Genetic analysis of osteoblast activity identifies Zbtb40 as a regulator of osteoblast activity and bone mass.

Osteoporosis is a genetic disease characterized by progressive reductions in bone mineral density (BMD) leading to an increased risk of fracture. Over the last decade, genome-wide association studies (GWASs) have identified over 1000 associations for BMD. However, as a phenotype BMD is challenging as bone is a multicellular tissue affected by both local and systemic physiology. Here, we focused on a single component of BMD, osteoblast-mediated bone formation in mice, and identified associations influencing osteoblast activity on mouse Chromosomes (Chrs) 1, 4, and 17. The locus on Chr. 4 was in an intergenic region between Wnt4 and Zbtb40, homologous to a locus for BMD in humans. We tested both Wnt4 and Zbtb40 for a role in osteoblast activity and BMD. Knockdown of Zbtb40, but not Wnt4, in osteoblasts drastically reduced mineralization. Additionally, loss-of-function mouse models for both genes exhibited reduced BMD. Our results highlight that investigating the genetic basis of in vitro osteoblast mineralization can be used to identify genes impacting bone formation and BMD.

Monitoring tacrolimus toxicity following Paxlovid administration in a liver transplant patient.

Maintaining therapeutic plasma tacrolimus concentrations is essential for mitigating potential solid organ transplant rejection and preventing toxic adverse side effects. While patients can benefit greatly from tacrolimus therapy, co-administration of drugs such as Paxlovid (nirmatrelvir/ritonavir) place patients at serious risk for drug interactions and harm. Here we present a case of tacrolimus toxicity following Paxlovid administration in a liver transplant patient. Therapeutic drug monitoring was further complicated by a limited upper reportable threshold for tacrolimus testing and highlights the value of validating a higher limit to the clinical reportable range to improve tacrolimus monitoring and meet clinical needs in the setting of drug toxicity.

Analytical Performance Evaluation of Three Point-of-Care CBC Analyzers for Management of Clozapine Therapy in Ambulatory Psychiatry Clinics.

BACKGROUND: Clozapine is a first-line therapy and the only FDA-approved drug for patients with treatment-resistant schizophrenia (TRS). However, frequent measurement of absolute neutrophil count (ANC) is required to monitor for potential adverse severe neutropenia from clozapine therapy. We evaluated 3 point-of-care (POC) instruments that perform the complete blood count (CBC) with differential to assess their analytical performance and potential to meet the clinical need for clozapine therapy management. METHODS: A CBC with differential was performed on 104 residual whole blood specimens using 3 CBC analyzers (Sight OLO, PixCell HemoScreen, and Sysmex pocH-100i) to assess analytical precision, linearity, and accuracy vs the ADVIA 2120i and manual differential reference methods. Clinical concordance of ANC between POC devices and manual differential at medical decision points for mild, moderate, or severe neutropenia, and the threshold for clozapine therapy discontinuation (1.0 × 109/L) were determined. RESULTS: For CBC parameters, a CV ≤ 6.4% was observed on the OLO, CV ≤ 6.2% for the HemoScreen, and CV ≤ 5.1% with the pocH-100i. Each device accurately identified ANC with the greatest mean bias ±0.42 × 109/L using the pocH-100i vs manual differential. For results near the medical decision points (ANC <1.5 × 109/L), clinical concordance of ANC results was 55.6% for the OLO, 89.5% for the HemoScreen, and 82.4% for the pocH-100i. CONCLUSIONS: The HemoScreen device demonstrated the best clinical concordance in ANC values at medical decision thresholds for clozapine therapy management.

Ski inhibits TGF-ß/phospho-Smad3 signaling and accelerates hypertrophic differentiation in chondrocytes.

Since transforming growing factor-ß (TGF-ß)/Smad signaling inhibits chondrocyte maturation, endogenous negative regulators of TGF-ß signaling are likely also important regulators of the chondrocyte differentiation process. One such negative regulator, Ski, is an oncoprotein that is known to inhibit TGF-ß/Smad3 signaling via its interaction with phospho-Smad3 and recruitment of histone deacetylases (HDACs) to the DNA binding complex. Based on this, we hypothesized that Ski inhibits TGF-ß signaling and accelerates maturation in chondrocytes via recruitment of HDACs to transcriptional complexes containing Smads. We tested this hypothesis in chick upper sternal chondrocytes (USCs), where gain and loss of Ski expression experiments were performed. Over-expression of Ski not only reversed the inhibitory effect of TGF-ß on the expression of hypertrophic marker genes such as type X collagen (colX) and osteocalcin, it induced these genes basally as well. Conversely, knockdown of Ski by RNA interference led to a reduction of colX and osteocalcin expression under basal conditions. Furthermore, Ski blocked TGF-ß induction of cyclinD1 and caused a basal up-regulation of Runx2, consistent with the observed acceleration of hypertrophy. Regarding mechanism, not only does Ski associate with phospho-Smad2 and 3, but its association with phospho-Smad3 is required for recruitment of HDAC4 and 5. Implicating this recruitment of HDACs in the phenotypic effects of Ski in chondrocytes, the HDAC inhibitor SAHA reversed the up-regulation of colX and osteocalcin in Ski over-expressing cells. These results suggest that inhibition of TGF-ß signaling by Ski, which involves its association with phospho-Smad3 and recruitment of HDAC4 and 5, leads to accelerated chondrocyte differentiation.

Surgical Induction of Posttraumatic Osteoarthritis in the Mouse.

Given the prevalence and the scope of the personal and societal burden of osteoarthritis (OA), investigators continue to be deeply interested in understanding the pathogenic basis of disease and developing novel disease modifying OA therapies. Because joint trauma/injury is considered a leading predisposing factor in the development of OA, and since posttraumatic OA is one of the most common forms of OA in general, large animal and rodent models of knee injury that accurately recapitulate the OA disease process have become increasingly widespread over the past decade. To enable study in the context of defined genetic backgrounds, investigative teams have developed standardized protocols for injuring the mouse knee that aim to induce a reproducible degenerative process both in terms of severity and temporal pacing of disease progression. The destabilization of the medial meniscus (DMM) is one of the most commonly employed surgical procedure in rodents that reproducibly models posttraumatic OA and allows for the study of disease progression from initiation to end-stage disease. The description provided here sets the stage for both inexperienced and established investigators to employ the DMM procedure, or other similar surgical destabilization methods, to initiate the development of posttraumatic OA in the mouse. Successful application of this method provides a preclinical platform to study the mechanisms driving the pathogenesis of posttraumatic OA and for testing therapeutic strategies to treat it.

IKKß-NF-κB signaling in adult chondrocytes promotes the onset of age-related osteoarthritis in mice.

Canonical nuclear factor κB (NF-κB) signaling mediated by homo- and heterodimers of the NF-κB subunits p65 (RELA) and p50 (NFKB1) is associated with age-related pathologies and with disease progression in posttraumatic models of osteoarthritis (OA). Here, we established that NF-κB signaling in articular chondrocytes increased with age, concomitant with the onset of spontaneous OA in wild-type mice. Chondrocyte-specific expression of a constitutively active form of inhibitor of κB kinase ß (IKKß) in young adult mice accelerated the onset of the OA-like phenotype observed in aging wild-type mice, including degenerative changes in the articular cartilage, synovium, and menisci. Both in vitro and in vivo, chondrocytes expressing activated IKKß had a proinflammatory secretory phenotype characterized by markers typically associated with the senescence-associated secretory phenotype (SASP). Expression of these factors was differentially regulated by p65, which contains a transactivation domain, and p50, which does not. Whereas the loss of p65 blocked the induction of genes encoding SASP factors in chondrogenic cells treated with interleukin-1ß (IL-1ß) in vitro, the loss of p50 enhanced the IL-1ß­induced expression of some SASP factors. The loss of p50 further exacerbated cartilage degeneration in mice with chondrocyte-specific IKKß activation. Overall, our data reveal that IKKß-mediated activation of p65 can promote OA onset and that p50 may limit cartilage degeneration in settings of joint inflammation including advanced age.

Mouse Models and Online Resources for Functional Analysis of Osteoporosis Genome-Wide Association Studies.

Osteoporosis is a complex genetic disease in which the number of loci associated with the bone mineral density, a clinical risk factor for fracture, has increased at an exponential rate in the last decade. The identification of the causative variants and candidate genes underlying these loci has not been able to keep pace with the rate of locus discovery. A large number of tools and data resources have been built around the use of the mouse as model of human genetic disease. Herein, we describe resources available for functional validation of human Genome Wide Association Study (GWAS) loci using mouse models. We specifically focus on large-scale phenotyping efforts focused on bone relevant phenotypes and repositories of genotype-phenotype data that exist for transgenic and mutant mice, which can be readily mined as a first step toward more targeted efforts designed to deeply characterize the role of a gene in bone biology.

Characterization of expression and alternative splicing of the gene cadherin-like and PC esterase domain containing 1 (Cped1).

Cadherin-like and PC-esterase domain containing 1 (CPED1) is an uncharacterized gene with no known function. Human genome wide association studies (GWAS) for bone mineral density (BMD) have repeatedly identified a significant locus on Chromosome 7 that contains the gene CPED1, but it remains unclear if this gene could be causative. While an open reading frame for this gene has been predicted, there has been no systematic exploration of expression or alternate splicing for CPED1 in humans or mice.Using mouse models, we demonstrate that Cped1 is alternately spliced whereby transcripts are generated with exon 3 or exons 16 and 17 removed. In calvarial-derived pre-osteoblasts, Cped1 utilizes the predicted promoter upstream of exon 1 as well as alternate promoters upstream of exon 3 and exon 12.Lastly, we have determined that some transcripts terminate at the end of exon 10 and therefore do not contain the cadherin like and the PC esterase domains.Together, these data suggest that multiple protein products may be produced by this gene, with some products either lacking or containing both the predicted functional domains. Our data provide a framework upon which future functional studies will be built to understand the role of this gene in bone biology.

Daily oral consumption of hydrolyzed type 1 collagen is chondroprotective and anti-inflammatory in murine posttraumatic osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease for which there are no disease modifying therapies. Thus, strategies that offer chondroprotective or regenerative capability represent a critical unmet need. Recently, oral consumption of a hydrolyzed type 1 collagen (hCol1) preparation has been reported to reduce pain in human OA and support a positive influence on chondrocyte function. To evaluate the tissue and cellular basis for these effects, we examined the impact of orally administered hCol1 in a model of posttraumatic OA (PTOA). In addition to standard chow, male C57BL/6J mice were provided a daily oral dietary supplement of hCol1 and a meniscal-ligamentous injury was induced on the right knee. At various time points post-injury, hydroxyproline (hProline) assays were performed on blood samples to confirm hCol1 delivery, and joints were harvested for tissue and molecular analyses were performed, including histomorphometry, OARSI and synovial scoring, immunohistochemistry and mRNA expression studies. Confirming ingestion of the supplements, serum hProline levels were elevated in experimental mice administered hCol1. In the hCol1 supplemented mice, chondroprotective effects were observed in injured knee joints, with dose-dependent increases in cartilage area, chondrocyte number and proteoglycan matrix at 3 and 12 weeks post-injury. Preservation of cartilage and increased chondrocyte numbers correlated with reductions in MMP13 protein levels and apoptosis, respectively. Supplemented mice also displayed reduced synovial hyperplasia that paralleled a reduction in Tnf mRNA, suggesting an anti-inflammatory effect. These findings establish that in the context of murine knee PTOA, daily oral consumption of hCol1 is chondroprotective, anti-apoptotic in articular chondrocytes, and anti-inflammatory. While the underlying mechanism driving these effects is yet to be determined, these findings provide the first tissue and cellular level information explaining the already published evidence of symptom relief supported by hCol1 in human knee OA. These results suggest that oral consumption of hCol1 is disease modifying in the context of PTOA.

Surgical induction of posttraumatic osteoarthritis in the mouse.

Given the prevalence and the scope of the personal and societal burden of OA, investigators have become increasingly interested in understanding the pathogenic basis of disease and developing novel disease-modifying OA therapies. Because of the well-documented central role that joint trauma plays in the initiation of knee OA, large animal and rodent models of knee injury that accurately recapitulate the OA disease process have become increasingly widespread over the past decade. To enable study in the context of defined genetic backgrounds, investigative teams have informally developed standardized protocols for injuring the mouse knee that aim to induce a reproducible degenerative process both in terms of severity and temporal pacing of disease progression. One such procedure, the meniscal/ligamentous injury (MLI) model of posttraumatic OA, is described in detail in this chapter. The description provided here sets the stage for both inexperienced and established investigators to employ the MLI procedure, or other similar surgical destabilization methods, to initiate the development of posttraumatic OA in the mouse. Successful application of this method provides a preclinical platform to study the mechanisms driving the pathogenesis of OA and to develop chondroprotective/regenerative strategies to treat it.