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1.
Nat Microbiol ; 8(8): 1468-1479, 2023 08.
Article in English | MEDLINE | ID: mdl-37429907

ABSTRACT

Although dengue virus (DENV) infection typically causes asymptomatic disease, DENV-infected patients can experience severe complications. A risk factor for symptomatic disease is pre-existing anti-DENV IgG antibodies. Cellular assays suggested that these antibodies can enhance viral infection of Fcγ receptor (FcγR)-expressing myeloid cells. Recent studies, however, revealed more complex interactions between anti-DENV antibodies and specific FcγRs by demonstrating that modulation of the IgG Fc glycan correlates with disease severity. To investigate the in vivo mechanisms of antibody-mediated dengue pathogenesis, we developed a mouse model for dengue disease that recapitulates the unique complexity of human FcγRs. In in vivo mouse models of dengue disease, we discovered that the pathogenic activity of anti-DENV antibodies is exclusively mediated through engagement of FcγRIIIa on splenic macrophages, resulting in inflammatory sequelae and mortality. These findings highlight the importance of IgG-FcγRIIIa interactions in dengue, with important implications for the design of safer vaccination approaches and effective therapeutic strategies.


Subject(s)
Dengue Virus , Dengue , Humans , Animals , Mice , Receptors, IgG , Macrophages , Immunoglobulin G
2.
Nat Commun ; 14(1): 2853, 2023 05 18.
Article in English | MEDLINE | ID: mdl-37202422

ABSTRACT

Immunoglobulin G (IgG) antibodies contain a complex N-glycan embedded in the hydrophobic pocket between its heavy chain protomers. This glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby dictating distinct cellular responses. The variable construction of this glycan structure leads to highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported synthetic nanobodies that distinguish IgG glycoforms. Here, we present the structure of one such nanobody, X0, in complex with the Fc fragment of afucosylated IgG1. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N-glycan and acts as a 'glycan sensor', forming hydrogen bonds with the afucosylated IgG N-glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.


Subject(s)
Immunoglobulin G , Receptors, IgG , Animals , Mice , Glycosylation , Receptors, IgG/metabolism , Immunoglobulin Fc Fragments/metabolism , Polysaccharides/chemistry
3.
bioRxiv ; 2023 Jan 24.
Article in English | MEDLINE | ID: mdl-36747840

ABSTRACT

Immunoglobulin G (IgG) antibodies contain a single, complex N -glycan on each IgG heavy chain protomer embedded in the hydrophobic pocket between its Cγ2 domains. The presence of this glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby determining distinct cellular responses. On the Fc, the variable construction of this glycan structure leads to a family of highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported the development of synthetic nanobodies that distinguish IgG glycoforms without cross-reactivity to off-target glycoproteins or free glycans. Here, we present the X-ray crystal structure of one such nanobody, X0, in complex with its specific binding partner, the Fc fragment of afucosylated IgG1. Two X0 nanobodies bind a single afucosylated Fc homodimer at the upper Cγ2 domain, making both protein-protein and protein-carbohydrate contacts and overlapping the binding site for Fcγ receptors. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N -glycan and acts as a 'glycan sensor', forming hydrogen bonds with the afucosylated IgG N -glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.

4.
Proc Natl Acad Sci U S A ; 119(48): e2212658119, 2022 11 29.
Article in English | MEDLINE | ID: mdl-36409896

ABSTRACT

Protein glycosylation is a crucial mediator of biological functions and is tightly regulated in health and disease. However, interrogating complex protein glycoforms is challenging, as current lectin tools are limited by cross-reactivity while mass spectrometry typically requires biochemical purification and isolation of the target protein. Here, we describe a method to identify and characterize a class of nanobodies that can distinguish glycoforms without reactivity to off-target glycoproteins or glycans. We apply this technology to immunoglobulin G (IgG) Fc glycoforms and define nanobodies that specifically recognize either IgG lacking its core-fucose or IgG bearing terminal sialic acid residues. By adapting these tools to standard biochemical methods, we can clinically stratify dengue virus and SARS-CoV-2 infected individuals based on their IgG glycan profile, selectively disrupt IgG-Fcγ receptor binding both in vitro and in vivo, and interrogate the B cell receptor (BCR) glycan structure on living cells. Ultimately, we provide a strategy for the development of reagents to identify and manipulate IgG Fc glycoforms.


Subject(s)
COVID-19 , Single-Domain Antibodies , Humans , Immunoglobulin G/metabolism , SARS-CoV-2 , Immunoglobulin Fc Fragments/metabolism , Polysaccharides/metabolism
5.
Gastroenterology ; 163(3): 723-731.e6, 2022 09.
Article in English | MEDLINE | ID: mdl-35580655

ABSTRACT

BACKGROUND & AIMS: The COVID-19 pandemic has affected clinical services globally, including colorectal cancer (CRC) screening and diagnostic testing. We investigated the pandemic's impact on fecal immunochemical test (FIT) screening, colonoscopy utilization, and colorectal neoplasia detection across 21 medical centers in a large integrated health care organization. METHODS: We performed a retrospective cohort study in Kaiser Permanente Northern California patients ages 18 to 89 years in 2019 and 2020 and measured changes in the numbers of mailed, completed, and positive FITs; colonoscopies; and cases of colorectal neoplasia detected by colonoscopy in 2020 vs 2019. RESULTS: FIT kit mailings ceased in mid-March through April 2020 but then rebounded and there was an 8.7% increase in kits mailed compared with 2019. With the later mailing of FIT kits, there were 9.0% fewer FITs completed and 10.1% fewer positive tests in 2020 vs 2019. Colonoscopy volumes declined 79.4% in April 2020 compared with April 2019 but recovered to near pre-pandemic volumes in September through December, resulting in a 26.9% decline in total colonoscopies performed in 2020. The number of patients diagnosed by colonoscopy with CRC and advanced adenoma declined by 8.7% and 26.9%, respectively, in 2020 vs 2019. CONCLUSIONS: The pandemic led to fewer FIT screenings and colonoscopies in 2020 vs 2019; however, after the lifting of shelter-in-place orders, FIT screenings exceeded, and colonoscopy volumes nearly reached numbers from those same months in 2019. Overall, there was an 8.7% reduction in CRC cases diagnosed by colonoscopy in 2020. These data may help inform the development of strategies for CRC screening and diagnostic testing during future national emergencies.


Subject(s)
COVID-19 , Colorectal Neoplasms , Adolescent , Adult , Aged , Aged, 80 and over , COVID-19/diagnosis , COVID-19/epidemiology , Colonoscopy/methods , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/epidemiology , Community Health Services , Early Detection of Cancer/methods , Feces , Humans , Mass Screening/methods , Middle Aged , Occult Blood , Pandemics , Retrospective Studies , United States/epidemiology , Young Adult
6.
Nature ; 599(7885): 465-470, 2021 11.
Article in English | MEDLINE | ID: mdl-34547765

ABSTRACT

Monoclonal antibodies with neutralizing activity against SARS-CoV-2 have demonstrated clinical benefits in cases of mild-to-moderate SARS-CoV-2 infection, substantially reducing the risk for hospitalization and severe disease1-4. Treatment generally requires the administration of high doses of these monoclonal antibodies and has limited efficacy in preventing disease complications or mortality among hospitalized patients with COVID-195. Here we report the development and evaluation of anti-SARS-CoV-2 monoclonal antibodies with optimized Fc domains that show superior potency for prevention or treatment of COVID-19. Using several animal disease models of COVID-196,7, we demonstrate that selective engagement of activating Fcγ receptors results in improved efficacy in both preventing and treating disease-induced weight loss and mortality, significantly reducing the dose required to confer full protection against SARS-CoV-2 challenge and for treatment of pre-infected animals. Our results highlight the importance of Fcγ receptor pathways in driving antibody-mediated antiviral immunity and exclude the possibility of pathogenic or disease-enhancing effects of Fcγ receptor engagement of anti-SARS-CoV-2 antibodies upon infection. These findings have important implications for the development of Fc-engineered monoclonal antibodies with optimal Fc-effector function and improved clinical efficacy against COVID-19 disease.


Subject(s)
Antibodies, Monoclonal/therapeutic use , COVID-19 Drug Treatment , COVID-19/immunology , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Fc Fragments/therapeutic use , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibodies, Neutralizing/therapeutic use , Cricetinae , Disease Models, Animal , Female , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/pharmacology , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Male , Mice , Pre-Exposure Prophylaxis , Receptors, IgG/chemistry , Receptors, IgG/immunology , Treatment Outcome
7.
Proc Natl Acad Sci U S A ; 118(29)2021 07 20.
Article in English | MEDLINE | ID: mdl-34257155

ABSTRACT

Trastuzumab, a targeted anti-human epidermal-growth-factor receptor-2 (HER2) monoclonal antibody, represents a mainstay in the treatment of HER2-positive (HER2+) breast cancer. Although trastuzumab treatment is highly efficacious for early-stage HER2+ breast cancer, the majority of advanced-stage HER2+ breast cancer patients who initially respond to trastuzumab acquire resistance to treatment and relapse, despite persistence of HER2 gene amplification/overexpression. Here, we sought to leverage HER2 overexpression to engage antibody-dependent cellular phagocytosis (ADCP) through a combination of trastuzumab and anti-CD47 macrophage checkpoint immunotherapy. We have previously shown that blockade of CD47, a surface protein expressed by many malignancies (including HER2+ breast cancer), is an effective anticancer therapy. CD47 functions as a "don't eat me" signal through its interaction with signal regulatory protein-α (SIRPα) on macrophages to inhibit phagocytosis. Hu5F9-G4 (magrolimab), a humanized monoclonal antibody against CD47, blocks CD47's "don't eat me" signal, thereby facilitating macrophage-mediated phagocytosis. Preclinical studies have shown that combining Hu5F9-G4 with tumor-targeting antibodies, such as rituximab, further enhances Hu5F9-G4's anticancer effects via ADCP. Clinical trials have additionally demonstrated that Hu5F9-G4, in combination with rituximab, produced objective responses in patients whose diffuse large B cell lymphomas had developed resistance to rituximab and chemotherapy. These studies led us to hypothesize that combining Hu5F9-G4 with trastuzumab would produce an anticancer effect in antibody-dependent cellular cytotoxicity (ADCC)-tolerant HER2+ breast cancer. This combination significantly suppressed the growth of ADCC-tolerant HER2+ breast cancers via Fc-dependent ADCP. Our study demonstrates that combining trastuzumab and Hu5F9-G4 represents a potential new treatment option for HER2+ breast cancer patients, even for patients whose tumors have progressed after trastuzumab.


Subject(s)
Antibodies, Monoclonal, Humanized/administration & dosage , Antineoplastic Agents, Immunological/administration & dosage , Breast Neoplasms/drug therapy , CD47 Antigen/immunology , Trastuzumab/administration & dosage , Animals , Antibody-Dependent Cell Cytotoxicity/drug effects , Breast Neoplasms/genetics , Breast Neoplasms/immunology , CD47 Antigen/antagonists & inhibitors , CD47 Antigen/genetics , Cell Line, Tumor , Drug Resistance, Neoplasm , Drug Therapy, Combination , Female , Humans , Immunotherapy , Macrophages/drug effects , Macrophages/immunology , Mice , Mice, Inbred C57BL , Receptor, ErbB-2/genetics , Receptor, ErbB-2/immunology
8.
Res Sq ; 2021 May 27.
Article in English | MEDLINE | ID: mdl-34075373

ABSTRACT

Monoclonal antibodies (mAbs) with neutralizing activity against SARS-CoV-2 have demonstrated clinical benefit in cases of mild to moderate SARS-CoV-2 infection, substantially reducing the risk for hospitalization and severe disease1-4. Treatment generally requires the administration of high doses of these mAbs with limited efficacy in preventing disease complications or mortality among hospitalized COVID-19 patients5. Here we report the development and evaluation of Fc-optimized anti-SARS-CoV-2 mAbs with superior potency to prevent or treat COVID-19 disease. In several animal models of COVID-19 disease6,7, we demonstrate that selective engagement of activating FcγRs results in improved efficacy in both preventing and treating disease-induced weight loss and mortality, significantly reducing the dose required to confer full protection upon SARS-CoV-2 challenge and treatment of pre-infected animals. Our results highlight the importance of FcγR pathways in driving antibody-mediated antiviral immunity, while excluding any pathogenic or disease-enhancing effects of FcγR engagement of anti-SARS-CoV-2 antibodies upon infection. These findings have important implications for the development of Fc-engineered mAbs with optimal Fc effector function and improved clinical efficacy against COVID-19 disease.

9.
Nature ; 590(7847): 624-629, 2021 02.
Article in English | MEDLINE | ID: mdl-33461211

ABSTRACT

In the type III CRISPR-Cas immune response of prokaryotes, infection triggers the production of cyclic oligoadenylates that bind and activate proteins that contain a CARF domain1,2. Many type III loci are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction  endonuclease-like domain3,4. However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases5,6, whether and how these inducible effectors provide defence is not known. Here we investigated a type III CRISPR accessory protein, which we name cyclic-oligoadenylate-activated single-stranded ribonuclease and single-stranded deoxyribonuclease 1 (Card1). Card1 forms a symmetrical dimer that has a large central cavity between its CRISPR-associated Rossmann fold and restriction endonuclease domains that binds cyclic tetra-adenylate. The binding of ligand results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold, in which a manganese cation coordinates the catalytic residues and activates the cleavage of single-stranded-but not double-stranded-nucleic acids (both DNA and RNA). In vivo, activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used in CRISPR systems to provide immunity.


Subject(s)
Adenine Nucleotides/metabolism , CRISPR-Cas Systems/immunology , DNA, Single-Stranded/metabolism , Deoxyribonucleases/metabolism , Endoribonucleases/metabolism , Oligoribonucleotides/metabolism , RNA/metabolism , Staphylococcus/enzymology , Staphylococcus/immunology , Adenine Nucleotides/immunology , Adenosine Triphosphate/metabolism , Bacteriophages/immunology , Bacteriophages/physiology , Biocatalysis , Catalytic Domain , Deoxyribonucleases/chemistry , Deoxyribonucleases/genetics , Endoribonucleases/chemistry , Endoribonucleases/genetics , Enzyme Activation , Ligands , Manganese/chemistry , Manganese/metabolism , Models, Molecular , Oligoribonucleotides/immunology , Plasmids/genetics , Plasmids/metabolism , Protein Multimerization , Rotation , Staphylococcus/growth & development , Staphylococcus/virology , Substrate Specificity
10.
Biochem Biophys Res Commun ; 539: 34-41, 2021 02 05.
Article in English | MEDLINE | ID: mdl-33418191

ABSTRACT

Self-renewal and multipotency are essential functions of hematopoietic stem cells (HSCs). To maintain homeostatic hematopoiesis, functionally uniform HSCs have been thought to be an ideal cell-of-origin. Recent technological advances in the field have allowed us to analyze HSCs with single cell resolution and implicate that functional heterogeneity may exist even within the highly purified HSC compartment. However, due in part to the technical limitations of analyzing extremely rare populations and our incomplete understanding of HSC biology, neither the biological meaning of why heterogeneity exists nor the precise mechanism of how heterogeneity is determined within the HSC compartment is entirely known. Here we show the first evidence that self-renewal capacity varies with the degree of replication stress dose and results in heterogeneity within the HSC compartment. Using the Hoxb5-reporter mouse line which enables us to distinguish between long-term (LT)-HSCs and short-term (ST)-HSCs, we have found that ST-HSCs quickly lose self-renewal capacity under high stress environments but can maintain self-renewal under low stress environments for long periods of time. Critically, exogeneous Hoxb5 expression confers protection against loss of self-renewal to Hoxb5-negative HSCs and can partially alter the cell fate of ST-HSCs to that of LT-HSCs. Our results demonstrate that Hoxb5 imparts functional heterogeneity in the HSC compartment by regulating self-renewal capacity. Additionally, Hoxb5-positive HSCs may exist as fail-safe system to protect from the exhaustion of HSCs throughout an organism's lifespan.


Subject(s)
Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Homeodomain Proteins/metabolism , Animals , Cell Differentiation/physiology , Cell Lineage , Cell Proliferation/physiology , Cell Self Renewal/physiology , Hematopoiesis , Homeodomain Proteins/genetics , Mice
11.
Pancreatology ; 21(1): 144-154, 2021 Jan.
Article in English | MEDLINE | ID: mdl-33309223

ABSTRACT

BACKGROUND: Discontinuation of branch-duct intraductal papillary mucinous neoplasm (BD-IPMN) surveillance after 5 years of no change remains controversial. Long-term outcomes of BD-IPMN without significant changes in the first 5 years were evaluated. METHODS: We performed a multi-center retrospective analysis of patients with BD-IPMN diagnosis from 2005 to 2011 (follow-up until 2017). Significant changes were defined as pancreatic cancer (PC), pancreatectomy, high-risk stigmata (HRS), worrisome features (WF) and worrisome EUS features (WEUS). RESULTS: Of 982 patients who had no significant changes, 5 (0.5%), 7 (0.7%), 99 (10.1%), 4 (0.4%) patients developed PC, HRS, WF, WEUS, respectively, post-5 years. PC and HRS/WF/WEUS incidences at 12 years were 1.0% and 29.0%, respectively. Patients that developed HRS/WF/WEUS had larger cyst size in first 5 years compared to those that did not [16 (12-23) vs. 12 (9-17) mm, p = 0.0001], cyst size of >15 mm having higher cumulative incidence of HRS/WF/WEUS. PC mortality was 0.8%; all-cause mortality was 32%. Incidence of mortality due to PC was higher in HRS/WF/WEUS group, p < 0.0001. The mortality rate at 12 years for ACCI (age-adjusted Charlson Comorbidity Index) of ≤3, 4-6, and ≥7 were 3.5%, 19.9%, and 57.6% (p < 0.0001), respectively. CONCLUSIONS: Incidence of PC in patients with BD-IPMN without significant changes in first 5 years of diagnosis remains low at 1.0%. Incidence of HRS/WF/WEUS was higher at 29.0%. PC-related mortality was higher in HRS/WF/WEUS group. These risks should be weighed against patients' overall mortality (utilizing scoring systems such as ACCI) when making surveillance decision of BD-IPMN beyond 5 years.


Subject(s)
Carcinoma, Pancreatic Ductal/pathology , Pancreatic Ducts/pathology , Pancreatic Neoplasms/pathology , Adenocarcinoma, Mucinous , Adult , Aged , Aged, 80 and over , Carcinoma, Pancreatic Ductal/mortality , Clinical Decision-Making , Female , Follow-Up Studies , Humans , Incidence , Male , Middle Aged , Pancreatectomy , Pancreatic Cyst/epidemiology , Pancreatic Cyst/pathology , Pancreatic Neoplasms/mortality , Retrospective Studies , Treatment Outcome , Young Adult
12.
Proc Natl Acad Sci U S A ; 117(45): 28046-28055, 2020 11 10.
Article in English | MEDLINE | ID: mdl-33093202

ABSTRACT

An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2-pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Drug Design , Protein Engineering/methods , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Antiviral Agents/metabolism , Binding Sites , HEK293 Cells , Humans , Molecular Docking Simulation , Mutation , Peptide Library , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae , Spike Glycoprotein, Coronavirus/chemistry
13.
bioRxiv ; 2020 Aug 04.
Article in English | MEDLINE | ID: mdl-32766586

ABSTRACT

An essential mechanism for SARS-CoV-1 and -2 infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human Fc domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2 pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50) in the 10-100 ng/ml range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-utilizing coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated or generated from convalescent patients.

14.
BMC Gastroenterol ; 20(1): 60, 2020 Mar 06.
Article in English | MEDLINE | ID: mdl-32143633

ABSTRACT

BACKGROUND: Endoscopic mucosal resection (EMR) is a minimally invasive procedure used for the treatment of lesions in the gastrointestinal (GI) tract. There is increased usage of hemoclips during EMR for the prevention of delayed bleeding. This study aimed to evaluate the effect of hemoclips in the prevention of delayed bleeding after EMR of upper and lower GI tract lesions. METHOD: This is a retrospective cohort study using the Kaiser Permanente Southern California (KPSC) EMR registry. Lesions in upper and lower GI tracts that underwent EMR between January 2012 and December 2015 were analyzed. Rates of delayed bleeding were compared between the hemoclip and no-hemoclip groups. Analysis was stratified by upper GI and lower GI lesions. Lower GI group was further stratified by right and left colon. We examined the relationship between clip use and several clinically-relevant variables among the patients who exhibited delayed bleeding. Furthermore, we explored possible procedure-level and endoscopist-level characteristics that may be associated with clip usage. RESULTS: A total of 18 out of 657 lesions (2.7%) resulted in delayed bleeding: 7 (1.1%) in hemoclip group and 11 (1.7%) in no-hemoclip group (p = 0.204). There was no evidence that clip use moderated the effects of the lesion size (p = 0.954) or lesion location (p = 0.997) on the likelihood of delayed bleed. In the lower GI subgroup, clip application did not alter the effect of polyp location (right versus left colon) on the likelihood of delayed bleed (p = 0.951). Logistic regression analyses showed that the clip use did not modify the likelihood of delayed bleeding as related to the following variables: use of aspirin/NSAIDs/anti-coagulants/anti-platelets, pathologic diagnoses (including different types of colon polypoid lesions), ablation, piecemeal resection. The total number of clips used was 901 at a minimum additional cost of $173,893. CONCLUSION: Prophylactic hemoclip application did not reduce delayed post-EMR bleed for upper and lower GI lesions in this retrospective study performed in a large-scale community practice setting. Routine prophylactic hemoclip application during EMR may lead to significantly higher healthcare cost without a clear clinical benefit.


Subject(s)
Endoscopic Mucosal Resection/adverse effects , Gastrointestinal Diseases/surgery , Hemostatic Techniques/instrumentation , Postoperative Hemorrhage/prevention & control , Aged , Cost-Benefit Analysis , Female , Health Care Costs , Hemostatic Techniques/economics , Humans , Male , Middle Aged , Retrospective Studies , Risk Factors , Time Factors
15.
Blood Adv ; 3(18): 2713-2721, 2019 09 24.
Article in English | MEDLINE | ID: mdl-31519647

ABSTRACT

Peritoneal adhesions are pathological fibroses that ensnare organs after abdominal surgery. This dense connective tissue can cause small bowel obstruction, female infertility, and chronic abdominal pain. The pathogenesis of adhesions is a fibrotic response to tissue damage coordinated between mesothelial cells, fibroblasts, and immune cells. We have previously demonstrated that peritoneal adhesions are a consequence of mechanical injury to the mesothelial layer sustained during surgery. Neutrophils are among the first leukocytes involved in the early response to tissue damage. Here, we show that when subjected to mechanical stress, activated mesothelial cells directly recruit neutrophils and monocytes through upregulation of chemokines such as CXCL1 and monocyte chemoattractant protein 1 (MCP-1). We find that neutrophils within the adhesion sites undergo cell death and form neutrophil extracellular traps (NETosis) that contribute to pathogenesis. Conversely, tissue-resident macrophages were profoundly depleted throughout the disease time course. We show that this is distinct from traditional inflammatory kinetics such as after sham surgery or chemically induced peritonitis, and suggest that adhesions result from a primary difference in inflammatory kinetics. We find that transient depletion of circulating neutrophils significantly decreases adhesion burden, and further recruitment of monocytes with thioglycolate or MCP-1 also improves outcomes. Our findings suggest that the combination of neutrophil depletion and monocyte recruitment is sufficient to prevent adhesion formation, thus providing insight for potential clinical interventions.


Subject(s)
Monocytes/metabolism , Neutrophils/metabolism , Tissue Adhesions/metabolism , Animals , Female , Humans , Mice
16.
Proc Natl Acad Sci U S A ; 116(37): 18416-18422, 2019 09 10.
Article in English | MEDLINE | ID: mdl-31451629

ABSTRACT

GABRR1 is a rho subunit receptor of GABA, the major inhibitory neurotransmitter in the mammalian brain. While most investigations of its function focused on the nervous system, its regulatory role in hematopoiesis has not been reported. In this study, we found GABRR1 is mainly expressed on subsets of human and mouse hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MkPs). GABRR1-negative (GR-) HSCs led to higher donor-derived hematopoietic chimerism than GABRR1-positive (GR+) HSCs. GR+ but not GR- HSCs and MkPs respond to GABA in patch clamp studies. Inhibition of GABRR1 via genetic knockout or antagonists inhibited MkP differentiation and reduced platelet numbers in blood. Overexpression of GABRR1 or treatment with agonists significantly promoted MkP generation and megakaryocyte colonies. Thus, this study identifies a link between the neural and hematopoietic systems and opens up the possibility of manipulating GABA signaling for platelet-required clinical applications.


Subject(s)
Hematopoietic Stem Cells/metabolism , Megakaryocyte Progenitor Cells/metabolism , Receptors, GABA-A/metabolism , Animals , Blood Platelets/cytology , Blood Platelets/metabolism , Cell Differentiation/physiology , Hematopoiesis , Hematopoietic Stem Cells/cytology , Humans , Male , Megakaryocyte Progenitor Cells/cytology , Megakaryocytes/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Receptors, GABA , Receptors, GABA-A/genetics , Transcriptome
17.
Cell Stem Cell ; 25(2): 185-192.e3, 2019 08 01.
Article in English | MEDLINE | ID: mdl-31204177

ABSTRACT

Hematopoietic cell transplantation can correct hematological and immunological disorders by replacing a diseased blood system with a healthy one, but this currently requires depleting a patient's existing hematopoietic system with toxic and non-specific chemotherapy, radiation, or both. Here we report an antibody-based conditioning protocol with reduced toxicity and enhanced specificity for robust hematopoietic stem cell (HSC) transplantation and engraftment in recipient mice. Host pre-treatment with six monoclonal antibodies targeting CD47, T cells, NK cells, and HSCs followed by donor HSC transplantation enabled stable hematopoietic system reconstitution in recipients with mismatches at half (haploidentical) or all major histocompatibility complex (MHC) genes. This approach allowed tolerance to heart tissue from HSC donor strains in haploidentical recipients, showing potential applications for solid organ transplantation without immune suppression. Fully mismatched chimeric mice developed antibody responses to nominal antigens, showing preserved functional immunity. These findings suggest approaches for transplanting immunologically mismatched HSCs and solid organs with limited toxicity.


Subject(s)
Hematopoietic Stem Cell Transplantation , Myocardium/immunology , Transplantation Conditioning/methods , Allografts/immunology , Animals , Antibodies, Monoclonal , Cells, Cultured , HLA Antigens/immunology , Humans , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Organ Transplantation , Radiation Chimera , Transplantation Tolerance , Transplantation, Haploidentical , Transplantation, Homologous
18.
Proc Natl Acad Sci U S A ; 115(41): 10333-10338, 2018 10 09.
Article in English | MEDLINE | ID: mdl-30181288

ABSTRACT

Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated, cation-selective channel, is a prokaryotic homolog of the pentameric Cys-loop receptor ligand-gated ion channel family. Despite large changes in ion conductance, small conformational changes were detected in X-ray structures of detergent-solubilized GLIC at pH 4 (active/desensitized state) and pH 7 (closed state). Here, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to perform structural titration experiments to visualize GLIC gating at the single-molecule level under native conditions. Reference-free 2D classification revealed channels in multiple conformational states during pH gating. We find changes of protein-protein interactions so far elusive and conformational dynamics much larger than previously assumed. Asymmetric pentamers populate early stages of activation, which provides evidence for an intermediate preactivated state.


Subject(s)
Bacterial Proteins/chemistry , Cysteine Loop Ligand-Gated Ion Channel Receptors/chemistry , Microscopy, Atomic Force/methods , Bacterial Proteins/metabolism , Cyanobacteria/chemistry , Cysteine Loop Ligand-Gated Ion Channel Receptors/metabolism , Hydrogen-Ion Concentration , Ion Channel Gating/physiology , Protein Conformation
19.
Nat Commun ; 9(1): 3194, 2018 08 10.
Article in English | MEDLINE | ID: mdl-30097573

ABSTRACT

Macrophage-mediated programmed cell removal (PrCR) is a process essential for the clearance of unwanted (damaged, dysfunctional, aged, or harmful) cells. The detection and recognition of appropriate target cells by macrophages is a critical step for successful PrCR, but its molecular mechanisms have not been delineated. Here using the models of tissue turnover, cancer immunosurveillance, and hematopoietic stem cells, we show that unwanted cells such as aging neutrophils and living cancer cells are susceptible to "labeling" by secreted calreticulin (CRT) from macrophages, enabling their clearance through PrCR. Importantly, we identified asialoglycans on the target cells to which CRT binds to regulate PrCR, and the availability of such CRT-binding sites on cancer cells correlated with the prognosis of patients in various malignancies. Our study reveals a general mechanism of target cell recognition by macrophages, which is the key for the removal of unwanted cells by PrCR in physiological and pathophysiological processes.


Subject(s)
Calreticulin/metabolism , Homeostasis , Neoplasms/metabolism , Phagocytosis , Adult , Aged , Aged, 80 and over , Animals , Binding Sites , Cell Line, Tumor , Cell Membrane/metabolism , Cell Survival , Cellular Senescence , Female , Hematopoiesis , Humans , Ligands , Macrophages/metabolism , Male , Mice , Middle Aged , Neoplasms/pathology , Neutrophils/metabolism , Polysaccharides/metabolism
20.
Antioxid Redox Signal ; 29(2): 191-204, 2018 07 10.
Article in English | MEDLINE | ID: mdl-29113449

ABSTRACT

SIGNIFICANCE: Hematopoietic stem cells (HSCs) can sustain the production of blood throughout one's lifetime. However, for proper self-renewal of its own population and differentiation to blood, the HSC requires a specialized microenvironment called the "niche." Recent Advances: Recent studies using novel mouse models have shed new light on the cellular architecture and function of the HSC niche. Here, we review the different cells that constitute the HSC niche and the molecular mechanisms that underlie HSC and niche interaction. We discuss the evidence and potential features that distinguish the HSC niche from other microenvironments in the bone marrow. The relevance of the niche in malignant transformation of the HSCs and harboring cancer metastasis to the bone is also outlined. In addition, we address how the niche may regulate reactive oxygen species levels surrounding the HSCs. Critical Issues and Future Directions: We propose future directions and remaining challenges in investigating the niche of HSCs. We discuss how a better understanding of the HSC niche may help in restoring an aged hematopoietic system, fighting against malignancies, and transplanting purified HSCs safely and effectively into patients. Antioxid. Redox Signal. 00, 000-000.


Subject(s)
Bone Marrow Cells/cytology , Hematopoietic Stem Cells/physiology , Stem Cell Niche , Animals , Bone Marrow Cells/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Leukemia/etiology , Mesenchymal Stem Cells , Mice , Reactive Oxygen Species/metabolism
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