) or medium containing 100 nM ND6 (ND6-ND6 group) and incubated for 90 minutes at 37° C. on a 10-rpm rotator. Serial changes in PMN calcium mobilization at 30, 60 and 90 minutes in response to 100 nM fMLF and serial changes in PMN chemotaxis at 30 and 90 minutes in response to 100 nM fMLF were compared between the ND6-DMSO and ND6-ND6 groups. PMN chemotaxis at 30 and 90 minutes was measured as the number of PMNs migrated during 60-minute incubation between 30 and 90 minutes and 90 and 150 minutes, respectively. Additionally, in order to estimate the capacity of FPR1-mediated PMN chemotaxis, PMNs from different groups were treated with 10 μM CsH 15 minutes before incubation.
ND6 treatment inhibited ER calcium depletion, calcium influx and PMN chemotaxis in both of ND6-ND6 and ND6-DMSO groups. However, the centrifugal removal of ND6 was restored in all ND6-DMSO groups (
In contrast to short-term ND6 treatment of less than 180 seconds (
When examining the correlation between PMN chemotaxis and the calcium mobilization step in PMNs sequentially stimulated by ND6 and fMLF, PMN chemotaxis was associated with fMLF-induced ER calcium depletion (AUC150 quadratic) (Spearman's rho=0.930) and subsequent calcium influx. (AUC100) (rho=0.853), but inversely correlated with ND6-induced ER calcium depletion (AUC150 1st) (rho=−0.755) (
However, in ND6-exposed PMNs, after 30 minutes of centrifugal ND6 removal, FPR1 expression was significantly correlated with ER calcium depletion (AUC150) (rho=0.950), but was not correlated with calcium influx (AUC100) (rho=0.583) and chemotaxis (rho=0.467) (
The above results indicate that recycled FPR1 was relocated to the PMN membrane immediately after the removal of circulating mtFP, but the recovery of calcium influx was delayed due to interfering molecules that regulate the pathway from ER calcium depletion to calcium influx. It also indicates that any therapy that eliminates circulating mtFP must be applied for at least 90 minutes to rescue PMN chemotaxis in patients recovering from septic shock.
In order to achieve clinical applicability, the inventors of the present disclosure attempted to eliminate circulating mtFP with anti-mtFP antibodies, particularly those specific for potent human mtFPs, including ND6, ND3, ND4 and ND5. In order to estimate the removal ability of the anti-mtFP antibody, the difference in ND6 levels in the plasma of septic shock patients before and after treatment with the anti-ND6 antibody was measured. The characteristics of patients who donated plasma are shown in Table 1. Blood samples were collected from the patients through an arterial catheter upon admission.
Direct anti-ND6 antibody treatment of plasma did not affect plasma ND6 levels (
Based on these results, the protein A/sepharose (320 μL) was combined with specific antibodies for 4 mtFPs: ND6, ND3, ND4 and ND5 in order to remove potent mtFPs from the plasma of septic shock patients at once (bead-anti-mtFP mixture). 2.5 μg of each antibody was administered, and the final concentration was adjusted to 5.0 μg/mL in 500 μL plasma. After 2 hours of incubation with plasma on a 10-rpm rotator at 37° C., the bead-anti-mtFP mixture reduced the plasma levels of ND6, ND3, ND4 and ND5 to 139 pM, 112 pM, 104 pM, and 135 pM, respectively (
The inventors of the present disclosure investigated the chemotactic restoration effect of the anti-mtFP cocktail on PMNs by exposing PMNs from healthy volunteers to the plasma of septic shock patients treated with the anti-mtFP cocktail and then comparing PMN chemotaxis with plasma that had not received the anti-mtFP cocktail treatment. After exposing PMNs from healthy volunteers to untreated plasma from septic shock patients for 30 minutes, PMNs were centrifuged at 220 g for 10 minutes at 4° C., and the supernatant was discarded. PMN pellets were resuspended in untreated plasma, direct anti-mtFP antibody treated plasma or the bead anti-mtFP mixture treated plasma and incubated for 90 minutes on a 10-rpm rotator at 37° C. Next, PMN chemotaxis for 100 nM fMLF was measured. Untreated plasma from septic shock patients inhibited healthy volunteer PMN chemotaxis in response to fMLF. Direct anti-mtFP antibody treatment could not restore PMN chemotaxis, but the bead-anti-mtFP mixture treatment significantly restored PMN chemotaxis (
Rescue of PMN Chemotaxis in Septic Shock Patients with Secondary Infection by Bead-Anti-mtFP Combination Treatment.
The inventors of the present disclosure studied whether the bead-anti-mtFP mixture removes circulating mtFP and restores PMN chemotaxis in septic shock patients with secondary infection to the level of patients negative for secondary infection. The characteristics of patients who donated PMN and plasma are shown in Table 3. Blood samples were collected through an arterial catheter from patients who were recognized to have developed a secondary infection between 10 and 14 days after admission to the ICU (n=3). Secondary infections, including ventilator-associated pneumonia (VAP), central line-associated blood stream infection (CLABSI) and catheter-associated urinary tract infection (CAUTI), were defined according to the 2019 National Healthcare Safety Network (NHSN) Patient Safety Components Manual published by the Centers for Disease Control and Prevention. In addition, blood samples were collected from septic shock patients who did not develop secondary infections between 10 and 14 days after hospitalization (n=3).
Acinetobacter
Enterococcus
Pseudomonas
baumanii
faecium
aeruginosa
Cell-free plasma was prepared by separating plasma from blood samples collected from septic shock patients. Next, half of the cell-free plasma was incubated with the bead-anti-mtFP mixture for 2 hours at 37° C. on a 10-rpm rotator, and the other half of the cell-free plasma was incubated without the bead-anti-mtFP mixture. Plasma from patients negative for secondary infection was not treated with the bead anti-mtFP mixture. During the plasma incubation period, PMNs from patients were isolated and incubated in untreated plasma for 30 minutes on a 10-rpm rotator at 37° C. After centrifugation at 220 g for 10 minutes at 4° C., the supernatant was discarded. PMN pellets were resuspended in untreated plasma or bead-anti-mtFP mixture-treated plasma and incubated for 90 minutes on a 10-rpm rotator at 37° C. Next, changes in PMN chemotaxis for fMLF were compared.
In the plasma of septic shock patients who developed secondary infections, the bead anti-mtFP mixture treatment reduced the plasma levels of ND6, ND3, ND4 and ND5 by 139 pM, 112 pM, 104 pM, and 135 pM, respectively (
In contrast to PMNs obtained from healthy volunteers, chemotaxis was shown to be restored in PMNs obtained from septic shock patients who developed secondary infections exposed to bead-anti-mtPF mixture-treated autologous plasma, but did not show statistical significance when compared to PMNs exposed to untreated plasma (
Therefore, we also investigated the effect of additional treatment with LTB4 on the chemotaxis of PMNs exposed to bead-anti-mtFP mixture-treated plasma in septic shock patients who developed secondary infections. 50 pg/mL of recombinant LTB4 was added to the bead-anti-mtFP mixture treated plasma at the start of incubation with PMNs obtained from the same patient. After 90 minutes of incubation, PMN chemotaxis was measured. A 90-minute exposure to bead-anti-mtFP mixture treated plasma with additional recombinant LTB4 effectively restored PMN chemotaxis compared to PMN exposed to untreated plasma (
In other words, the removal of circulating potent mtFP rescued FPR1-mediated PMN chemotaxis in patients recovering from septic shock who developed secondary infection. In particular, the bead-anti-mtFP mixture prepared by binding the protein A/sepharose to anti-mtFP antibodies specific for ND6, ND3, ND4 and ND5 successfully removed circulating potent mtFP and restored PMN chemotaxis. In clinical settings, the removal of circulating mtFP through blood perfusion using extracorporeal membranes attaching anti-mtFP antibodies could be considered as an adjuvant therapeutic strategy to inhibit PMN chemotactic anergy. Inhibiting the development of secondary nosocomial infections in patients who survive the hyperinflammatory phase may improve the long-term clinical outcome of patients with septic shock.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2022-0176528 | Dec 2022 | KR | national |
This application is the U.S. national stage application of International Patent Application No. PCT/KR2023/019443, filed Nov. 29, 2023, which claims the benefit under 35 U.S.C. § 119 of Korean Application No. 10-2022-0176528, filed Dec. 16, 2022, the disclosures of each of which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2023/019443 | 11/29/2023 | WO |
