Implications of an Altered Gut Microbiome in Rat Experimental Vascularized Composite Transplantation.

OBJECTIVES: Vascularized composite allotransplantation is a reconstructive option after severe injury but is fraught with complications, including transplant rejection due to major histocompatibility complex mismatch in the context of allogeneic transplant, which in turn is due to altered immuno-inflammation secondary to transplant. The immunosuppressant tacrolimus can prevent rejection. Because tacrolimus is metabolized predominantly by the gut, this immunosuppressant alters the gut microbiome in multiple ways, thereby possibly affecting immunoinflammation. MATERIALS AND METHODS: We performed either allogeneic or syngeneic transplant with or without tacrolimus in rats. We quantified protein-level inflammatory mediators in the skin, muscle, and plasma and assessed the diversity of the gut microbiome through 16S RNA analysis at several timepoints over 31 days posttransplant. RESULTS: Statistical analysis highlighted a complex interaction between major histocompatibility complex and tacrolimus therapy on the relative diversity of the microbiome. Time-interval principal component analysis indicated numerous significant differences in the tissue characteristics of inflammation and gut microbiome that varied over time and across experimental conditions. Classification and regression tree analysis suggested that both inflammatory mediators in specific tissues and changes in the gut microbiome are useful in characterizing the temporal dynamics of posttransplant inflammation. Dynamic network analysis highlighted unique changes in Methanosphaera that were correlated with Peptococcusin allogeneic transplants with and without tacrolimus versus Prevotella in syngeneic transplant with tacrolimus, suggesting that alterations in Methanosphaera might be a biomarker of vascularized composite allotransplant rejection. CONCLUSIONS: Our results suggest a complex interaction among major histocompatibility complex, local and systemic immuno-inflammation, and tacrolimus therapy and highlight the potential for novel insights into vascularized composite allotransplant from computational approaches.

A common single nucleotide polymorphism is associated with inflammation and critical illness outcomes.

Acute inflammation is heterogeneous in critical illness and predictive of outcome. We hypothesized that genetic variability in novel, yet common, gene variants contributes to this heterogeneity and could stratify patient outcomes. We searched algorithmically for significant differences in systemic inflammatory mediators associated with any of 551,839 SNPs in one derivation (n = 380 patients with blunt trauma) and two validation (n = 75 trauma and n = 537 non-trauma patients) cohorts. This analysis identified rs10404939 in the LYPD4 gene. Trauma patients homozygous for the A allele (rs10404939AA; 27%) had different trajectories of systemic inflammation along with persistently elevated multiple organ dysfunction (MOD) indices vs. patients homozygous for the G allele (rs10404939GG; 26%). rs10404939AA homozygotes in the trauma validation cohort had elevated MOD indices, and non-trauma patients displayed more complex inflammatory networks and worse 90-day survival compared to rs10404939GG homozygotes. Thus, rs10404939 emerged as a common, broadly prognostic SNP in critical illness.

Defining spatiotemporal gene modules in liver regeneration using Analytical Dynamic Visual Spatial Omics Representation (ADViSOR).

BACKGROUND: The liver is the only organ with the ability to regenerate following surgical or toxicant insults, and partial hepatectomy serves as an experimental model of liver regeneration (LR). Dynamic changes in gene expression occur from the periportal to pericentral regions of the liver following partial hepatectomy; thus, spatial transcriptomics, combined with a novel computational pipeline (ADViSOR [Analytic Dynamic Visual Spatial Omics Representation]), was employed to gain insights into the spatiotemporal molecular underpinnings of LR. METHODS: ADViSOR, comprising Time-Interval Principal Component Analysis and sliding dynamic hypergraphs, was applied to spatial transcriptomics data on 100 genes assayed serially through LR, including key components of the Wnt/ß-catenin pathway at critical timepoints after partial hepatectomy. RESULTS: This computational pipeline identified key functional modules demonstrating cell signaling and cell-cell interactions, inferring shared regulatory mechanisms. Specifically, ADViSOR analysis suggested that macrophage-mediated inflammation is a critical component of early LR and confirmed prior studies showing that Ccnd1, a hepatocyte proliferative gene, is regulated by the Wnt/ß-catenin pathway. These findings were subsequently validated through protein localization, which provided further confirmation and novel insights into the spatiotemporal changes in the Wnt/ß-catenin pathway during LR. CONCLUSIONS: Thus, ADViSOR may yield novel insights in other complex, spatiotemporal contexts.

Functional Asymmetry During Working Memory and Its Association with Markers of Alzheimer's Disease in Cognitively Normal Older Adults.

BACKGROUND: Amyloid-ß (Aß) deposits asymmetrically early in Alzheimer's disease (AD). This process is variable and has been associated with asymmetric hypometabolism. OBJECTIVE: We investigated whether neural asymmetry during working memory and executive function processing was associated with AD genetic risk and markers of AD as well as other brain neuropathology biomarkers, cognitive function, and cognitive reserve in cognitively normal older adults. METHODS: We analyzed data from 77 cognitively healthy, older adults who completed functional magnetic resonance imaging, positron emission tomography, and cognitive testing. We identified regions of significant activation and asymmetry during the Digital Symbol Substitution Task (DSST). We examined associations between regions with significant hemispheric asymmetry (directional and absolute) and global cerebral Aß, cerebral glucose metabolism, white matter hyperintensities, APOE ɛ4 allele status, DSST reaction time, age, sex, education, and cognitive function. RESULTS: Asymmetry was not associated with several factors including cognitive function, Aß, and white matter hyperintensities. The presence of at least one ɛ4 APOE allele in participants was associated with less asymmetric activation in the angular gyrus (right dominant activation). Greater education was associated with less asymmetric activation in mediodorsal thalamus (left dominant activation). CONCLUSIONS: Genetic risk of AD was associated with lower asymmetry in angular gyrus activation, while greater education was associated with lower asymmetry in mediodorsal thalamus activation. Changes in asymmetry may reflect components of compensation or cognitive reserve. Asymmetric neural recruitment during working memory may be related to maintenance of cognitive function in cognitively normal older adults.

Temporally complex inflammatory networks in an animal model reveal signatures for interstitial cystitis and bladder pain syndrome phenotype.

INTRODUCTION AND OBJECTIVE: Interstitial cystitis and bladder pain syndrome (IC/BPS) presents with symptoms of debilitating bladder pain and is typically a diagnosis of exclusion. The cystoscopic detection of Hunner's lesions increases the likelihood of detecting tissue inflammation on bladder biopsy and increases the odds of therapeutic success with anti-inflammatory drugs. However, the identification of this subgroup remains challenging with the current lack of surrogate biomarkers of IC/BPS. On the path towards identifying biomarkers of IC/BPS, we modeled the dynamic evolution of inflammation in an experimental IC/BPS rodent model using computational biological network analysis of inflammatory mediators (cytokines and chemokines) released into urine. The use of biological network analysis allows us to identify urinary proteins that could be drivers of inflammation and could therefore serve as therapeutic targets for the treatment of IC/BPS. METHODS: Rats subjected to cyclophosphamide (CYP) injection (150 mg/kg) were used as an experimental model for acute IC/BPS (n = 8). Urine from each void was collected from the rats over a 12-h period and was assayed for 13 inflammatory mediators using Luminex™. Time-interval principal component analysis (TI-PCA) and dynamic network analysis (DyNA), two biological network algorithms, were used to identify biomarkers of inflammation characteristic of IC/BPS over time. RESULTS: Compared to vehicle-treated rats, nearly all inflammatory mediators were elevated significantly (p < 0.05) in the urine of CYP treated rats. TI-PCA highlighted that GRO-KC, IL-5, IL-18, and MCP-1 account for the greatest variance in the inflammatory response. At early time points, DyNA indicated a positive correlation between IL-4 and IL-1ß and between TNF-α and IL-1ß. Analysis of TI-PCA and DyNA at later time points showed the emergence of IL-5, IL-6, and IFNγ as additional key mediators of inflammation. Furthermore, DyNA network complexity rose and fell before peaking at 9.5 h following CYP treatment. This pattern of inflammation may mimic the fluctuating severity of inflammation associated with IC/BPS flares. CONCLUSIONS: Computational analysis of inflammation networks in experimental IC/BPS analysis expands on the previously accepted inflammatory signatures of IC by adding IL-5, IL-18, and MCP-1 to the prior studies implicating IL-6 and GRO as IC/BPS biomarkers. This analysis supports a complex evolution of inflammatory networks suggestive of the rise and fall of inflammation characteristic of IC/BPS flares.

Peripheral nerve repair is associated with augmented cross-tissue inflammation following vascularized composite allotransplantation.

Introduction: Vascularized composite allotransplantation (VCA), with nerve repair/coaptation (NR) and tacrolimus (TAC) immunosuppressive therapy, is used to repair devastating traumatic injuries but is often complicated by inflammation spanning multiple tissues. We identified the parallel upregulation of transcriptional pathways involving chemokine signaling, T-cell receptor signaling, Th17, Th1, and Th2 pathways in skin and nerve tissue in complete VCA rejection compared to baseline in 7 human hand transplants and defined increasing complexity of protein-level dynamic networks involving chemokine, Th1, and Th17 pathways as a function of rejection severity in 5 of these patients. We next hypothesized that neural mechanisms may regulate the complex spatiotemporal evolution of rejection-associated inflammation post-VCA. Methods: For mechanistic and ethical reasons, protein-level inflammatory mediators in tissues from Lewis rats (8 per group) receiving either syngeneic (Lewis) or allogeneic (Brown-Norway) orthotopic hind limb transplants in combination with TAC, with and without sciatic NR, were compared to human hand transplant samples using computational methods. Results: In cross-correlation analyses of these mediators, VCA tissues from human hand transplants (which included NR) were most similar to those from rats undergoing VCA + NR. Based on dynamic hypergraph analyses, NR following either syngeneic or allogeneic transplantation in rats was associated with greater trans-compartmental localization of early inflammatory mediators vs. no-NR, and impaired downregulation of mediators including IL-17A at later times. Discussion: Thus, NR, while considered necessary for restoring graft function, may also result in dysregulated and mis-compartmentalized inflammation post-VCA and therefore necessitate mitigation strategies. Our novel computational pipeline may also yield translational, spatiotemporal insights in other contexts.

Central role for neurally dysregulated IL-17A in dynamic networks of systemic and local inflammation in combat casualties.

Dynamic Network Analysis (DyNA) and Dynamic Hypergraphs (DyHyp) were used to define protein-level inflammatory networks at the local (wound effluent) and systemic circulation (serum) levels from 140 active-duty, injured service members (59 with TBI and 81 non-TBI). Interleukin (IL)-17A was the only biomarker elevated significantly in both serum and effluent in TBI vs. non-TBI casualties, and the mediator with the most DyNA connections in TBI wounds. DyNA combining serum and effluent data to define cross-compartment correlations suggested that IL-17A bridges local and systemic circulation at late time points. DyHyp suggested that systemic IL-17A upregulation in TBI patients was associated with tumor necrosis factor-α, while IL-17A downregulation in non-TBI patients was associated with interferon-γ. Correlation analysis suggested differential upregulation of pathogenic Th17 cells, non-pathogenic Th17 cells, and memory/effector T cells. This was associated with reduced procalcitonin in both effluent and serum of TBI patients, in support of an antibacterial effect of Th17 cells in TBI patients. Dysregulation of Th17 responses following TBI may drive cross-compartment inflammation following combat injury, counteracting wound infection at the cost of elevated systemic inflammation.

Interleukin-17 as a spatiotemporal bridge from acute to chronic inflammation: Novel insights from computational modeling.

A systematic review of several acute inflammatory diseases ranging from sepsis and trauma/hemorrhagic shock to the relevant pathology of the decade, COVID-19, points to the cytokine interleukin (IL)-17A as being centrally involved in the propagation of inflammation. We summarize the role of IL-17A in acute inflammation, leveraging insights made possible by biological network analysis and novel computational methodologies aimed at defining the spatiotemporal spread of inflammation in both experimental animal models and humans. These studies implicate IL-17A in the cross-tissue spread of inflammation, a process that appears to be in part regulated through neural mechanisms. Although acute inflammatory diseases are currently considered distinct from chronic inflammatory pathologies, we suggest that chronic inflammation may represent repeated, cyclical episodes of acute inflammation driven by mechanisms involving IL-17A. Thus, insights from computational modeling of acute inflammatory diseases may improve diagnosis and treatment of chronic inflammation; in turn, therapeutics developed for chronic/autoimmune disease may be of benefit in acute inflammation. This article is categorized under: Immune System Diseases > Computational Models.

Large Variations in the Prices of Urologic Procedures at Academic Medical Centers 1 Year After Implementation of the Price Transparency Final Rule.

Importance: Patients with urologic diseases often experience financial toxicity, defined as high levels of financial burden and concern, after receiving care. The Price Transparency Final Rule, which requires hospitals to disclose both the commercial and cash prices for at least 300 services, was implemented to facilitate price shopping, decrease price dispersion, and lower health care costs. Objective: To evaluate compliance with the Price Transparency Final Rule and to quantify variations in the price of urologic procedures among academic hospitals and by insurance class. Design, Setting, and Participants: This was a cross-sectional study that determined the prices of 5 common urologic procedures among academic medical centers and by insurance class. Prices were obtained from the Turquoise Health Database on March 24, 2022. Academic hospitals were identified from the Association of American Medical Colleges website. The 5 most common urologic procedures were cystourethroscopy, prostate biopsy, laparoscopic radical prostatectomy, transurethral resection of the prostate, and ureteroscopy with laser lithotripsy. Using the corresponding Current Procedural Terminology codes, the Turquoise Health Database was queried to identify the cash price, Medicare price, Medicaid price, and commercial insurance price for these procedures. Exposures: The Price Transparency Final Rule, which went into effect January 1, 2021. Main Outcomes and Measures: Variability in procedure price among academic medical centers and by insurance class (Medicare, Medicaid, commercial, and cash price). Results: Of 153 hospitals, only 20 (13%) listed a commercial price for all 5 procedures. The commercial price was reported most often for cystourethroscopy (86 hospitals [56%]) and least often for laparoscopic radical prostatectomy (45 hospitals [29%]). The cash price was lower than the Medicare, Medicaid, and commercial price at 24 hospitals (16%). Prices varied substantially across hospitals for all 5 procedures. There were significant variations in the prices of cystoscopy (χ23 = 85.9; P = .001), prostate biopsy (χ23 = 64.6; P = .001), prostatectomy (χ23 = 24.4; P = .001), transurethral resection of the prostate (χ23 = 51.3; P = .001), and ureteroscopy with laser lithotripsy (χ23 = 63.0; P = .001) by insurance type. Conclusions and Relevance: These findings suggest that, more than 1 year after the implementation of the Price Transparency Final Rule, there are still large variations in the prices of urologic procedures among academic hospitals and by insurance class. Currently, in certain situations, health care costs could be reduced if patients paid out of pocket. The Centers for Medicare & Medicaid Services may improve price transparency by better enforcing penalties for noncompliance, increasing penalties, and ensuring that hospitals report prices in a way that is easy for patients to access and understand.

Inferring Tissue-Specific, TLR4-Dependent Type 17 Immune Interactions in Experimental Trauma/Hemorrhagic Shock and Resuscitation Using Computational Modeling.

Trauma/hemorrhagic shock followed by resuscitation (T/HS-R) results in multi-system inflammation and organ dysfunction, in part driven by binding of damage-associated molecular pattern molecules to Toll-like Receptor 4 (TLR4). We carried out experimental T/HS-R (pseudo-fracture plus 2 h of shock followed by 0-22 h of resuscitation) in C57BL/6 (wild type [WT]) and TLR4-null (TLR4-/-) mice, and then defined the dynamics of 20 protein-level inflammatory mediators in the heart, gut, lung, liver, spleen, kidney, and systemic circulation. Cross-correlation and Principal Component Analysis (PCA) on data from the 7 tissues sampled suggested that TLR4-/- samples express multiple inflammatory mediators in a small subset of tissue compartments as compared to the WT samples, in which many inflammatory mediators were localized non-specifically to nearly all compartments. We and others have previously defined a central role for type 17 immune cells in human trauma. Accordingly, correlations between IL-17A and GM-CSF (indicative of pathogenic Th17 cells); between IL-17A and IL-10 (indicative of non-pathogenic Th17 cells); and IL-17A and TNF (indicative of memory/effector T cells) were assessed across all tissues studied. In both WT and TLR4-/- mice, positive correlations were observed between IL-17A and GM-CSF, IL-10, and TNF in the kidney and gut. In contrast, the variable and dynamic presence of both pathogenic and non-pathogenic Th17 cells was inferred in the systemic circulation of TLR4-/- mice over time, suggesting a role for TLR4 in efflux of these cells into peripheral tissues. Hypergraph analysis - used to define dynamic, cross compartment networks - in concert with PCA-suggested that IL-17A was present persistently in all tissues at all sampled time points except for its absence in the plasma at 0.5h in the WT group, supporting the hypothesis that T/HS-R induces efflux of Th17 cells from the circulation and into specific tissues. These analyses suggest a complex, context-specific role for TLR4 and type 17 immunity following T/HS-R.

Fixed and flexible: Dynamic prefrontal activations and working memory capacity relationships vary with memory demand.

Prefrontal cortex (PFC) activation during encoding of memoranda (proactive responses) is associated with better working memory (WM) compared to reactive/retrieval-based activation. This suggests that dynamic PFC activation patterns may be fixed, based upon one's WM ability, with individuals who have greater WM ability relying more on proactive processes and individuals with lesser WM ability relying more on reactive processes. We newly tested whether this heuristic applied when challenging an individual's WM capacity. Twenty-two participants (N = 22) underwent functional near-infrared spectroscopy (fNIRS) during a modified Sternberg WM paradigm. We tested whether the relationship between dynamic PFC activation patterns and WM capacity changed, as a function of WM demands (N = 14 after quality control). Here, higher-WM capacity was associated with more proactive PFC patterns, but only when WM capacity was overloaded. Lower-WM capacity was associated with these same patterns, but only when WM demand was low. Findings are inconsistent with a purely fixed view of dynamic PFC activation patterns and suggest higher- and lower-WM-capacity individuals flexibly engage PFC processes in a fundamentally different manner, dependent upon current WM demands.