Bacteria make T cell memories in utero.

Education of the human immune system begins in utero via T cell activation and memory development. However, whether part of the education is provided by exposure to microbes in utero remains controversial and unclear. In this issue of Cell, Mishra et al. provide new evidence that the fetal gut may be colonized by bacteria that prime T cell memories.

Bethlem Myopathy (Collagen VI-Related Dystrophies): A Retrospective Cohort Study on Musculoskeletal Pathologies and Clinical Course.

BACKGROUND: Collagen VI-related myopathies with pathologic COL6A1, COL6A2, and COL6A3 variants manifest as a phenotypic continuum of rare disorders, including Bethlem myopathy (BM), characterized by early onset muscle weakness, proximal joint contractures, and distal joint laxity. Herein we discuss the concomitant orthopedic manifestations of BM, potential management strategies, and patient outcomes. METHODS: An IRB-approved retrospective cohort study (n=23) from 2 pediatric institutions with a confirmed diagnosis of BM. Charts were reviewed for demographic data, age of disease presentation and diagnosis, COL6 genotype, diagnosis method, ambulation status, need for assistance, musculoskeletal abnormalities, other systemic comorbidities, advanced imaging and screening diagnostics, previous surgical interventions, and progression of the disease. RESULTS: The mean age was 11.65 years (range 3 to 19 y). Mean age at initial presentation with symptoms was 4.18 years old, whereas diagnosis was delayed until 8.22 years old on average. Muscle weakness was the most common presenting symptom (65.2%), and 73.9% of patients required some use of assistive or mobility devices. Overall, 30.4% of patients were diagnosed with scoliosis; 57.1% required operative intervention for their scoliosis; 43.5% of patients had acetabular dysplasia; 10% required open reduction of a dislocated hip; 10% required closed reduction with hip spica application; 10% required bilateral periacetabular osteotomies for instability; 91.3% of patients developed foot and ankle deformities; 33.3% of patients underwent posteromedial-lateral equinovarus releases; 28.6% required an Achilles tendon lengthening, and 86.9% of patients had muscle tendon contractures, the most common locations being the ankle (55%) and elbow (40%). CONCLUSION: Although often less severe than other more common neuropathies and myopathies like Charcot-Marie-Tooth disease and Duchenne muscular dystrophy, BM does lead to progressive musculoskeletal deformity and disability. Its relative rarity makes it less familiar to providers and likely contributes to delays in diagnosis. Scoliosis, hip dysplasia, and equinus and varus ankle deformities are the most common musculoskeletal deformities. Physicians and surgeons should appropriately counsel patients and families about the clinical course of this disorder and the potential need for mobility assistance or surgical procedures. LEVEL OF EVIDENCE: III, Prognostic. study.

Radiographic outcomes of cortical screw fixation as an alternative to Kirschner wire fixation for temporary lateral column stabilization in displaced Lisfranc joint fracture-dislocations: a retrospective cohort analysis.

BACKGROUND: Injuries of the tarsometatarsal joint complex ranging from purely ligamentous to multidirectionally unstable midfoot fracture-dislocations are anatomically fixed to minimize long-term sequelae including post-traumatic arthritis, pes planus deformity, and chronic pain. Lateral column disruption is commonly treated with temporary Kirschner wire (K-wire) fixation, maintaining alignment during healing and allowing resumption of physiologic motion after hardware removal. More unstable fracture patterns may require temporary cortical screw fixation to maintain adequate reduction. We evaluated the efficacy of temporary lateral column screw fixation compared to K-wire fixation for Lisfranc fracture-dislocation treatment. METHODS: This retrospective cohort study reviewed 45 patients over fourteen years who underwent Lisfranc fracture-dislocation fixation at a level-one trauma center. All patients underwent medial and middle column fixation; 31 underwent lateral column fixation. Twenty six patients remained after excluding those without electronic records or follow-up. The primary outcome was radiographic lateral column healing before and after hardware removal; secondary outcomes included pain, ambulation, and return to normal shoe wear. RESULTS: Twenty patients were male, with mean age 41 years. Thirteen patients underwent cortical screw fixation and twelve K-wire fixation. One had both implants. Twenty four patients underwent lateral column hardware removal; all had radiographic evidence of bony healing before hardware removal. Mean follow-up was 88.2 ± 114 weeks for all patients. The cortical screw cohort had significantly longer mean time to hardware removal (p = 0.002). The K-wire cohort had significantly more disuse osteopenia (p = 0.045) and postoperative pain (p = 0.019). CONCLUSIONS: Radiographic and clinical outcomes of unstable Lisfranc fracture-dislocation treatment support temporary lateral column screw fixation as an alternate technique. LEVEL OF CLINICAL EVIDENCE: 3 (retrospective cohort study).

Tenacious D: Symbiodinium in clade D remain in reef corals at both high and low temperature extremes despite impairment.

Reef corals are sensitive to thermal stress, which induces coral bleaching (the loss of algal symbionts), often leading to coral mortality. However, corals hosting certain symbionts (notably some members of Symbiodinium clade D) resist bleaching when exposed to high temperatures. To determine whether these symbionts are also cold tolerant, we exposed corals hosting either Symbiodinium C3 or D1a to incremental warming (+1°C week-1 to 35°C) and cooling (-1°C week-1 to 15°C), and measured photodamage and symbiont loss. During warming to 33°C, C3 corals were photodamaged and lost >99% of symbionts, while D1a corals experienced photodamage but did not bleach. During cooling, D1a corals suffered more photodamage than C3 corals but still did not bleach, while C3 corals lost 94% of symbionts. These results indicate that photodamage does not always lead to bleaching, suggesting alternate mechanisms exist by which symbionts resist bleaching, and helping explain the persistence of D1a symbionts on recently bleached reefs, with implications for the future of these ecosystems.

Change in algal symbiont communities after bleaching, not prior heat exposure, increases heat tolerance of reef corals.

Mutualistic organisms can be particularly susceptible to climate change stress, as their survivorship is often limited by the most vulnerable partner. However, symbiotic plasticity can also help organisms in changing environments by expanding their realized niche space. Coral-algal (Symbiodinium spp.) symbiosis exemplifies this dichotomy: the partnership is highly susceptible to 'bleaching' (stress-induced symbiosis breakdown), but stress-tolerant symbionts can also sometimes mitigate bleaching. Here, we investigate the role of diverse and mutable symbiotic partnerships in increasing corals' ability to thrive in high temperature conditions. We conducted repeat bleaching and recovery experiments on the coral Montastraea cavernosa, and used quantitative PCR and chlorophyll fluorometry to assess the structure and function of Symbiodinium communities within coral hosts. During an initial heat exposure (32 °C for 10 days), corals hosting only stress-sensitive symbionts (Symbiodinium C3) bleached, but recovered (at either 24 °C or 29 °C) with predominantly (>90%) stress-tolerant symbionts (Symbiodinium D1a), which were not detected before bleaching (either due to absence or extreme low abundance). When a second heat stress (also 32 °C for 10 days) was applied 3 months later, corals that previously bleached and were now dominated by D1a Symbiodinium experienced less photodamage and symbiont loss compared to control corals that had not been previously bleached, and were therefore still dominated by Symbiodinium C3. Additional corals that were initially bleached without heat by a herbicide (DCMU, at 24 °C) also recovered predominantly with D1a symbionts, and similarly lost fewer symbionts during subsequent thermal stress. Increased thermotolerance was also not observed in C3-dominated corals that were acclimated for 3 months to warmer temperatures (29 °C) before heat stress. These findings indicate that increased thermotolerance post-bleaching resulted from symbiont community composition changes, not prior heat exposure. Moreover, initially undetectable D1a symbionts became dominant only after bleaching, and were critical to corals' resilience after stress and resistance to future stress.

Pediatric distal third tibial shaft fractures: a comparison of surgical fixation methods and incidence of concomitant physeal fractures.

The purposeof this study was to compare outcomes of operatively treated pediatric distal third tibial shaft fractures fixed with elastic nailing or plate fixation and to evaluate the incidence of concurrent distal tibia physeal fractures. Retrospective review identified skeletally immature patients that underwent operative fixation of distal third tibia fractures at a level 1 children's hospital from 2010 to 2020. Patient and fracture characteristics were recorded. Analysis of treatment outcomes was performed and rates of concurrent distal tibia physeal fractures were evaluated. Of the 214 surgically treated tibial shaft fractures, 43 were distal third fractures. A concurrent distal tibia physeal fracture was present in 32.6% of patients. These were significantly associated with spiral distal third tibial shaft fractures. The presence of concurrent physeal fractures did not affect patient treatment outcomes. Comparing elastic nailing versus open reduction and plating revealed no difference with time to fracture union, time of postoperative immobilization, or time to full weight-bearing. While elastic nailing was associated with increased coronal angulation, translation, and shortening of fractures on initial postoperative imaging, there was no difference in rates of malunion at final follow-up. In our series, there were no differences in treatment outcomes based on fixation method. Our operatively treated distal third tibial shaft fractures had a higher rate of associated distal tibial physeal fractures than previously published in the pediatric orthopedic literature. We recommend careful evaluation of the ankle for concurrent physeal injuries in patients with distal third tibial shaft fractures indicated for operative treatment. Level of evidence: level III therapeutic study - retrospective comparative study.

Precise DNA cleavage using CRISPR-SpRYgests.

Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR-Cas9 or CRISPR-Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks.

PAM-Flexible Genome Editing with an Engineered Chimeric Cas9.

CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN PAM preference, with the N-terminus of Sc++, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse NNN PAMs and disease-related loci for potential therapeutic applications. In total, the unique approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.

Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR-Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.

PAM-flexible genome editing with an engineered chimeric Cas9.

CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.

Relationship Between Age and Pathology With Treatment of Pediatric and Adolescent Discoid Lateral Meniscus: A Report From the SCORE Multicenter Database.

BACKGROUND: Surgical treatment options of discoid lateral meniscus in pediatric patients consist of saucerization with or without meniscal repair, meniscocapular stabilization, and, less often, subtotal meniscectomy. PURPOSE: To describe a large, prospectively collected multicenter cohort of discoid menisci undergoing surgical intervention, and further investigate corresponding treatment of discoid menisci. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A multicenter quality improvement registry (16 institutions, 26 surgeons), Sports Cohort Outcomes Registry, was queried. Patient characteristics, discoid type, presence and type of intrasubstance meniscal tear, peripheral rim instability, repair technique, and partial meniscectomy/debridement beyond saucerization were reviewed. Discoid meniscus characteristics were compared between age groups (<14 and >14 years old), based on receiver operating characteristic curve, and discoid morphology (complete and incomplete). RESULTS: In total, 274 patients were identified (mean age, 12.4 years; range, 3-18 years), of whom 55.6% had complete discoid. Meniscal repairs were performed in 55.1% of patients. Overall, 48.5% of patients had rim instability and 36.8% had >1 location of peripheral rim instability. Of the patients, 21.5% underwent meniscal debridement beyond saucerization, with 8.4% undergoing a subtotal meniscectomy. Patients <14 years of age were more likely to have a complete discoid meniscus (P < .001), peripheral rim instability (P = .005), and longitudinal tears (P = .015) and require a meniscal repair (P < .001). Patients ≥14 years of age were more likely to have a radial/oblique tear (P = .015) and require additional debridement beyond the physiologic rim (P = .003). Overall, 70% of patients <14 years of age were found to have a complete discoid meniscus necessitating saucerization, and >50% in this young age group required peripheral stabilization/repair. CONCLUSION: To preserve physiological "normal" meniscus, a repair may be indicated in >50% of patients <14 years of age but occurred in <50% of those >14 years. Additional resection beyond the physiological rim may be needed in 15% of younger patients and 30% of those aged >14 years.

Specificity is rarely absolute in coral-algal symbiosis: implications for coral response to climate change.

Some reef-building corals have been shown to respond to environmental change by shifting the composition of their algal symbiont (genus Symbiodinium) communities. These shifts have been proposed as a potential mechanism by which corals might survive climate stressors, such as increased temperatures. Conventional molecular methods suggest this adaptive capacity may not be widespread because few (∼25%) coral species have been found to associate with multiple Symbiodinium clades. However, these methods can fail to detect low abundance symbionts (typically less than 10-20% of the total algal symbiont community). To determine whether additional Symbiodinium clades are present, but are not detected using conventional techniques, we applied a high-resolution, real-time PCR assay to survey Symbiodinium (in clades A-D) from 39 species of phylogenetically and geographically diverse scleractinian corals. This survey included 26 coral species thought to be restricted to hosting a single Symbiodinium clade ('symbiotic specialists'). We detected at least two Symbiodinium clades (C and D) in at least one sample of all 39 coral species tested; all four Symbiodinium clades were detected in over half (54%) of the 26 symbiotic specialist coral species. Furthermore, on average, 68 per cent of all sampled colonies within a given coral species hosted two or more symbiont clades. We conclude that the ability to associate with multiple symbiont clades is common in scleractinian (stony) corals, and that, in coral-algal symbiosis, 'specificity' and 'flexibility' are relative terms: specificity is rarely absolute. The potential for reef corals to adapt or acclimatize to environmental change via symbiont community shifts may therefore be more phylogenetically widespread than has previously been assumed.

Constraints on the use of lifespan-shortening Wolbachia to control dengue fever.

Dengue fever, a viral disease spread by the mosquito Aedes aegypti, affects 50-100 million people a year in many tropical countries. Because the virus must incubate within mosquito hosts for two weeks before being able to transmit the infection, shortening the lifespan of mosquitoes may curtail dengue transmission. We developed a continuous time reaction-diffusion model of the spatial spread of Wolbachia through a population of A. aegypti. This model incorporates the lifespan-shortening effects of Wolbachia on infected A. aegypti and the fitness advantage to infected females due to cytoplasmic incompatibility (CI). We found that local establishment of the Wolbachia infection can occur if the fitness advantage due to CI exceeds the fitness reduction due to lifespan-shortening effects, in accordance with earlier results concerning fecundity reduction. However, spatial spread is possible only if the fitness advantage due to CI is twice as great as the fitness reduction due to lifespan shortening effects. Moreover, lifespan-shortening and fecundity-reduction can have different effects on the speed of wave-retreat. Using data from the literature, we estimated all demographic parameters for infected and uninfected mosquitoes and computed the velocities of spread of infection. Our most optimistic estimates suggest that the spatial spread of lifespan-shortening Wolbachia may be so slow that efficient spatial spread would require a prohibitively large number of point releases. However, as these estimates of demographic parameters may not accurately reflect natural conditions, further research is necessary to corroborate these predictions.

Arthroscopic Repair of Radial Tears in the Junction of the Anterior Horn and Body of the Lateral Meniscus Using an All-Inside Device.

Surgical treatment of radial tears in the junction of the anterior horn and body of the lateral meniscus is indicated in the acute setting and with failure of nonoperative therapy. There are several options for arthroscopic repair; however, these are more technically difficult to perform because of the current surgical instruments available to address these tears. An outside-in technique is the current standard of care for this tear location and pattern. We present a technique for an all-inside side-to-side repair of the lateral meniscus for radial tears in the junction of the anterior horn and body.

Range of motion following percutaneous fixation of pediatric supracondylar humerus fracture is independent of anterior osseous fragment resorption.

The objective of this study was to understand postoperative resorption of the anterior osseous fragment following closed reduction and percutaneous pinning (CRPP) of pediatric supracondylar humerus fractures and its effect on final range of motion (ROM). Eighty-six patients that underwent CRPP had sagittal and or axial plane deformities resulting in an anterior fragment. Humerocapitellar angle (HCA), anterior humeral line (AHL) and angle of rotation (AoR) were measured. A total of 11 (12.8%) patients failed to resorb the anterior fragment, 10 (90.9%) had satisfactory ROM. HCA initially was acceptable in 40 (46.5%) patients, and 37 (92.5%) demonstrated acceptable ROM. Final HCA was acceptable in 44 (51.2%) patients and 42 (95.4%) had acceptable final ROM. AHL was in the anterior third of the capitellum in 35 (40.6%) patients and 33 (94.3%) had acceptable ROM. Final AHL was in the anterior third of the capitellum in 43 (50.0%) patients and 41 (95.3%) had acceptable final ROM. No difference was found between acceptable ROM and HCA or AHL at either follow-up. Sixty-five and 21 patients had an AoR of 0° and between 23 and 36°, respectively. A total of 59 (90.7%) patients with an AoR of 0°, and 18 (85.7%) patients with an AoR of 23-36° displayed acceptable ROM. A total of 57 (87.7%) patients with an AoR of 0° and 18 (85.7%) with an AoR of 23-36° resorbed the anterior fragment. No association was found between rotational deformity and postoperative ROM or fragment resorption. Postoperative sagittal and axial plane alignment, HCA, AHL, AoR and resorption of the anterior osseous fragment does not correlate with final ROM.

A potent tumor-selective ERK pathway inactivator with high therapeutic index.

FDA-approved BRAF and MEK small molecule inhibitors have demonstrated some level of efficacy in patients with metastatic melanomas. However, these "targeted" therapeutics have a very low therapeutic index, since these agents affect normal cells, causing undesirable, even fatal, side effects. To address these significant drawbacks, here, we have reengineered the anthrax toxin-based protein delivery system to develop a potent, tumor-selective MEK inactivator. This toxin-based MEK inactivator exhibits potent activity against a wide range of solid tumors, with the highest activity seen when directed toward tumors containing the BRAFV600E mutation. We demonstrate that this reengineered MEK inactivator also exhibits an extremely high therapeutic index (>15), due to its in vitro and in vivo activity being strictly dependent on the expression of multiple tumor-associated factors including tumor-associated proteases matrix metalloproteinase, urokinase plasminogen activator, and anthrax toxin receptor capillary morphogenesis protein-2. Furthermore, we have improved the specificity of this MEK inactivator, restricting its enzymatic activity to only target the ERK pathway, thereby greatly diminishing off-target toxicity. Together, these data suggest that engineered bacterial toxins can be modified to have significant in vitro and in vivo therapeutic effects with high therapeutic index.

Group therapy on in utero colonization: seeking common truths and a way forward.

The human microbiome refers to the genetic composition of microorganisms in a particular location in the human body. Emerging evidence over the past many years suggests that the microbiome constitute drivers of human fate almost at par with our genome and epigenome. It is now well accepted after decades of disbelief that a broad understanding of human development, health, physiology, and disease requires understanding of the microbiome along with the genome and epigenome. We are learning daily of the interdependent relationships between microbiome/microbiota and immune responses, mood, cancer progression, response to therapies, aging, obesity, antibiotic usage, and overusage and much more. The next frontier in microbiome field is understanding when does this influence begin? Does the human microbiome initiate at the time of birth or are developing human fetuses already primed with microbes and their products in utero. In this commentary, we reflect on evidence gathered thus far on this question and identify the unknown common truths. We present a way forward to continue understanding our microbial colleagues and our interwoven fates.

Distal Radius Fractures in Patients Aged 50 Years or Older: Obstacles to Bone Health Analysis and Follow-Up in a Community Setting.

Purpose: To determine barriers to implementing an osteoporosis protocol in a community institution following distal radius (DR) fragility fracture to help prevent subsequent fragility fractures. Methods: This cross-sectional study included elderly patients with DR fractures that occurred between 2016 and 2018. Exclusion criteria were age under 50 years, high-energy mechanism, and inability to follow-up locally. Patients were directed to follow-up with the hospital's osteoporosis center (OC) or an endocrinologist. Patients were contacted to identify if care was established with the OC and screened for potential barriers in evaluation for bone health. Primary outcomes included the completion of a follow-up visit with an osteoporosis care provider and identification of barriers for patients who did not complete this visit. Secondary outcomes included whether or not patients obtained bone health labs, dual-energy x-ray absorptiometry (DEXA) scans, and/or underwent medical treatment for osteoporosis. Results: One hundred seventy-five patients met final inclusion criteria and were contacted after discharge. Fifty patients agreed to follow-up with the OC, voicemails were left for 66 patients, only 70 (60.3%) patients actually followed up for bone health analysis. Patients were lost to follow-up due to lack of accessibility (32 patients; death, incorrect phone number, no voicemail, or impaired cognition), and lack of interest (27 patients). Ninety-six (54.9%) patients received appropriate treatment based on bone health labs and/or DEXA scan. Ninety (51.4%) patients had chemical treatment for osteoporosis. Fifty-five patients underwent DEXA scans with equal distribution of patients with normal, osteopenic, and osteoporotic bone. Forty-three (78%) patients who had DEXA scans underwent treatment. Conclusions: Establishing a protocol for follow-up for bone health assessment following a DR fracture is challenging. Only half of the patients underwent evaluation and management of their bone health. It is imperative to understand the barriers for at-risk patients to provide them with care that will improve their quality of life. Type of study/level of evidence: Diagnostic III.

SARS-CoV-2 colonization of maternal and fetal cells of the human placenta promotes alteration of local renin-angiotensin system.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection appears to increase the risk of adverse pregnancy outcomes, such as pre-eclampsia in pregnant women. The mechanism(s) by which this occurs remains unclear. METHODS: We investigated the pathophysiology of SARS-CoV-2 at maternal-fetal interface in pregnant women who tested positive for the virus using RNA in situ hybridization (viral RNA), immunohistochemistry, and hematoxylin and eosin staining. To investigate whether viral infection alters the renin angiotensin system (RAS) in placenta, which controls blood pressure, we treated human trophoblasts with recombinant spike protein or a live modified virus with a vesicular stomatitis viral backbone expressing spike protein (VSV-S). FINDINGS: Viral colonization was highest in maternal decidua, fetal trophoblasts, Hofbauer cells, and in placentas delivered prematurely. We localized SARS-CoV-2 to cells expressing angiotensin-converting enzyme 2 (ACE2) and demonstrate that infected placentas had significantly reduced ACE2. In response to both spike protein and VSV-S, cellular ACE2 decreased although angiotensin II receptor type 1 (AT1R) increased with concomitant increase in soluble fms-like tyrosine kinase-1 (sFlt1). Viral infection decreased pro-angiogenic factors, AT2R, and placental growth factor, which competitively binds to sFlt1. Sera from infected pregnant women had elevated levels of sFlt1 and angiotensin II type 1-receptor autoantibodies prior to delivery, both signatory markers of pre-eclampsia. CONCLUSIONS: SARS-CoV-2 colonizes ACE2-expressing maternal and fetal cells in the placenta. Infection in pregnant women correlates with alteration of placental RAS. As RAS regulates blood pressure, SARS-CoV-2 infection may thus increase adverse hemodynamic outcomes, such as pre-eclampsia in pregnant women. FUNDING: NIH/NICHD grants R01 HD091218 and 3R01HD091218-04S1 (RADx-UP Supplement).

Viral-Immune Cell Interactions at the Maternal-Fetal Interface in Human Pregnancy.

The human decidua and placenta form a distinct environment distinguished for its promotion of immunotolerance to infiltrating semiallogeneic trophoblast cells to enable successful pregnancy. The maternal-fetal interface also successfully precludes transmission of most pathogens. This barrier function occurs in conjunction with a diverse influx of decidual immune cells including natural killer cells, macrophages and T cells. However, several viruses, among other microorganisms, manage to escape destruction by the host adaptive and innate immune system, leading to congenital infection and adverse pregnancy outcomes. In this review, we describe mechanisms of pathogenicity of two such viral pathogens, Human cytomegalovirus (HCMV) and Zika virus (ZIKV) at the maternal-fetal interface. Host decidual immune cell responses to these specific pathogens will be considered, along with their interactions with other cell types and the ways in which these immune cells may both facilitate and limit infection at different stages of pregnancy. Neither HCMV nor ZIKV naturally infect commonly used animal models [e.g., mice] which makes it challenging to understand disease pathogenesis. Here, we will highlight new approaches using placenta-on-a-chip and organoids models that are providing functional and physiologically relevant ways to study viral-host interaction at the maternal-fetal interface.