Publications by authors named "Michael Gale"

309 Publications

T-cell receptor sequencing identifies prior SARS-CoV-2 infection and correlates with neutralizing antibodies and disease severity.

JCI Insight 2022 Apr 19. Epub 2022 Apr 19.

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, United States of America.

Background: Measuring the immune response to SARS-CoV-2 enables assessment of past infection and protective immunity. SARS-CoV-2 infection induces humoral and T-cell responses, but these responses vary with disease severity and individual characteristics.

Methods: A T-cell receptor (TCR) immunosequencing assay was conducted using small-volume blood samples from 302 individuals recovered from COVID-19. Correlations between the magnitude of the T-cell response and neutralizing antibody (nAb) titers or indicators of disease severity were evaluated. Sensitivity of T-cell testing was assessed and compared to serologic testing.

Results: SARS-CoV-2-specific T-cell responses were significantly correlated with nAb titers and clinical indicators of disease severity, including hospitalization, fever, and difficulty breathing. Despite modest declines in depth and breadth of T-cell responses during convalescence, high sensitivity was observed until at least 6 months after infection, with overall sensitivity ~5% greater than serology tests for identifying prior SARS-CoV-2 infection. Improved performance of T-cell testing was most apparent in recovered, non-hospitalized individuals sampled >150 days after initial illness, suggesting greater sensitivity than serology at later timepoints and in individuals with less severe disease. T-cell testing identified SARS-CoV-2 infection in 68% (55/81) of samples with undetectable nAb titers (<1:40) and in 37% (13/35) of samples negative by 3 antibody assays.

Conclusion: These results support TCR-based testing as a scalable, reliable measure of past SARS-CoV-2 infection with clinical value beyond serology.

Funding: Adaptive Biotechnologies, Frederick National Laboratory for Cancer Research, National Institutes of Allergy and Infectious Diseases, Fred Hutchinson Joel Meyers Endowment; Fast Grants, American Society for Transplantation and Cell Therapy.
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http://dx.doi.org/10.1172/jci.insight.150070DOI Listing
April 2022

Imprinted SARS-CoV-2-specific memory lymphocytes define hybrid immunity.

Cell 2022 Apr 17;185(9):1588-1601.e14. Epub 2022 Mar 17.

Department of Immunology, University of Washington School of Medicine, Seattle, WA 98109, USA. Electronic address:

Immune memory is tailored by cues that lymphocytes perceive during priming. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic created a situation in which nascent memory could be tracked through additional antigen exposures. Both SARS-CoV-2 infection and vaccination induce multifaceted, functional immune memory, but together, they engender improved protection from disease, termed hybrid immunity. We therefore investigated how vaccine-induced memory is shaped by previous infection. We found that following vaccination, previously infected individuals generated more SARS-CoV-2 RBD-specific memory B cells and variant-neutralizing antibodies and a distinct population of IFN-γ and IL-10-expressing memory SARS-CoV-2 spike-specific CD4 T cells than previously naive individuals. Although additional vaccination could increase humoral memory in previously naive individuals, it did not recapitulate the distinct CD4 T cell cytokine profile observed in previously infected subjects. Thus, imprinted features of SARS-CoV-2-specific memory lymphocytes define hybrid immunity.
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http://dx.doi.org/10.1016/j.cell.2022.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8926873PMC
April 2022

Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice.

Sci Transl Med 2022 Apr 12:eabn1252. Epub 2022 Apr 12.

Department of Biochemistry, University of Washington, Seattle, WA, 98195, USA.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy with a homo-trimeric version of the 75-residue angiotensin converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. In the cryo-electron microscopy structure, TRI2 formed a tripod on top of the spike protein which engaged all three receptor binding domains (RBDs) simultaneously as in the design model. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than that of monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies and native receptor traps. By comparison, the designed proteins have resistance to viral escape and antigenic drift by construction, precisely tuned avidity, and greatly reduced chance of autoimmune responses.
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http://dx.doi.org/10.1126/scitranslmed.abn1252DOI Listing
April 2022

Validation of Home Visual Acuity Tests for Telehealth in the COVID-19 Era.

JAMA Ophthalmol 2022 May;140(5):465-471

Casey Eye Institute, Oregon Health & Science University, Portland.

Importance: Visual acuity (VA) is one of the most important clinical data points in ophthalmology. However, few options for validated at-home VA assessments are currently available.

Objective: To validate 3 at-home visual acuity tests in comparison with in-office visual acuity.

Design, Setting, And Participants: Between July 2020 and April 2021, eligible participants with VA of 20/200 or better were recruited from 4 university-based ophthalmology clinics (comprehensive, cornea, glaucoma, and retina clinics). Participants were prospectively randomized to self-administer 2 of 3 at-home VA tests (printed chart, mobile phone app, and website) within 3 days before their standard-of-care clinic visit. Participants completed a survey assessing usability of the at-home tests. At the clinic visit, best-corrected Snellen distance acuity was measured as the reference standard.

Main Outcomes And Measures: The at-home VA test results were compared with the in-office VA test results using paired and unpaired t tests, Pearson correlation coefficients, analysis of variance, χ2 tests, and Cohen κ agreement. The sensitivity, specificity, positive predictive value, and negative predictive value of each at-home test were calculated to detect significant VA changes (≥0.2 logMAR) from the in-office baseline.

Results: A total of 121 participants with a mean (SD) age of 63.8 (13.0) years completed the study. The mean in-office VA was 0.11 logMAR (Snellen equivalent 20/25) with similar numbers of participants from the 4 clinics. Mean difference (logMAR) between the at-home test and in-office acuity was -0.07 (95% CI, -0.10 to -0.04) for the printed chart, -0.12 (95% CI, -0.15 to -0.09) for the mobile phone app, and -0.13 (95% CI, -0.16 to -0.10) for the website test. The Pearson correlation coefficient for the printed chart was 0.72 (95% CI, 0.62-0.79), mobile phone app was 0.58 (95% CI, 0.46-0.69), and website test was 0.64 (95% CI, 0.53-0.73).

Conclusions And Relevance: The 3 at-home VA test results (printed chart, mobile phone app, and website) appeared comparable within 1 line to in-office VA measurements. Older participants were more likely to have limited access to digital tools. Further development and validation of at-home VA testing modalities is needed with the expansion of teleophthalmology care.
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http://dx.doi.org/10.1001/jamaophthalmol.2022.0396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8972145PMC
May 2022

ZIKV can infect human term placentas in the absence of maternal factors.

Commun Biol 2022 03 18;5(1):243. Epub 2022 Mar 18.

Center for Innate Immunity and Immune Disease, Department of Immunology, University of Washington School of Medicine, Seattle, WA, USA.

Zika virus infection can result in devastating pregnancy outcomes when it crosses the placental barrier. For human pregnancies, the mechanisms of vertical transmission remain enigmatic. Utilizing a human placenta-cotyledon perfusion model, we examined Zika virus exposure in the absence of maternal factors. To distinguish responses related to viral infection vs. recognition, we evaluated cotyledons perfused with either active or inactivated Zika virus. Active Zika virus exposure resulted in infection, cell death and syncytium injury. Pathology corresponded with transcriptional changes related to inflammation and innate immunity. Inactive Zika virus exposure also led to syncytium injury and related changes in gene expression but not cell death. Our observations reveal pathologies and innate immune responses that are dependent on infection or virus placenta interactions independent of productive infection. Importantly, our findings indicate that Zika virus can infect and compromise placentas in the absence of maternal humoral factors that may be protective.
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http://dx.doi.org/10.1038/s42003-022-03158-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8933440PMC
March 2022

Biological functions of DEAD/DEAH-box RNA helicases in health and disease.

Nat Immunol 2022 03;23(3):354-357

Center for Innate Immunity and Immune Disease, Department of Immunology, University of Washington, Seattle, WA, USA.

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http://dx.doi.org/10.1038/s41590-022-01149-7DOI Listing
March 2022

SARS-CoV2 variant-specific replicating RNA vaccines protect from disease following challenge with heterologous variants of concern.

Elife 2022 Feb 22;11. Epub 2022 Feb 22.

HDT Bio, Seattle, United States.

Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late 2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoCs) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second-generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered in vivo via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform can be updated to target emergent VoCs, elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.
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http://dx.doi.org/10.7554/eLife.75537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8983041PMC
February 2022

Performance of anterior nares and tongue swabs for nucleic acid, Nucleocapsid, and Spike antigen testing for detecting SARS-CoV-2 against nasopharyngeal PCR and viral culture.

Int J Infect Dis 2022 Apr 9;117:287-294. Epub 2022 Feb 9.

Department of Global Health, School of Public Health, University of Washington, Seattle, WA.

Objectives: This study assesses and compares the performance of different swab types and specimen collection sites for SARS-CoV-2 testing, to reference standard real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and viral culture.

Methods: Symptomatic adults with COVID-19 who visited routine COVID-19 testing sites used spun polyester and FLOQSwabs to self-collect specimens from the anterior nares and tongue. We evaluated the self-collected specimen from anterior nares and tongue swabs for the nucleocapsid (N) or spike (S) antigen of SARS-CoV-2 by RT-PCR and then compared these results with results from RT-PCR and viral cultures from nurse-collected nasopharyngeal swabs.

Results: Diagnostic sensitivity was highest for RT-PCR testing conducted using specimens from the anterior nares collected on FLOQSwabs (84%; 95% CI 68-94%) and spun polyester swabs (82%; 95% CI 66-92%), compared to RT-PCR tests conducted using specimens from nasopharyngeal swabs. Relative to viral culture from nasopharyngeal swabs, diagnostic sensitivities were higher for RT-PCR and antigen testing of anterior nares swabs (91-100%) than that of tongue swabs (18-81%). Antigen testing of anterior nares swabs had higher sensitivities against viral culture (91%) than against nasopharyngeal RT-PCR (38-70%). All investigational tests had high specificity compared with nasopharyngeal RT-PCR. Spun polyester swabs are equally effective as FLOQSwabs for anterior nasal RT-PCR testing.

Conclusions: We found that anterior nares specimens were more sensitive than tongue swab specimens or antigen testing for detecting SARS-CoV-2 by RT-PCR. Thus, self-collected anterior nares specimens may represent an alternative method for diagnostic SARS-CoV-2 testing in some settings.
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http://dx.doi.org/10.1016/j.ijid.2022.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8827388PMC
April 2022

T cell response to intact SARS-CoV-2 includes coronavirus cross-reactive and variant-specific components.

JCI Insight 2022 03 22;7(6). Epub 2022 Mar 22.

Department of Medicine.

SARS-CoV-2 provokes a robust T cell response. Peptide-based studies exclude antigen processing and presentation biology, which may influence T cell detection studies. To focus on responses to whole virus and complex antigens, we used intact SARS-CoV-2 and full-length proteins with DCs to activate CD8 and CD4 T cells from convalescent people. T cell receptor (TCR) sequencing showed partial repertoire preservation after expansion. Resultant CD8 T cells recognize SARS-CoV-2-infected respiratory tract cells, and CD4 T cells detect inactivated whole viral antigen. Specificity scans with proteome-covering protein/peptide arrays show that CD8 T cells are oligospecific per subject and that CD4 T cell breadth is higher. Some CD4 T cell lines enriched using SARS-CoV-2 cross-recognize whole seasonal coronavirus (sCoV) antigens, with protein, peptide, and HLA restriction validation. Conversely, recognition of some epitopes is eliminated for SARS-CoV-2 variants, including spike (S) epitopes in the Alpha, Beta, Gamma, and Delta variant lineages.
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http://dx.doi.org/10.1172/jci.insight.158126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8986086PMC
March 2022

T cell response to intact SARS-CoV-2 includes coronavirus cross-reactive and variant-specific components.

medRxiv 2022 Jan 25. Epub 2022 Jan 25.

SARS-CoV-2 provokes a brisk T cell response. Peptide-based studies exclude antigen processing and presentation biology and may influence T cell detection studies. To focus on responses to whole virus and complex antigens, we used intact SARS-CoV-2 and full-length proteins with DC to activate CD8 and CD4 T cells from convalescent persons. T cell receptor (TCR) sequencing showed partial repertoire preservation after expansion. Resultant CD8 T cells recognize SARS-CoV-2-infected respiratory cells, and CD4 T cells detect inactivated whole viral antigen. Specificity scans with proteome-covering protein/peptide arrays show that CD8 T cells are oligospecific per subject and that CD4 T cell breadth is higher. Some CD4 T cell lines enriched using SARS-CoV-2 cross-recognize whole seasonal coronavirus (sCoV) antigens, with protein, peptide, and HLA restriction validation. Conversely, recognition of some epitopes is eliminated for SARS-CoV-2 variants, including spike (S) epitopes in the alpha, beta, gamma, and delta variant lineages.
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http://dx.doi.org/10.1101/2022.01.23.22269497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8811910PMC
January 2022

Liver Bacterial Dysbiosis With Non-Tuberculosis Mycobacteria Occurs in SIV-Infected Macaques and Persists During Antiretroviral Therapy.

Front Immunol 2021 10;12:793842. Epub 2022 Jan 10.

Seattle Children's Research Institute, Center for Global Infectious Disease Research, Seattle, WA, United States.

Liver disease is a significant contributor to morbidity and mortality in HIV-infected individuals, even during successful viral suppression with combination antiretroviral therapy (cART). Similar to HIV infection, SIV infection of rhesus macaques is associated with gut microbiome dysbiosis and microbial translocation that can be detected systemically in the blood. As microbes leaving the intestines must first pass through the liver the portal vein, we evaluated the livers of both SIV-infected (SIV+) and SIV-infected cART treated (SIV+cART) rhesus macaques for evidence of microbial changes compared to uninfected macaques. Dysbiosis was observed in both the SIV+ and SIV+cART macaques, encompassing changes in the relative abundance of several genera, including a reduction in the levels of and . Most strikingly, we found an increase in the relative abundance and absolute quantity of bacteria within the genus in both SIV+ and SIV+cART macaques. Multi-gene sequencing identified a species of atypical mycobacteria similar to the opportunistic pathogen . Phosphatidyl inositol lipoarabinomannan (PILAM) (a glycolipid cell wall component found in atypical mycobacteria) stimulation in primary human hepatocytes resulted in an upregulation of inflammatory transcriptional responses, including an increase in the chemokines associated with neutrophil recruitment (CXCL1, CXCL5, and CXCL6). These studies provide key insights into SIV associated changes in hepatic microbial composition and indicate a link between microbial components and immune cell recruitment in SIV+ and SIV+cART treated macaques.
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http://dx.doi.org/10.3389/fimmu.2021.793842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8784802PMC
February 2022

Single-Cell-Based High-Throughput Ig and TCR Repertoire Sequencing Analysis in Rhesus Macaques.

J Immunol 2022 02 5;208(3):762-771. Epub 2022 Jan 5.

Department of Molecular Biomedical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC;

Recent advancements in microfluidics and high-throughput sequencing technologies have enabled recovery of paired H and L chains of Igs and VDJ and VJ chains of TCRs from thousands of single cells simultaneously in humans and mice. Despite rhesus macaques being one of the most well-studied model organisms for the human adaptive immune response, high-throughput single-cell immune repertoire sequencing assays are not yet available due to the complexity of these polyclonal receptors. We used custom primers that capture all known rhesus macaque Ig and TCR isotypes and chains that are fully compatible with a commercial solution for single-cell immune repertoire profiling. Using these rhesus-specific assays, we sequenced Ig and TCR repertoires in >60,000 cells from cryopreserved rhesus PBMCs, splenocytes, and FACS-sorted B and T cells. We were able to recover every Ig isotype and TCR chain, measure clonal expansion in proliferating T cells, and pair Ig and TCR repertoires with gene expression profiles of the same single cells. Our results establish the ability to perform high-throughput immune repertoire analysis in rhesus macaques at the single-cell level.
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http://dx.doi.org/10.4049/jimmunol.2100824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8820446PMC
February 2022

SARS-CoV2 variant-specific replicating RNA vaccines protect from disease and pathology and reduce viral shedding following challenge with heterologous SARS-CoV2 variants of concern.

bioRxiv 2021 Dec 13. Epub 2021 Dec 13.

HDT Bio, Seattle, WA 98102, USA.

Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late-2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoC) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.
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http://dx.doi.org/10.1101/2021.12.10.472134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8687464PMC
December 2021

Myelin Protein Zero Immunohistochemistry Is Not a Reliable Marker of Extrinsic Mucosal Innervation in Patients With Hirschsprung Disease.

Pediatr Dev Pathol 2021 Dec 14:10935266211059395. Epub 2021 Dec 14.

Center for Innate Immunity and Immune Disease, Department of Immunology, 7284University of Washington, Seattle, WA, USA.

Background: Innervation of aganglionic rectum in Hirschsprung disease derives from extrinsic nerves which project from cell bodies located outside the bowel wall and markers that distinguish extrinsic from intrinsic innervation are diagnostically useful. Myelin protein zero (MPZ) is a putative marker of extrinsic glial cells which could distinguish mucosal innervation in aganglionic vs ganglionic colon.

Methods: Sections and protein blots from ganglionic and aganglionic colon were immunolabeled with MPZ-specific antibodies.

Results: Immunolabeling of MPZ with a chicken polyclonal or mouse monoclonal antibody confirmed glial specificity and reliably labeled hypertrophic submucosal nerves in Hirschsprung disease. In contrast, a rabbit polyclonal antibody strongly labeled extrinsic and intrinsic nerves, including most mucosal branches. Immunoblots showed MPZ is expressed in mucosal glial cells, albeit at lower levels than in extrinsic nerves, and that the rabbit antibody is more sensitive that the other two probes. Unfortunately, none of these antibodies consistently distinguished mucosal innervation in aganglionic vs ganglionic rectum.

Conclusions: The results suggest that (a) glial cell myelin protein zero expression is influenced more by location (mucosa vs submucosa) than the extrinsic vs intrinsic origin of the accompanied nerves and (b) myelin protein zero immunohistochemistry has limited value as a diagnostic adjunct for Hirschsprung disease.
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http://dx.doi.org/10.1177/10935266211059395DOI Listing
December 2021

Cross-validation of SARS-CoV-2 responses in kidney organoids and clinical populations.

JCI Insight 2021 12 22;6(24). Epub 2021 Dec 22.

Department of Medicine.

Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome-edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules among diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflects proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants alpha, beta, gamma, kappa, and delta exhibit comparable levels of infection in organoids. Infection is ameliorated in ACE2-/- organoids and blocked via treatment with de novo-designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.
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http://dx.doi.org/10.1172/jci.insight.154882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8783682PMC
December 2021

Recurrent bacterial vaginosis following metronidazole treatment is associated with microbiota richness at diagnosis.

Am J Obstet Gynecol 2022 02 22;226(2):225.e1-225.e15. Epub 2021 Sep 22.

Division of Surgical Outcomes and Precision Medicine Research, Department of Surgery, University of Minnesota Medical School, Minneapolis, MN; Department of Pediatrics, University of Miami, Miami, FL. Electronic address:

Background: Bacterial vaginosis-a condition defined by a shift from Lactobacillus dominance to a polymicrobial, anaerobic bacterial community-increases the risk of acquiring sexually transmitted infections and other complications of the female reproductive tract. Antibiotic treatment frequently fails to return the microbiome to an optimal Lactobacillus-dominated state. No criteria currently exist to identify the patients likely to experience treatment failure.

Objective: We sought to identify the pretreatment community signatures associated with treatment failure through 16S ribosomal RNA gene analysis.

Study Design: Twenty-eight women who were enrolled in an oral metronidazole treatment trial of bacterial vaginosis were studied. Cervicovaginal lavage samples were collected before metronidazole treatment and at 7 and 30 days posttreatment. Cervicovaginal lavage DNA was amplified and sequenced using a paired-end, V4 region 2×150 MiSeq run.

Results: Of the 28 women, 25% failed to clear bacterial vaginosis; 35.7% demonstrated a transient clearance, shifting to community-type 2 (Lactobacillus iners dominant) at visit 2 only; 7.1% demonstrated a delayed clearance, reaching community-type 2 at the final visit only; and 32.1% of patients experienced sustained bacterial vaginosis clearance. Examination of the community composition and structure demonstrated that both the richness and the evenness were significantly lower for the women who experienced sustained clearance, whereas the women who failed to clear bacterial vaginosis possessed the highest median levels of richness, evenness, and diversity pretreatment. Soluble immune factors in the lower reproductive tract improved significantly following a shift from community-type 4 to a Lactobacillus-dominant microbiome, with the samples categorized as community-type 2 possessing significantly higher levels of secretory leukocyte protease inhibitor, growth-regulated alpha protein, and macrophage inflammatory protein-3 and significantly lower levels of intercellular adhesion molecule-1. Although the shifts to Lactobacillus dominance improved the markers of mucosal tissue health, these gains were only temporary among the women who experienced recurrence.

Conclusion: Assemblies of highly diverse microbiota are associated with the enhanced resilience of bacterial vaginosis to standard metronidazole treatment. These communities may be foundational to treatment resistance or simply an indication of a well-established community made possible by canonical biofilm-forming taxa. Future studies must target the transcriptional activity of these communities under the pressure of antibiotic treatment to resolve the mechanisms of their resistance.
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http://dx.doi.org/10.1016/j.ajog.2021.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8887553PMC
February 2022

Durability of mRNA-1273 vaccine-induced antibodies against SARS-CoV-2 variants.

Science 2021 Sep 13;373(6561):1372-1377. Epub 2021 Aug 13.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations may diminish vaccine-induced protective immune responses, particularly as antibody titers wane over time. Here, we assess the effect of SARS-CoV-2 variants B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.429 (Epsilon), B.1.526 (Iota), and B.1.617.2 (Delta) on binding, neutralizing, and angiotensin-converting enzyme 2 (ACE2)–competing antibodies elicited by the messenger RNA (mRNA) vaccine mRNA-1273 over 7 months. Cross-reactive neutralizing responses were rare after a single dose. At the peak of response to the second vaccine dose, all individuals had responses to all variants. Binding and functional antibodies against variants persisted in most subjects, albeit at low levels, for 6 months after the primary series of the mRNA-1273 vaccine. Across all assays, B.1.351 had the lowest antibody recognition. These data complement ongoing studies to inform the potential need for additional boost vaccinations.
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http://dx.doi.org/10.1126/science.abj4176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8691522PMC
September 2021

Single-cell transcriptomic analyses of T cells in chronic HCV-infected patients dominated by DAA-induced interferon signaling changes.

PLoS Pathog 2021 08 9;17(8):e1009799. Epub 2021 Aug 9.

Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.

Chronic infection with HCV is manifested by dysregulation of innate immune responses and impaired T cell function at multiple levels. These changes may impact susceptibility to other infections, responsiveness to antiviral therapies, vaccine responsiveness, and development of complications such as hepatocellular carcinoma. Highly effective direct-acting antiviral (DAA) therapy has revolutionized the management of chronic HCV, with expected cure rates exceeding 95%. DAA treatment represents a unique opportunity to investigate to what extent elimination of viral replication and chronic antigen stimulation can restore immunologic phenotype. In this study we interrogated the global transcriptional profile of isolated peripheral blood T cells before, during and after IFN-free DAA therapy using single-cell mRNA sequencing. Our results demonstrate that T cells mapped at single-cell resolution have dramatic transcriptomic changes early after initiation of DAA and many of these changes are sustained after completion of DAA therapy. Specifically, we see a significant reduction in transcripts associated with innate immune activation and interferon signaling such as ISG15, ISG20, IFIT3, OAS and MX1 in many different T cell subsets. Furthermore, we find an early upregulation of a gene involved in suppression of immune activation, DUSP1, in circulating T cells. Conclusion: This study provides the first in-depth transcriptomic analysis at the single-cell level of patients undergoing DAA therapy, demonstrating that IFN-free antiviral therapy in chronic HCV infection induces hitherto unrecognized shifts in innate immune and interferon signaling within T cell populations early, during, and long-term after treatment. The present study provides a rich data source to explore the effects of DAA treatment on bulk T cells.
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http://dx.doi.org/10.1371/journal.ppat.1009799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8376199PMC
August 2021

Endomembrane targeting of human OAS1 p46 augments antiviral activity.

Elife 2021 08 3;10. Epub 2021 Aug 3.

Department of Immunology, School of Medicine, University of Washington, Seattle, United States.

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from cellular innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flaviviruses, picornaviruses, and SARS-CoV-2. Finally, our human genetic analysis shows that the splice-site SNP responsible for production of the OAS1 p46 isoform correlates with protection from severe COVID-19. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests that early control of SARS-CoV-2 replication through OAS1 p46 is an important determinant of COVID-19 severity.
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http://dx.doi.org/10.7554/eLife.71047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8357416PMC
August 2021

Multivalent designed proteins protect against SARS-CoV-2 variants of concern.

bioRxiv 2021 Jul 7. Epub 2021 Jul 7.

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness.

One-sentence Summary: We designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.
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http://dx.doi.org/10.1101/2021.07.07.451375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8282097PMC
July 2021

Interleukin-15 response signature predicts RhCMV/SIV vaccine efficacy.

PLoS Pathog 2021 07 6;17(7):e1009278. Epub 2021 Jul 6.

Department of Pathology, Case Western Reserve University, Cleveland, Ohio, United States of America.

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with strain 68-1 Rhesus Cytomegalovirus (RhCMV) vectors expressing SIV proteins (RhCMV/SIV) results in a binary outcome: stringent control and subsequent clearance of highly pathogenic SIV in ~55% of vaccinated RMs with no protection in the remaining 45%. Although previous work indicates that unconventionally restricted, SIV-specific, effector-memory (EM)-biased CD8+ T cell responses are necessary for efficacy, the magnitude of these responses does not predict efficacy, and the basis of protection vs. non-protection in 68-1 RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that 68-1 RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including immune cell, toll-like receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly correlating with subsequent vaccine efficacy. Treatment of a separate RM cohort with IL-15 confirmed the central involvement of this cytokine in the protection signature, linking the major innate and adaptive immune gene expression networks that correlate with RhCMV/SIV vaccine efficacy. This change-from-baseline IL-15 response signature was also demonstrated to significantly correlate with vaccine efficacy in an independent validation cohort of vaccinated and challenged RMs. The differential IL-15 gene set response to vaccination strongly correlated with the pre-vaccination activity of this pathway, with reduced baseline expression of IL-15 response genes significantly correlating with higher vaccine-induced induction of IL-15 signaling and subsequent vaccine protection, suggesting that a robust de novo vaccine-induced IL-15 signaling response is needed to program vaccine efficacy. Thus, the RhCMV/SIV vaccine imparts a coordinated and persistent induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ T cell function, that support the ability of vaccine-elicited unconventionally restricted CD8+ T cells to mediate protection against SIV challenge.
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http://dx.doi.org/10.1371/journal.ppat.1009278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284654PMC
July 2021

Correlation of Regulatory T Cell Numbers with Disease Tolerance upon Virus Infection.

Immunohorizons 2021 04 23;5(4):157-169. Epub 2021 Apr 23.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA;

The goal of a successful immune response is to clear the pathogen while sparing host tissues from damage associated with pathogen replication and active immunity. Regulatory T cells (Treg) have been implicated in maintaining this balance as they contribute both to the organization of immune responses as well as restriction of inflammation and immune activation to limit immunopathology. To determine if Treg abundance prior to pathogen encounter can be used to predict the success of an antiviral immune response, we used genetically diverse mice from the collaborative cross infected with West Nile virus (WNV). We identified collaborative cross lines with extreme Treg abundance at steady state, either high or low, and used mice with these extreme phenotypes to demonstrate that baseline Treg quantity predicted the magnitude of the CD8 T cell response to WNV infection, although higher numbers of baseline Tregs were associated with reduced CD8 T cell functionality in terms of TNF and granzyme B expression. Finally, we found that abundance of CD44 Tregs in the spleen at steady state was correlated with an increased early viral load within the spleen without an association with clinical disease. Thus, we propose that Tregs participate in disease tolerance in the context of WNV infection by tuning an appropriately focused and balanced immune response to control the virus while at the same time minimizing immunopathology and clinical disease. We hypothesize that Tregs limit the antiviral CD8 T cell function to curb immunopathology at the expense of early viral control as an overall host survival strategy.
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http://dx.doi.org/10.4049/immunohorizons.2100009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8281504PMC
April 2021

SARS-CoV-2 ORF6 Disrupts Bidirectional Nucleocytoplasmic Transport through Interactions with Rae1 and Nup98.

mBio 2021 04 13;12(2). Epub 2021 Apr 13.

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA

RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The accessory protein ORF6 has previously been shown to be a major inhibitor of interferon production in both severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show SARS-CoV-2-infected cells display an elevated level of nuclear mRNA accumulation compared to mock-infected cells. We demonstrate that ORF6 is responsible for this nuclear imprisonment of host mRNA, and using a cotransfected reporter assay, we show this nuclear retention of mRNA blocks expression of newly transcribed mRNAs. ORF6's nuclear entrapment of host mRNA is associated with its ability to copurify with the mRNA export factors, Rae1 and Nup98. These protein-protein interactions map to the C terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly copurifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and presymptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. We also show that both SARS-CoV and SARS-CoV-2 ORF6 block nuclear import of a broad range of host proteins. Together, these data support a model in which ORF6 clogs the nuclear pore through its interactions with Rae1 and Nup98 to prevent both nuclear import and export, rendering host cells incapable of responding to SARS-CoV-2 infection. SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), is an RNA virus with a large genome that encodes multiple accessory proteins. While these accessory proteins are not required for growth , they can contribute to the pathogenicity of the virus. We demonstrate that SARS-CoV-2-infected cells accumulate poly(A) mRNA in the nucleus, which is attributed to the accessory protein ORF6. Nuclear entrapment of mRNA and reduced expression of newly transcribed reporter proteins are associated with ORF6's interactions with the mRNA export proteins Rae1 and Nup98. SARS-CoV ORF6 also shows the same interactions with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly represses reporter expression and copurifies with Rae1 and Nup98 compared to SARS-CoV ORF6. Both SARS-CoV ORF6 and SARS-CoV-2 ORF6 block nuclear import of a wide range of host factors through interactions with Rae1 and Nup98. Together, our results suggest ORF6's disruption of nucleocytoplasmic transport prevents infected cells from responding to the invading virus.
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http://dx.doi.org/10.1128/mBio.00065-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092196PMC
April 2021

The herpesvirus accessory protein γ134.5 facilitates viral replication by disabling mitochondrial translocation of RIG-I.

PLoS Pathog 2021 03 26;17(3):e1009446. Epub 2021 Mar 26.

Department of Microbiology and Immunology University of Illinois College of Medicine, Chicago, Illinois, United States of America.

RIG-I and MDA5 are cytoplasmic RNA sensors that mediate cell intrinsic immunity against viral pathogens. While it has been well-established that RIG-I and MDA5 recognize RNA viruses, their interactive network with DNA viruses, including herpes simplex virus 1 (HSV-1), remains less clear. Using a combination of RNA-deep sequencing and genetic studies, we show that the γ134.5 gene product, a virus-encoded virulence factor, enables HSV growth by neutralization of RIG-I dependent restriction. When expressed in mammalian cells, HSV-1 γ134.5 targets RIG-I, which cripples cytosolic RNA sensing and subsequently suppresses antiviral gene expression. Rather than inhibition of RIG-I K63-linked ubiquitination, the γ134.5 protein precludes the assembly of RIG-I and cellular chaperone 14-3-3ε into an active complex for mitochondrial translocation. The γ134.5-mediated inhibition of RIG-I-14-3-3ε binding abrogates the access of RIG-I to mitochondrial antiviral-signaling protein (MAVS) and activation of interferon regulatory factor 3. As such, unlike wild type virus HSV-1, a recombinant HSV-1 in which γ134.5 is deleted elicits efficient cytokine induction and replicates poorly, while genetic ablation of RIG-I expression, but not of MDA5 expression, rescues viral growth. Collectively, these findings suggest that viral suppression of cytosolic RNA sensing is a key determinant in the evolutionary arms race of a large DNA virus and its host.
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http://dx.doi.org/10.1371/journal.ppat.1009446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996975PMC
March 2021

SARS-CoV-2 Infects Human Pluripotent Stem Cell-Derived Cardiomyocytes, Impairing Electrical and Mechanical Function.

Stem Cell Reports 2021 03 13;16(3):478-492. Epub 2021 Feb 13.

Department of Laboratory Medicine and Pathology, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195, USA; Center for Cardiovascular Biology, University of Washington, 850 Republican Street, Brotman Building, Seattle, WA 98109, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, 850 Republican Street, Seattle, WA 98109, USA; Sana Biotechnology, 188 E Blaine Street, Seattle, WA 98102, USA; Department of Bioengineering, University of Washington, 3720 15th Avenue NE, Seattle, WA 98105, USA; Department of Medicine/Cardiology, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195, USA. Electronic address:

COVID-19 patients often develop severe cardiovascular complications, but it remains unclear if these are caused directly by viral infection or are secondary to a systemic response. Here, we examine the cardiac tropism of SARS-CoV-2 in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and smooth muscle cells (hPSC-SMCs). We find that that SARS-CoV-2 selectively infects hPSC-CMs through the viral receptor ACE2, whereas in hPSC-SMCs there is minimal viral entry or replication. After entry into cardiomyocytes, SARS-CoV-2 is assembled in lysosome-like vesicles and egresses via bulk exocytosis. The viral transcripts become a large fraction of cellular mRNA while host gene expression shifts from oxidative to glycolytic metabolism and upregulates chromatin modification and RNA splicing pathways. Most importantly, viral infection of hPSC-CMs progressively impairs both their electrophysiological and contractile function, and causes widespread cell death. These data support the hypothesis that COVID-19-related cardiac symptoms can result from a direct cardiotoxic effect of SARS-CoV-2.
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http://dx.doi.org/10.1016/j.stemcr.2021.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881699PMC
March 2021

Innate cell microenvironments in lymph nodes shape the generation of T cell responses during type I inflammation.

Sci Immunol 2021 02;6(56)

Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington School of Medicine, Seattle, WA 98109, USA.

Microanatomical organization of innate immune cells within lymph nodes (LNs) is critical for the generation of adaptive responses. In particular, steady-state LN-resident dendritic cells (Res cDCs) are strategically localized to intercept lymph-draining antigens. Whether myeloid cell organization changes during inflammation and how that might affect the generation of immune responses are unknown. Here, we report that during type I, but not type II, inflammation after adjuvant immunization or viral infection, antigen-presenting Res cDCs undergo CCR7-dependent intranodal repositioning from the LN periphery into the T cell zone (TZ) to elicit T cell priming. Concurrently, inflammatory monocytes infiltrate the LNs via local blood vessels, enter the TZ, and cooperate with Res cDCs by providing polarizing cytokines to optimize T cell effector differentiation. Monocyte infiltration is nonuniform across LNs, generating distinct microenvironments with varied local innate cell composition. These spatial microdomains are associated with divergent early T cell effector programming, indicating that innate microenvironments within LNs play a critical role in regulating the quality and heterogeneity of T cell responses. Together, our findings reveal that dynamic modulation of innate cell microenvironments during type I inflammation leads to optimized generation of adaptive immune responses to vaccines and infections.
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http://dx.doi.org/10.1126/sciimmunol.abb9435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8274717PMC
February 2021

Suppression of hepatitis B virus through therapeutic activation of RIG-I and IRF3 signaling in hepatocytes.

iScience 2021 Jan 4;24(1):101969. Epub 2021 Jan 4.

Center for Innate Immunity and Immune Disease, Department of Immunology, University of Washington, Seattle, WA 98109, USA.

Hepatitis B virus (HBV) mediates persistent infection, chronic hepatitis, and liver disease. HBV covalently closed circular (ccc)DNA is central to viral persistence such that its elimination is considered the cornerstone for HBV cure. Inefficient detection by pathogen recognition receptors (PRRs) in the infected hepatocyte facilitates HBV persistence via avoidance of innate immune activation and interferon regulatory factor (IRF)3 induction of antiviral gene expression. We evaluated a small molecule compound, F7, and 5'-triphosphate-poly-U/UC pathogen-associated-molecular-pattern (PAMP) RNA agonists of RIG-I, a PRR that signals innate immunity, for ability to suppress cccDNA. F7 and poly-U/UC PAMP treatment of HBV-infected cells induced RIG-I signaling of IRF3 activation to induce antiviral genes for suppression of cccDNA formation and accelerated decay of established cccDNA, and were additive to the actions of entecavir. Our study shows that activation of the RIG-I pathway and IRF3 to induce innate immune actions offers therapeutic benefit toward elimination of cccDNA.
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http://dx.doi.org/10.1016/j.isci.2020.101969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7797372PMC
January 2021

Diabetic eye disease: A review of screening and management recommendations.

Clin Exp Ophthalmol 2021 Mar 10;49(2):128-145. Epub 2021 Jan 10.

Casey Eye Institute, Department of Ophthalmology, Oregon Health & Science University, Portland, Oregon, USA.

The prevalence of diabetes is rapidly increasing, and it is now the leading cause of blindness worldwide. Although early detection of diabetic retinopathy is key to preventing vision loss, many patients do not receive appropriate examinations. Using a multidisciplinary approach, primary care physicians and eye care providers should follow evidence-based recommendations for screening and monitoring diabetic patients while working to improve patients' glycaemic index, blood pressure, and metabolic risk factors. Anti-vascular endothelial growth factor intravitreal injections in combination with panretinal photocoagulation and focal laser treatment remain the cornerstones of modern therapy. The many landmark studies for diabetic eye disease management should guide counselling and decision making for treating diabetic macular oedema, proliferative retinopathy and other diabetes-related eye diseases.
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http://dx.doi.org/10.1111/ceo.13894DOI Listing
March 2021
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