Oncogenic plasmid DNA and liver injury agent dictates liver cancer development in a mouse model.

Primary liver cancer is an increasing problem worldwide and is associated with significant mortality. A popular method of modeling liver cancer in mice is plasmid hydrodynamic tail vein injection (HTVI). However, plasmid-HTVI models rarely recapitulate the chronic liver injury which precedes the development of most human liver cancer. We sought to investigate how liver injury using thioacetamide contributes to the pathogenesis and progression of liver cancer in two oncogenic plasmid-HTVI-induced mouse liver cancer models. Fourteen-week-old male mice received double-oncogene plasmid-HTVI (SB/AKT/c-Met and SB/AKT/NRas) and then twice-weekly intraperitoneal injections of thioacetamide for 6 weeks. Liver tissue was examined for histopathological changes, including fibrosis and steatosis. Further characterization of fibrosis and inflammation was performed with immunostaining and real-time quantitative PCR. RNA sequencing with pathway analysis was used to explore novel pathways altered in the cancer models. Hepatocellular and cholangiocellular tumors were observed in mice injected with double-oncogene plasmid-HTVI models (SB/AKT/c-Met and SB/AKT/NRas). Thioacetamide induced mild fibrosis and increased alpha smooth muscle actin-expressing cells. However, the combination of plasmids and thioacetamide did not significantly increase tumor size, but increased multiplicity of small neoplastic lesions. Cancer and/or liver injury up-regulated profibrotic and proinflammatory genes while metabolic pathway genes were mostly down-regulated. We conclude that the liver injury microenvironment can interact with liver cancer and alter its presentation. However, the effects on cancer development vary depending on the genetic drivers with differing active oncogenic pathways. Therefore, the choice of plasmid-HTVI model and injury agent may influence the extent to which injury promotes liver cancer development.

Disease heterogeneity and molecular classification of inflammatory palmoplantar diseases.

BACKGROUND: Palmoplantar pustulosis (PPP) is an inflammatory disease characterized by relapsing eruptions of neutrophil-filled, sterile pustules on the palms and soles that can be clinically difficult to differentiate from non-pustular palmoplantar psoriasis (palmPP) and dyshidrotic palmoplantar eczema (DPE). OBJECTIVE: We sought to identify overlapping and unique PPP, palmPP, and DPE drivers to provide molecular insight into their pathogenesis. METHODS: We performed bulk RNA sequencing of lesional PPP (n = 33), palmPP (n = 5), and DPE (n = 28) samples, as well as 5 healthy nonacral and 10 healthy acral skin samples. RESULTS: Acral skin showed a unique immune environment, likely contributing to a unique niche for palmoplantar inflammatory diseases. Compared to healthy acral skin, PPP, palmPP, and DPE displayed a broad overlapping transcriptomic signature characterized by shared upregulation of proinflammatory cytokines (TNF, IL-36), chemokines, and T-cell-associated genes, along with unique disease features of each disease state, including enriched neutrophil processes in PPP and to a lesser extent in palmPP, and lipid antigen processing in DPE. Strikingly, unsupervised clustering and trajectory analyses demonstrated divergent inflammatory profiles within the 3 disease states. These identified putative key upstream immunologic switches, including eicosanoids, interferon responses, and neutrophil degranulation, contributing to disease heterogeneity. CONCLUSION: A molecular overlap exists between different inflammatory palmoplantar diseases that supersedes clinical and histologic assessment. This highlights the heterogeneity within each condition, suggesting limitations of current disease classification and the need to move toward a molecular classification of inflammatory acral diseases.

Protocol to create phenotypic primary human hepatocyte cultures using the RASTRUM 3D cell model platform.

To improve human hepatotoxicity prediction, in vitro liver cell models replicating hepatocyte function, drug metabolism, and toxicity are required. Here, we present a protocol for creating 3D primary human hepatocyte (PHH) cell models using the RASTRUM Platform. We describe the process for PHH model generation; procedures for characterizing the PHH model, including viability, albumin production, and CYP450 inducibility; and drug treatment using acetaminophen and troglitazone. This protocol has applications in upscaling phenotypic hepatotoxicity applications.

Telehealth Access and Substitution in the VHA.

BACKGROUND: In response to COVID-19, the Veterans Health Administration (VHA) expanded telehealth availability, allowing veterans to receive care at home. We explore the extent of substitution of telehealth for in-person care among medical centers (facilities), providers, and patients. We explore the extent to which patient preferences drive telehealth utilization, and compare access to care (as measured by waiting times) for telehealth and in-person visits. METHODS: We use VHA electronic health records to identify scheduled outpatient mental health (MH) appointments from January 2019 through February 2023 focusing on care delivered by social workers, psychologists, and psychiatrists. For each quarter, we compute the proportion of completed appointments that were delivered via phone or video by each facility, provider, and patient and show the changes in these proportions before, during, and after the onset of COVID-19. To explore patient preferences, we match providers of patients with high rates of telehealth utilization and examine the extent to which those providers deliver in-person care. To examine access to care, we compute waiting times for in-person, video, and phone new patient appointments. We investigate differences between urban and rural patients, and patients of different ages. KEY RESULTS: Telehealth for MH grew dramatically in the VHA after the onset of COVID-19. While some facilities provided more telehealth than others, all facilities (as of early 2023) provided some telehealth MH services. Approximately 86% of individual providers provided telehealth, with 27% scheduling MH appointments almost exclusively as telehealth appointments and 59% providing a mix. Patients exhibited more polarization, with 36% scheduling only in-person visits for almost all their MH visits and 56% of them scheduling exclusively telehealth, and only 8% of them utilizing a mix of modalities. Of those who exclusively received telehealth care, a majority of them utilized video (80%) over phone (20%). Take-up of MH among younger patients was higher relative to older patients. Urban patients used telehealth more than rural patients. Patient preferences rather than provider preferences drove utilization of patients who almost exclusively utilized telehealth. Between April 2021 and February 2023, the average difference in waiting time for in-person and video appointments was less than 1 day, with comparable appointment volumes, suggesting that the supply of and demand for in-person and video were not different enough to merit waiting longer. Telehealth was chosen over in-person more among urban and younger patients, as older and rural patients exhibited higher willingness to wait for in-person over video appointments. By contrast, appointment volumes and waiting times for phone appointments were lower across all groups, suggesting that phone may not be as substitutable for in-person visits in MH. CONCLUSIONS: We find that the VHA has made telehealth widely available, providing access to many veterans. While telehealth utilization has increased, face-to-face care persists for MH services, suggesting that one modality may not serve all purposes and preferences for care. Patient preferences drive the modality decision among those who exclusively use MH care via telehealth. For those who persist in mostly utilizing in-person care, there may be various factors influencing those preferences such as issues with limited internet connectivity, language barriers, and digital literacy, especially for older and rural patients who utilize in-person care more than those who are younger and more urban. Further investigation is required to investigate the optimal mix of modalities which may allow for potential increases in patient satisfaction, quality of care, and clinic efficiency.

Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development.

Virus-like particles (VLPs) have been proposed as an attractive tool in SARS-CoV-2 vaccine development, both as (1) a vaccine candidate with high immunogenicity and low reactogenicity and (2) a substitute for live virus in functional and neutralization assays. Though multiple SARS-CoV-2 VLP designs have already been explored in Sf9 insect cells, a key parameter ensuring VLPs are a viable platform is the VLP spike yield (i.e., spike protein content in VLP), which has largely been unreported. In this study, we show that the common strategy of producing SARS-CoV-2 VLPs by expressing spike protein in combination with the native coronavirus membrane and/or envelope protein forms VLPs, but at a critically low spike yield (~0.04-0.08 mg/L). In contrast, fusing the spike ectodomain to the influenza HA transmembrane domain and cytoplasmic tail and co-expressing M1 increased VLP spike yield to ~0.4 mg/L. More importantly, this increased yield translated to a greater VLP spike antigen density (~96 spike monomers/VLP) that more closely resembles that of native SARS-CoV-2 virus (~72-144 Spike monomers/virion). Pseudotyping further allowed for production of functional alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) SARS-CoV-2 VLPs that bound to the target ACE2 receptor. Finally, we demonstrated the utility of pseudotyped VLPs to test neutralizing antibody activity using a simple, acellular ELISA-based assay performed at biosafety level 1 (BSL-1). Taken together, this study highlights the advantage of pseudotyping over native SARS-CoV-2 VLP designs in achieving higher VLP spike yield and demonstrates the usefulness of pseudotyped VLPs as a surrogate for live virus in vaccine and therapeutic development against SARS-CoV-2 variants.

A comparative analysis of 2D and 3D experimental data for the identification of the parameters of computational models.

Computational models are becoming an increasingly valuable tool in biomedical research. Their accuracy and effectiveness, however, rely on the identification of suitable parameters and on appropriate validation of the in-silico framework. Both these steps are highly dependent on the experimental model used as a reference to acquire the data. Selecting the most appropriate experimental framework thus becomes key, together with the analysis of the effect of combining results from different experimental models, a common practice often necessary due to limited data availability. In this work, the same in-silico model of ovarian cancer cell growth and metastasis, was calibrated with datasets acquired from traditional 2D monolayers, 3D cell culture models or a combination of the two. The comparison between the parameters sets obtained in the different conditions, together with the corresponding simulated behaviours, is presented. It provides a framework for the study of the effect of the different experimental models on the development of computational systems. This work also provides a set of general guidelines for the comparative testing and selection of experimental models and protocols to be used for parameter optimization in computational models.

Targeting Homologous Recombination Deficiency in Ovarian Cancer with PARP Inhibitors: Synthetic Lethal Strategies That Impact Overall Survival.

The advent of molecular targeted therapies has made a significant impact on survival of women with ovarian cancer who have defects in homologous recombination repair (HRR). High-grade serous ovarian cancer (HGSOC) is the most common histological subtype of ovarian cancer, with over 50% displaying defective HRR. Poly ADP ribose polymerases (PARPs) are a family of enzymes that catalyse the transfer of ADP-ribose to target proteins, functioning in fundamental cellular processes including transcription, chromatin remodelling and DNA repair. In cells with deficient HRR, PARP inhibitors (PARPis) cause synthetic lethality leading to cell death. Despite the major advances that PARPis have heralded for women with ovarian cancer, questions and challenges remain, including: can the benefits of PARPis be brought to a wider range of women with ovarian cancer; can other drugs in clinical use function in a similar way or with greater efficacy than currently clinically approved PARPis; what can we learn from long-term responders to PARPis; can PARPis sensitise ovarian cancer cells to immunotherapy; and can synthetic lethal strategies be employed more broadly to develop new therapies for women with ovarian cancer. We examine these, and other, questions with focus on improving outcomes for women with ovarian cancer.

Imaging Mass Cytometry Reveals Predominant Innate Immune Signature and Endothelial-Immune Cell Interaction in Juvenile Myositis Compared to Lupus Skin.

OBJECTIVE: Cutaneous inflammation can signal disease in juvenile dermatomyositis (DM) and childhood-onset systemic lupus erythematosus (cSLE), but we do not fully understand cellular mechanisms of cutaneous inflammation. In this study, we used imaging mass cytometry to characterize cutaneous inflammatory cell populations and cell-cell interactions in juvenile DM as compared to cSLE. METHODS: We performed imaging mass cytometry analysis on skin biopsy samples from juvenile DM patients (n = 6) and cSLE patients (n = 4). Tissue slides were processed and incubated with metal-tagged antibodies for CD14, CD15, CD16, CD56, CD68, CD11c, HLA-DR, blood dendritic cell antigen 2, CD20, CD27, CD138, CD4, CD8, E-cadherin, CD31, pan-keratin, and type I collagen. Stained tissue was ablated, and raw data were acquired using the Hyperion imaging system. We utilized the Phenograph unsupervised clustering algorithm to determine cell marker expression and permutation test by histoCAT to perform neighborhood analysis. RESULTS: We identified 14 cell populations in juvenile DM and cSLE skin, including CD14+ and CD68+ macrophages, myeloid and plasmacytoid dendritic cells (pDCs), CD4+ and CD8+ T cells, and B cells. Overall, cSLE skin had a higher inflammatory cell infiltrate, with increased CD14+ macrophages, pDCs, and CD8+ T cells and immune cell-immune cell interactions. Juvenile DM skin displayed a stronger innate immune signature, with a higher overall percentage of CD14+ macrophages and prominent endothelial cell-immune cell interaction. CONCLUSION: Our findings identify immune cell population differences, including CD14+ macrophages, pDCs, and CD8+ T cells, in juvenile DM skin compared to cSLE skin, and highlight a predominant innate immune signature and endothelial cell-immune cell interaction in juvenile DM, providing insight into candidate cell populations and interactions to better understand disease-specific pathophysiology.

Nonlesional lupus skin contributes to inflammatory education of myeloid cells and primes for cutaneous inflammation.

Cutaneous lupus erythematosus (CLE) is a disfiguring and poorly understood condition frequently associated with systemic lupus. Previous studies suggest that nonlesional keratinocytes play a role in disease predisposition, but this has not been investigated in a comprehensive manner or in the context of other cell populations. To investigate CLE immunopathogenesis, normal-appearing skin, lesional skin, and circulating immune cells from lupus patients were analyzed via integrated single-cell RNA sequencing and spatial RNA sequencing. We demonstrate that normal-appearing skin of patients with lupus represents a type I interferon-rich, prelesional environment that skews gene transcription in all major skin cell types and markedly distorts predicted cell-cell communication networks. We also show that lupus-enriched CD16+ dendritic cells undergo robust interferon education in the skin, thereby gaining proinflammatory phenotypes. Together, our data provide a comprehensive characterization of lesional and nonlesional skin in lupus and suggest a role for skin education of CD16+ dendritic cells in CLE pathogenesis.

Provider supply and access to primary care.

Resource-constrained delivery systems often have access issues, causing patients to wait a long time to see a provider. We develop theoretical and empirical models of wait times and apply them to primary care delivery by the U.S. Veterans Health Administration (VHA). Using instrumental variables to handle simultaneity issues, we estimate the effect of clinician supply on new patient wait times. We find that it has a sizable impact. A 10% increase in capacity reduces wait times by 2.1%. Wait times are also associated with clinician productivity, scheduling protocols, and patient access to alternative sources of care. The VHA has adopted our models to identify underserved areas as specified by the MISSION Act of 2018.

Dually-enrolled patients choose providers with lower wait times: Budgetary implications for the VHA.

OBJECTIVE: To estimate the effect of wait times on patients' choice of provider and simulate changes in choice of provider due to compliance with VA MISSION Act wait time targets. DATA SOURCES: We use nationwide administrative data (2014-2017) on Veterans who are enrolled in Medicare and the Veterans Health Administration (VHA), the Survey of VHA Enrollees, Area Health Resource Files, and other data provided by the Centers for Medicare & Medicaid Services. STUDY DESIGN: We use an instrumental variables approach to identify the effect of VHA wait times on the proportion of total (Medicare and VHA) services that are paid for by the VHA ("reliance"). We exploit shocks to VHA provider supply to isolate supply-driven changes in wait times and estimate the effect on VHA reliance. We control for market and time fixed effects and local demand factors. DATA COLLECTION/EXTRACTION METHODS: We use monthly aggregated data on 140 markets (groups of counties). VHA reliance is computed among patients aged 65 years or older who are dually enrolled in VHA and Medicare. VHA wait times and reliance are calculated for multiple specialties: cardiology, gastroenterology, orthopedics, urology, dermatology, and ophthalmology/optometry. PRINCIPAL FINDINGS: A 10% increase in the mean wait time (+2.8 days) reduces VHA reliance by 2.3 percentage points (95% CI: 2.3, 2.7), or 7.9% of the sample mean. This implies that meeting the MISSION Act wait time targets may have multi-billion-dollar budgetary impacts. Effects vary across specialties. For example, a 10% increase in the mean wait time for cardiology services (+2.0 days) reduces reliance by 1.8 percentage points (95% CI: 1.6, 2.1), or 6.3% of the sample mean for cardiology services. CONCLUSIONS: Meeting statutory wait time targets may have substantial unforeseen impacts on federal health care spending as patients sort to providers who have lower wait times.

Three-Dimensional Modelling of Ovarian Cancer: From Cell Lines to Organoids for Discovery and Personalized Medicine.

Ovarian cancer has the highest mortality of all of the gynecological malignancies. There are several distinct histotypes of this malignancy characterized by specific molecular events and clinical behavior. These histotypes have differing responses to platinum-based drugs that have been the mainstay of therapy for ovarian cancer for decades. For histotypes that initially respond to a chemotherapeutic regime of carboplatin and paclitaxel such as high-grade serous ovarian cancer, the development of chemoresistance is common and underpins incurable disease. Recent discoveries have led to the clinical use of PARP (poly ADP ribose polymerase) inhibitors for ovarian cancers defective in homologous recombination repair, as well as the anti-angiogenic bevacizumab. While predictive molecular testing involving identification of a genomic scar and/or the presence of germline or somatic BRCA1 or BRCA2 mutation are in clinical use to inform the likely success of a PARP inhibitor, no similar tests are available to identify women likely to respond to bevacizumab. Functional tests to predict patient response to any drug are, in fact, essentially absent from clinical care. New drugs are needed to treat ovarian cancer. In this review, we discuss applications to address the currently unmet need of developing physiologically relevant in vitro and ex vivo models of ovarian cancer for fundamental discovery science, and personalized medicine approaches. Traditional two-dimensional (2D) in vitro cell culture of ovarian cancer lacks critical cell-to-cell interactions afforded by culture in three-dimensions. Additionally, modelling interactions with the tumor microenvironment, including the surface of organs in the peritoneal cavity that support metastatic growth of ovarian cancer, will improve the power of these models. Being able to reliably grow primary tumoroid cultures of ovarian cancer will improve the ability to recapitulate tumor heterogeneity. Three-dimensional (3D) modelling systems, from cell lines to organoid or tumoroid cultures, represent enhanced starting points from which improved translational outcomes for women with ovarian cancer will emerge.

Sitagliptin Is More Effective Than Gliclazide in Preventing Pro-Fibrotic and Pro-Inflammatory Changes in a Rodent Model of Diet-Induced Non-Alcoholic Fatty Liver Disease.

A diet-induced non-alcoholic fatty liver disease (NAFLD) model causing obesity in rodents was used to examine whether sitagliptin and gliclazide therapies have similar protective effects on pathological liver change. METHODS: Male mice were fed a high-fat diet (HFD) or standard chow (Chow) ad libitum for 25 weeks and randomly allocated to oral sitagliptin or gliclazide treatment for the final 10 weeks. Fasting blood glucose and circulating insulin were measured. Inflammatory and fibrotic liver markers were assessed by qPCR. The second messenger ERK and autophagy markers were examined by Western immunoblot. F4/80, collagens and CCN2 were assessed by immunohistochemistry (IHC). RESULTS: At termination, HFD mice were obese, hyperinsulinemic and insulin-resistant but non-diabetic. The DPP4 inhibitor sitagliptin prevented intrahepatic induction of pro-fibrotic markers collagen-IV, collagen-VI, CCN2 and TGF-ß1 and pro-inflammatory markers TNF-α and IL-1ß more effectively than sulfonylurea gliclazide. By IHC, liver collagen-VI and CCN2 induction by HFD were inhibited only by sitagliptin. Sitagliptin had a greater ability than gliclazide to normalise ERK-protein liver dysregulation. CONCLUSION: These data indicate that sitagliptin, compared with gliclazide, exhibits greater inhibition of pro-fibrotic and pro-inflammatory changes in an HFD-induced NAFLD model. Sitagliptin therapy, even in the absence of diabetes, may have specific benefits in diet-induced NAFLD.

Targeting CCN2 protects against progressive non-alcoholic steatohepatitis in a preclinical model induced by high-fat feeding and type 2 diabetes.

Type 2 diabetes is an independent risk factor for non-alcoholic steatohepatitis (NASH) progression and its mediators have not been resolved. In this study, a pathogenic role of cellular communication network factor 2 (CCN2) protein in NASH pathology, was investigated in an established preclinical NASH model. Male wild type C57BL/6 mice received either Chow or high fat diet (HFD) for 26 weeks, with some mice in each group randomly selected to receive low dose streptozotocin (STZ: 3 i.p. injections, 65 mg/kg) at 15 weeks to induce type 2 diabetes. In the final 10 of the 26 weeks mice from each group were administered i.p. either rabbit anti-CCN2 neutralizing antibody (CCN2Ab) or as control normal rabbit IgG, at a dose of 150 µg per mouse twice/week. NASH developed in the HFD plus diabetes (HFD+DM) group. Administration of CCN2Ab significantly downregulated collagen I and collagen III mRNA induction and prevented pro-inflammatory MCP-1 mRNA induction in HFD+DM mice. At the protein level, CCN2Ab significantly attenuated collagen accumulation by PSR stain and collagen I protein induction in HFD+DM. Phosphorylation of the pro-fibrotic ERK signalling pathway in liver in HFD+DM was attenuated by CCN2Ab treatment. Intrahepatic CCN1 mRNA was induced, whereas CCN3 was downregulated at both the mRNA and protein levels in HFD+DM. CCN3 down-regulation was prevented by CCN2Ab treatment. This in vivo study indicates that CCN2 is a molecular target in NASH with high fat diet and diabetes, and that regulation of ERK signalling is implicated in this process.

B Cell Signatures Distinguish Cutaneous Lupus Erythematosus Subtypes and the Presence of Systemic Disease Activity.

Cutaneous lupus erythematosus (CLE) is a chronic inflammatory skin disease characterized by a diverse cadre of clinical presentations. CLE commonly occurs in patients with systemic lupus erythematosus (SLE), and CLE can also develop in the absence of systemic disease. Although CLE is a complex and heterogeneous disease, several studies have identified common signaling pathways, including those of type I interferons (IFNs), that play a key role in driving cutaneous inflammation across all CLE subsets. However, discriminating factors that drive different phenotypes of skin lesions remain to be determined. Thus, we sought to understand the skin-associated cellular and transcriptional differences in CLE subsets and how the different types of cutaneous inflammation relate to the presence of systemic lupus disease. In this study, we utilized two distinct cohorts comprising a total of 150 CLE lesional biopsies to compare discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE), and acute cutaneous lupus erythematosus (ACLE) in patients with and without associated SLE. Using an unbiased approach, we demonstrated a CLE subtype-dependent gradient of B cell enrichment in the skin, with DLE lesions harboring a more dominant skin B cell transcriptional signature and enrichment of B cells on immunostaining compared to ACLE and SCLE. Additionally, we observed a significant increase in B cell signatures in the lesional skin from patients with isolated CLE compared with similar lesions from patients with systemic lupus. This trend was driven primarily by differences in the DLE subgroup. Our work thus shows that skin-associated B cell responses distinguish CLE subtypes in patients with and without associated SLE, suggesting that B cell function in skin may be an important link between cutaneous lupus and systemic disease activity.

The Effect of a Sustained High-Fat Diet on the Metabolism of White and Brown Adipose Tissue and Its Impact on Insulin Resistance: A Selected Time Point Cross-Sectional Study.

(1) Background: studies on the long-term dynamic changes in fat depot metabolism in response to a high-fat diet (HFD) on hepatic lipid deposition and insulin resistance are sparse. This study investigated the dynamic changes produced by HFD and the production of dysfunctional fat depots on insulin resistance and liver lipid metabolism. (2) Methods: mice fed a chow or HFD (45% kcal fat) diet had three fat depots, liver, and blood collected at 6, 10, 20, and 30 weeks. Anthropometric changes and gene markers for adipogenesis, thermogenesis, ECM remodeling, inflammation, and tissue insulin resistance were measured. (3) Results: early responses to the HFD were increased body weight, minor deposition of lipid in liver, increased adipocyte size, and adipogenesis. Later changes were dysfunctional adipose depots, increased liver fat, insulin resistance (shown by changes in ITT) accompanied by increased inflammatory markers, increased fibrosis (fibrosis > 2-fold, p < 0.05 from week 6), and the presence of crown cells in white fat depots. Later, changes did not increase thermogenic markers in response to the increased calories and decreased UCP1 and PRDM16 proteins in WAT. (4) Conclusions: HFD feeding initially increased adipocyte diameter and number, but later changes caused adipose depots to become dysfunctional, restricting adipose tissue expansion, changing the brown/beige ratios in adipose depots, and causing ectopic lipid deposition and insulin resistance.

Generation and analysis of 3D cell culture models for drug discovery.

Successful preclinical drug testing relies in part on data generated using in vitro cell culture models that recapitulate the structure and function of tumours and other tissues in vivo. The growing evidence that 3D cell models can more accurately predict the efficacy of drug responses compared to traditionally utilised 2D cell culture systems has led to continuous scientific and technological advances that enable better physiologically representative in vitro modelling of in vivo tissues. This review will provide an overview of the utility of current 3D cell models from a drug screening perspective and explore the future of 3D cell models for drug discovery applications.

Contribution of plasma cells and B cells to hidradenitis suppurativa pathogenesis.

Hidradenitis suppurativa (HS) is a debilitating chronic inflammatory skin disease characterized by chronic abscess formation and development of multiple draining sinus tracts in the groin, axillae, and perineum. Using proteomic and transcriptomic approaches, we characterized the inflammatory responses in HS in depth, revealing immune responses centered on IFN-γ, IL-36, and TNF, with lesser contribution from IL-17A. We further identified B cells and plasma cells, with associated increases in immunoglobulin production and complement activation, as pivotal players in HS pathogenesis, with Bruton's tyrosine kinase (BTK) and spleen tyrosine kinase (SYK) pathway activation as a central signal transduction network in HS. These data provide preclinical evidence to accelerate the path toward clinical trials targeting BTK and SYK signaling in moderate-to-severe HS.

Medicare's Bundled Payment Initiatives for Hospital-Initiated Episodes: Evidence and Evolution.

Policy Points Evidence suggests that bundled payment contracting can slow the growth of payer costs relative to fee-for-service contracting, although bundled payment models may not reduce absolute costs. Bundled payments may be more effective than fee-for-service payments in containing costs for certain medical conditions. For the most part, Medicare's bundled payment initiatives have not been associated with a worsening of quality in terms of readmissions, emergency department use, and mortality. Some evidence suggests a worsening of other quality measures for certain medical conditions. Bundled payment contracting involves trade-offs: Expanding a bundle's scope and duration may better contain costs, but a more comprehensive bundle may be less attractive to providers, reducing their willingness to accept it as an alternative to fee-for-service payment. CONTEXT: Bundled payments have been promoted as an alternative to fee-for-service payments that can mitigate the incentives for service volume under the fee-for-service model. As Medicare has gained experience with bundled payments, it has widened their scope and increased their duration. However, there have been few reviews of the empirical literature on the impact of Medicare's bundled payment programs on cost, resource use, utilization, and quality. METHODS: We examined the history and features of 16 of Medicare's bundled payment programs involving hospital-initiated episodes of care and conducted a literature review of articles about those programs. Database and additional searches yielded 1,479 articles. We evaluate the studies' methodological quality and summarize the quantitative findings about Medicare expenditures and quality of care from 37 studies that used higher-quality research designs. FINDINGS: Medicare's bundled payment initiatives have varied in their design features, such as episode scope and duration. Many initiatives were associated with little to no reduction in Medicare expenditures, unless large pricing discounts for providers were negotiated in advance. Initiatives that included post-acute care services were associated with lower expenditures for certain conditions. Hospitals may have been able to reduce internal production costs with help from physicians via gainsharing. Most initiatives were not associated with significant changes in quality of care, as measured by readmission and mortality rates. Of the significant changes in readmission rates, the results were mixed, showing increases and decreases associated with bundled payments. Some evidence suggested that worse patient outcomes were associated bundled payments, although most results were not statistically significant. Results on case-mix selection were mixed: Several initiatives were associated with reductions in episode severity, whereas others were associated with little change. CONCLUSIONS: Bundled payments for hospital-initiated episodes may be a good alternative to fee-for-service payments. Bundled payments can help slow the growth of payer spending, although they do not necessarily reduce absolute spending. They are associated with lower provider production costs, and there is no overwhelming evidence of compromised quality. However, designing a bundled payment contract that is attractive to both providers and payers proves to be a challenge.