Risk of myopericarditis following COVID-19 mRNA vaccination in a large integrated health system: A comparison of completeness and timeliness of two methods.

PURPOSE: How completely do hospital discharge diagnoses identify cases of myopericarditis after an mRNA vaccine? METHODS: We assembled a cohort 12-39 year-old patients, insured by Kaiser Permanente Northwest, who received at least one dose of an mRNA vaccine (Pfizer-BioNTech or Moderna) between December 2020 and October 2021. We followed them for up to 30 days after their second dose of an mRNA vaccine to identify encounters for myocarditis, pericarditis or myopericarditis. We compared two identification methods: A method that searched all encounter diagnoses using a brief text description (e.g., ICD-10-CM code I40.9 is defined as 'acute myocarditis, unspecified'). We searched the text description of all inpatient or outpatient encounter diagnoses (in any position) for "myocarditis" or "pericarditis." The other method was developed by the Centers for Disease Control and Prevention's Vaccine Safety Datalink (VSD), which searched for emergency department visits or hospitalizations with a select set of discharge ICD-10-CM diagnosis codes. For both methods, two physicians independently reviewed the identified patient records and classified them as confirmed, probable or not cases using the CDC's case definition. RESULTS: The encounter methodology identified 14 distinct patients who met the confirmed or probable CDC case definition for acute myocarditis or pericarditis with an onset within 21 days of receipt of COVID-19 vaccination. When we extended the search for relevant diagnoses to 30 days since vaccination, we identified two additional patients (for a total of 16 patients) who met the case definition for acute myocarditis or pericarditis, but those patients had been misdiagnosed at the time of their original presentation. Three of these patients had an ICD-10-CM code of I51.4 "Myocarditis, Unspecified;" that code was omitted by the VSD algorithm (in the late fall of 2021). The VSD methodology identified 11 patients who met the CDC case definition for acute myocarditis or pericarditis. Seven (64%) of the 11 patients had initial care for myopericarditis outside of a KPNW facility and their diagnosis could not be ascertained by the VSD methodology until claims were submitted (median delay of 33 days; range of 12-195 days). Among those who received a second dose of vaccine (n = 146 785), we estimated a risk as 95.4 cases of myopericarditis per million second doses administered (95% CI, 52.1-160.0). CONCLUSION: We identified additional valid cases of myopericarditis following an mRNA vaccination that would be missed by the VSD's search algorithm, which depends on select hospital discharge diagnosis codes. The true incidence of myopericarditis is markedly higher than the incidence reported to US advisory committees in the fall of 2021. The VSD should validate its search algorithm to improve its sensitivity for myopericarditis.

Clinical management of septic arthritis.

Septic arthritis is a rheumatologic emergency as joint destruction occurs rapidly and can lead to significant morbidity and mortality. Accurate diagnosis can be particularly challenging in patients with underlying inflammatory joint disease. This review outlines the risk factors for septic arthritis and summarizes the causative bacterial organisms. We highlight advances in antibiotic management with a focus on new drugs for methicillin-resistant Staphylococcus aureus (MRSA) and discuss the use of adjunctive therapies for treatment of septic arthritis in adults.

Cardiac events following JYNNEOS vaccination for prevention of Mpox.

The incidence of cardiac adverse events following JYNNEOS vaccination for prevention of mpox is unknown, however the Advisory Committee on Immunization Practices states that people with underlying cardiac risk factors should be counseled about the theoretical risk for myopericarditis following vaccination. We conducted a retrospective cohort study of 2,126 patients who were vaccinated with at least 1 dose of JYNNEOS vaccine and searched the Kaiser Permanente Northwest databases, including the electronic health record, to evaluate for cardiac adverse events of special interest (AESI). After physician adjudication, there were 10 confirmed cardiac AESI for an incidence of 3.1 per 1000 doses (exact 95% CI, 1.5 to 5.7), however none of these events could be directly attributed to vaccination. This retrospective cohort study of JYNNEOS vaccination for prevention of mpox identified 10 cardiac events that all had alternative explanations; and no hospitalizations or serious adverse outcomes were attributed to vaccination.

Genotypic resistance testing creates new treatment challenges: two cases of oxacillin-susceptible methicillin-resistant Staphylococcus aureus.

Oxacillin-susceptible, mecA-positive Staphylococcus aureus isolates create a treatment challenge for the clinician. In this article, we describe two cases of bacteremia from isolates that carried the mecA gene but were susceptible to oxacillin (oxacillin-susceptible methicillin-resistant S. aureus [OS-MRSA]). DNA microarray analysis was used to characterize these isolates as a mecA-positive, clonal complex 5, pediatric strain and a mecA-positive, clonal complex 8, USA300 strain.

BMP-9-induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/beta-catenin signalling.

Bone morphogenetic protein 9 (BMP-9) is a member of the transforming growth factor (TGF)-beta/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/beta-catenin signalling plays an important role in BMP-9-induced osteogenic differentiation of MSCs. Wnt3A and BMP-9 enhanced each other's ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP-9-induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR-5 or low-density lipoprotein receptor-related protein (LRP)-6 exerted no inhibitory effect on BMP-9-induced ALP activity. beta-Catenin knockdown in MSCs and MEFs diminished BMP-9-induced ALP activity, and led to a decrease in BMP-9-induced osteocalcin reporter activity and BMP-9-induced expression of late osteogenic markers. Furthermore, beta-catenin knockdown or FrzB overexpression inhibited BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP-9 induced recruitment of both Runx2 and beta-catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between beta-catenin and Runx2, plays an important role in BMP-9-induced osteogenic differentiation of MSCs.

Osteogenic BMPs promote tumor growth of human osteosarcomas that harbor differentiation defects.

Osteosarcoma (OS) is the most common primary malignancy of bone. Here, we investigated a possible role of defective osteoblast differentiation in OS tumorigenesis. We found that basal levels of the early osteogenic marker alkaline phosphatase (ALP) activity were low in OS lines. Osteogenic regulators Runx2 and OSX, and the late marker osteopontin (OPN) expressed at low levels in most OS lines, indicating that most OS cells fail to undergo terminal differentiation. Furthermore, OS cells were refractory to osteogenic BMP-induced increases in ALP activity. Osteogenic BMPs were shown to upregulate early target genes, but not late osteogenic markers OPN and osteocalcin (OC). Furthermore, osteogenic BMPs failed to induce bone formation from human OS cells, rather effectively promoted OS tumor growth in an orthotopic OS model. Exogenous expression of early target genes enhanced BMP-stimulated OS tumor growth, whereas osteogenic BMP-promoted OS tumor growth was inhibited by exogenous Runx2 expression. These results suggest that alterations in osteoprogenitors may disrupt osteogenic differentiation pathway. Thus, identifying potential differentiation defects in OS tumors would allow us to reconstruct the tumorigenic events in osteoprogenitors and to develop rational differentiation therapies for clinical OS management.

Regulation of osteogenic differentiation during skeletal development.

Bone formation during skeletal development involves a complex coordination among multiple cell types and tissues. Bone is of crucial importance for the human body, providing skeletal support, and serving as a home for the formation of hematopoietic cells and as a reservoir for calcium and phosphate. Bone is also continuously remodeled in vertebrates throughout life. Osteoblasts and osteoclasts are specialized cells responsible for bone formation and resorption, respectively. Early development of the vertebrate skeleton depends on genes that control the distribution and proliferation of cells from cranial neural crest, sclerotomes, and lateral plate mesoderm into mesenchymal condensations, where cells differentiate to osteoblasts. Significant progress has been made over the past decade in our understanding of the molecular framework that controls osteogenic differentiation. A large number of morphogens, signaling molecules, and transcriptional regulators have been implicated in regulating bone development. A partial list of these factors includes the Wnt/beta-catenin, TGF-beta/BMP, FGF, Notch and Hedgehog signaling pathways, and Runx2, Osterix, ATF4, TAZ, and NFATc1 transcriptional factors. A better understanding of molecular mechanisms behind osteogenic differentiation would not only help us to identify pathogenic causes of bone and skeletal diseases but also lead to the development of targeted therapies for these diseases.

S100A6 expression and function in human osteosarcoma.

There is a critical need to identify markers that can accurately identify existing or predict future metastatic disease in patients with osteosarcoma since the majority of patients present with undetectable micrometastatic disease. We previously reported S100A6 is overexpressed in human osteosarcoma and increased expression of S100A6 by immunohistochemistry correlated with decreased clinical metastasis. We have established 11 primary cultures from biopsies of patients with osteosarcoma and ten of the 11 primary cultures have increased expression of S100A6 relative to normal human osteoblasts. To further explore possible mechanisms for metastasis suppression previously reported, we used in this report siRNA-mediated knockdown of S100A6 in four commonly used human osteosarcoma lines, then examined their cell adhesion, migration, and invasion properties. Knockdown of S100A6 expression inhibited cell adhesion and promoted cell migration and invasion in these lines. Conversely, S100A6 overexpression enhanced cell adhesion and inhibited cell invasion. Our data demonstrate S100A6 is commonly overexpressed in human osteosarcoma. S100A6 may inhibit osteosarcoma metastasis by promoting cell adhesion and inhibiting cell motility and invasion. Thus, S100A6 may be considered a potential marker for human osteosarcoma with prognostic value for identifying patients without metastases.

Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing.

RNA interference (RNAi)-mediated gene silencing has become a valuable tool for functional studies, reverse genomics, and drug discoveries. One major challenge of using RNAi is to identify the most effective short interfering RNAs (siRNAs) sites of a given gene. Although several published bioinformatic prediction models have proven useful, the process to select and validate optimal siRNA sites for a given gene remains empirical and laborious. Here, we developed a fluorescence-based selection system using a retroviral vector backbone, namely pSOS, which was based on the premise that candidate siRNAs would knockdown the chimeric transcript between GFP and target gene. The expression of siRNA was driven by the opposing convergent H1 and U6 promoters. This configuration simplifies the cloning of duplex siRNA oligonucleotide cassettes. We demonstrated that GFP signal reduction was closely correlated with siRNA knockdown efficiency of human beta-catenin, as well as with the inhibition of beta-catenin/Tcf4 signaling activity. The pSOS should not only facilitate the selection and validation of candidate siRNA sites, but also provide efficient delivery tools of siRNAs via viral vectors in mammalian cells. Thus, the pSOS system represents an efficient and user-friendly strategy to select and validate siRNA target sites.

Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells.

Efficacious bone regeneration could revolutionize the clinical management of many bone and musculoskeletal disorders. Bone morphogenetic proteins (BMPs) can regulate the differentiation of mesenchymal stem cells into cartilage, bone, tendon/ligament, and fat lineages. Early data documented the osteogenic potential of rhBMP2 and rhBMP7/OP-1. However, prior to this work that summarized several of our recent studies, no comprehensive analysis had been undertaken to characterize relative osteogenic activity of all BMPs. Using recombinant adenoviruses expressing 14 BMPs, we have demonstrated that, besides BMP2 and BMP7, BMP6 and BMP9 exhibit the highest osteogenic activity both in vitro and in vivo. We further demonstrated that several BMPs may exert synergistic effect on osteogenic differentiation, and that osteogenic BMPs produce a distinct set of molecular fingerprints during osteogenic differentiation. The reported work should expand our current understanding of BMP functions during osteogenic differentiation. It is conceivable that osteogenic BMPs (i.e., BMP2, 4, 6, 7, and 9) may be used to formulate synergistic pairs among themselves and/or with other less osteogenic BMPs for efficacious bone regeneration in clinical settings.

Hey1 basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells.

Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We previously demonstrated that bone morphogenetic protein (BMP) 9 is one of the most potent and yet least characterized BMPs that are able to induce osteogenic differentiation of MSCs both in vitro and in vivo. Here, we conducted gene expression-profiling analysis and identified that Hey1 of the hairy/Enhancer of split-related repressor protein basic helix-loop-helix family was among the most significantly up-regulated early targets in BMP9-stimulated MSCs. We demonstrated that Hey1 expression was up-regulated at the immediate early stage of BMP9-induced osteogenic differentiation. Chromatin immunoprecipitation analysis indicated that Hey1 may be a direct target of the BMP9-induced Smad signaling pathway. Silencing Hey1 expression diminished BMP9-induced osteogenic differentiation both in vitro and in vivo and led to chondrogenic differentiation. Likewise, constitutive Hey1 expression augmented BMP9-mediated bone matrix mineralization. Hey1 and Runx2 were shown to act synergistically in BMP9-induced osteogenic differentiation, and Runx2 expression significantly decreased in the absence of Hey1, suggesting that Runx2 may function downstream of Hey1. Accordingly, the defective osteogenic differentiation caused by Hey1 knockdown was rescued by exogenous Runx2 expression. Thus, our findings suggest that Hey1, through its interplay with Runx2, may play an important role in regulating BMP9-induced osteoblast lineage differentiation of MSCs.

Wnt signaling and human diseases: what are the therapeutic implications?

Wnt signaling plays an important role in regulating cell proliferation and differentiation. De-regulation of these signaling pathways has been implicated in many human diseases, ranging from cancers to skeletal disorders. Wnt proteins are a large family of secreted factors that bind to the Frizzled receptors and LRP5/6 co-receptors and initiate complex signaling cascades. Over the past two decades, our understanding of Wnt signaling has been significantly improved due to the identification of many key regulators and mediators of these pathways. Given that Wnt signaling is tightly regulated at multiple cellular levels, these pathways themselves offer ample nodal points for targeted therapeutics. Here, we focus on our current understanding of these pathways, the associations of Wnt signaling with human disorders, and the opportunities to target key components of Wnt signaling for rational drug discovery.

A protocol for rapid generation of recombinant adenoviruses using the AdEasy system.

Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We have developed an approach that simplifies the generation and production of such viruses called the AdEasy system. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, employing homologous recombination in bacteria rather than in eukaryotic cells. After transfection of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of GFP encoded by a gene incorporated into the viral backbone. This system has expedited the process of generating and testing recombinant adenoviruses for a variety of purposes. In this protocol, we describe the practical aspects of using the AdEasy system for generating recombinant adenoviruses. The full protocol usually takes 4-5 weeks to complete.