Supplementary Material for: Correlation of Neuroendocrine Differentiation with A Distinctively Suppressive Immune Microenvironment in Gastric Cancer

Introduction Neuroendocrine neoplasms (NENs) harbored significantly suppressive tumor immune microenvironments (TIMEs). However, the immunological effects of neuroendocrine differentiation (NED) on non-NE neoplasms, such as gastric cancer (GC), were unknown. Methods Between pure gastric cancer (PGC) and GC-NED, TIME features were scored based on expression data and validated on serial whole-tissue sections of surgical samples, with tertiary lymphoid structures (TLSs) and the extra-TLS zone evaluated independently using multi-marker immunohistochemical staining. Risk analyses of TIME features on tumor behaviors were performed in GC-NED. The universal immunological effects of NED were explored preliminarily in adenocarcinomas arising in other organs. Results Based on over 11,500 annotated TLSs and 2,700 extra-TLS zones, compared with PGC, GC-NED harbored a distinctively more suppressive TIME characterized by increased but immature TLSs, with higher naïve B cell and follicular regulatory T cell densities and downregulated TLS maturation-related cell ratios inside TLSs; increased naïve B cell and regulatory T cell densities and a high proportion of exhausted T cells in the extra-TLS zone. The upregulated tumor PD-L1 expression and its close correlations with TLS formation and maturation were remarkable exclusively in GC-NED. TIME features, especially those regarding TLSs, were significantly correlated with tumor growth and invasion. The desynchrony between TLS formation and maturation and increased naïve or regulatory immune cell infiltration was observed in adenocarcinomas of the colorectum, pancreas, lung, and prostate. Conclusion NED highlighted a distinct GC entity with more suppressive TIME features correlated with tumor behaviors, indicating a cohort that would benefit more from immunotherapies

Supplementary Material for: Correlation of Neuroendocrine Differentiation with A Distinctively Suppressive Immune Microenvironment in Gastric Cancer

Introduction Neuroendocrine neoplasms (NENs) harbored significantly suppressive tumor immune microenvironments (TIMEs). However, the immunological effects of neuroendocrine differentiation (NED) on non-NE neoplasms, such as gastric cancer (GC), were unknown. Methods Between pure gastric cancer (PGC) and GC-NED, TIME features were scored based on expression data and validated on serial whole-tissue sections of surgical samples, with tertiary lymphoid structures (TLSs) and the extra-TLS zone evaluated independently using multi-marker immunohistochemical staining. Risk analyses of TIME features on tumor behaviors were performed in GC-NED. The universal immunological effects of NED were explored preliminarily in adenocarcinomas arising in other organs. Results Based on over 11,500 annotated TLSs and 2,700 extra-TLS zones, compared with PGC, GC-NED harbored a distinctively more suppressive TIME characterized by increased but immature TLSs, with higher naïve B cell and follicular regulatory T cell densities and downregulated TLS maturation-related cell ratios inside TLSs; increased naïve B cell and regulatory T cell densities and a high proportion of exhausted T cells in the extra-TLS zone. The upregulated tumor PD-L1 expression and its close correlations with TLS formation and maturation were remarkable exclusively in GC-NED. TIME features, especially those regarding TLSs, were significantly correlated with tumor growth and invasion. The desynchrony between TLS formation and maturation and increased naïve or regulatory immune cell infiltration was observed in adenocarcinomas of the colorectum, pancreas, lung, and prostate. Conclusion NED highlighted a distinct GC entity with more suppressive TIME features correlated with tumor behaviors, indicating a cohort that would benefit more from immunotherapies

Supplementary Material for: Correlation of Neuroendocrine Differentiation with A Distinctively Suppressive Immune Microenvironment in Gastric Cancer

Introduction Neuroendocrine neoplasms (NENs) harbored significantly suppressive tumor immune microenvironments (TIMEs). However, the immunological effects of neuroendocrine differentiation (NED) on non-NE neoplasms, such as gastric cancer (GC), were unknown. Methods Between pure gastric cancer (PGC) and GC-NED, TIME features were scored based on expression data and validated on serial whole-tissue sections of surgical samples, with tertiary lymphoid structures (TLSs) and the extra-TLS zone evaluated independently using multi-marker immunohistochemical staining. Risk analyses of TIME features on tumor behaviors were performed in GC-NED. The universal immunological effects of NED were explored preliminarily in adenocarcinomas arising in other organs. Results Based on over 11,500 annotated TLSs and 2,700 extra-TLS zones, compared with PGC, GC-NED harbored a distinctively more suppressive TIME characterized by increased but immature TLSs, with higher naïve B cell and follicular regulatory T cell densities and downregulated TLS maturation-related cell ratios inside TLSs; increased naïve B cell and regulatory T cell densities and a high proportion of exhausted T cells in the extra-TLS zone. The upregulated tumor PD-L1 expression and its close correlations with TLS formation and maturation were remarkable exclusively in GC-NED. TIME features, especially those regarding TLSs, were significantly correlated with tumor growth and invasion. The desynchrony between TLS formation and maturation and increased naïve or regulatory immune cell infiltration was observed in adenocarcinomas of the colorectum, pancreas, lung, and prostate. Conclusion NED highlighted a distinct GC entity with more suppressive TIME features correlated with tumor behaviors, indicating a cohort that would benefit more from immunotherapies

Supplementary Material for: Biofilm Formation Restrained by Subinhibitory Concentrations of Tigecyclin in Acinetobacter baumannii Is Associated with Downregulation of Efflux Pumps

<i>Background:</i> Tigecycline, one of the few therapeutic options against multidrug-resistant <i>Acinetobacter baumannii,</i> reaches subinhibitory serum concentrations only with cautious clinical dosing and pharmacokinetics. Subinhibitory concentrations of tigecycline might induce an <i>A. baumannii</i> biofilm. <i>Methods:</i> Biofilm formation was assessed via the crystal violet staining method. We further analyzed the main biofilm components with NaIO4, proteinase K, and DNase. Real-time RT-PCR was applied for quantitative detection of biofilm potential-associated genes. <i>Results:</i> In this study, <i>A. baumannii </i>proved to be a strong biofilm producer, and we found that proteins and extracellular DNA are crucial components of the <i>A. baumannii </i>biofilm. Quantitative real-time RT-PCR revealed positive correlations between biofilm formation restrained by subinhibitory concentrations of tigecycline and the expression of biofilm potential-associated genes, especially the <i>AdeFGH</i> efflux pump gene. <i>Conclusion:</i> Our results suggest that downregulation of efflux pumps, especially the <i>AdeFGH</i> efflux pump, is probably responsible for the decline in biofilm formation in <i>A. baumannii</i> treated with subinhibitory concentrations of tigecyclin

Supplementary Material for: Fibroblast Growth Factor 23 Predicts All-Cause Mortality in a Dose-Response Fashion in Pre-Dialysis Patients with Chronic Kidney Disease

<p><b><i>Background:</i></b> Quantitative dose-response associations between fibroblast growth factor 23 (FGF23) and risks of mortality, cardiovascular disease (CVD), and renal events in chronic kidney disease (CKD) are not known. This study aimed to summarize and quantify the predictive effects of FGF23 among the pre-dialysis CKD stages 1-5 population. <b><i>Methods:</i></b> Data sources included PubMed, EMBASE, and Web of Science. Prospective cohort studies assessing the associations between FGF23 and all-cause mortality, CVD, and renal events in CKD patients were selected. Summary risk ratios (RRs) and 95% confidence intervals (CIs) were calculated using the random-effects model. The composite higher or the highest level in FGF23 categories of each study was considered the high level. The reference level was regarded as the low level in the overall analysis. The restricted cubic spline model was used to estimate dose-response associations. <b><i>Results:</i></b> Fifteen prospective cohort studies centered around 15,355 subjects were analyzed. A high FGF23 level was associated with increased risks of all-cause mortality (RR 1.46, 95% CI 1.38-1.55, <i>p</i> < 0.001), CVD (RR 1.37, 95% CI 1.15-1.63, <i>p</i> < 0.001), and renal events (RR 1.31, 95% CI 1.07-1.59, <i>p </i>= 0.008), respectively. There was a positive, nonlinear, dose-response relationship between FGF23 and all-cause mortality. The reference level in dose-response analysis was defined as 51 RU/mL of c-terminal FGF23. We then calculated RRs for increments of 20 RU/mL, which was associated with increased risks of mortality (RR 1.04, 95% CI 1.00-1.07, <i>p</i> = 0.038), CVD (RR 1.02, <i>p</i> < 0.001), and renal events (RR 1.01, <i>p</i> < 0.001), respectively. <b><i>Conclusions:</i></b> There may be positive dose-response predictive effects of FGF23 on all-cause mortality, CVD, and renal events in patients with CKD.</p

PowerPoint Slides for: Fibroblast Growth Factor 23 Predicts All-Cause Mortality in a Dose-Response Fashion in Pre-Dialysis Patients with Chronic Kidney Disease

<p><b><i>Background:</i></b> Quantitative dose-response associations between fibroblast growth factor 23 (FGF23) and risks of mortality, cardiovascular disease (CVD), and renal events in chronic kidney disease (CKD) are not known. This study aimed to summarize and quantify the predictive effects of FGF23 among the pre-dialysis CKD stages 1-5 population. <b><i>Methods:</i></b> Data sources included PubMed, EMBASE, and Web of Science. Prospective cohort studies assessing the associations between FGF23 and all-cause mortality, CVD, and renal events in CKD patients were selected. Summary risk ratios (RRs) and 95% confidence intervals (CIs) were calculated using the random-effects model. The composite higher or the highest level in FGF23 categories of each study was considered the high level. The reference level was regarded as the low level in the overall analysis. The restricted cubic spline model was used to estimate dose-response associations. <b><i>Results:</i></b> Fifteen prospective cohort studies centered around 15,355 subjects were analyzed. A high FGF23 level was associated with increased risks of all-cause mortality (RR 1.46, 95% CI 1.38-1.55, <i>p</i> < 0.001), CVD (RR 1.37, 95% CI 1.15-1.63, <i>p</i> < 0.001), and renal events (RR 1.31, 95% CI 1.07-1.59, <i>p </i>= 0.008), respectively. There was a positive, nonlinear, dose-response relationship between FGF23 and all-cause mortality. The reference level in dose-response analysis was defined as 51 RU/mL of c-terminal FGF23. We then calculated RRs for increments of 20 RU/mL, which was associated with increased risks of mortality (RR 1.04, 95% CI 1.00-1.07, <i>p</i> = 0.038), CVD (RR 1.02, <i>p</i> < 0.001), and renal events (RR 1.01, <i>p</i> < 0.001), respectively. <b><i>Conclusions:</i></b> There may be positive dose-response predictive effects of FGF23 on all-cause mortality, CVD, and renal events in patients with CKD.</p

Supplementary Material for: Differentiating Glomerular Inflammation from Fibrosis in a Bone Marrow Chimera for Rat Anti-Glomerular Basement Membrane Glomerulonephritis

<b><i>Background:</i></b> Many types of glomerulonephritis (GN) undergo tandem connected phases: inflammation and fibrosis. Fibrosis in human GNs leads to irreversible end-stage disease. This study investigated how these 2 phases were controlled. <b><i>Methods:</i></b> Using a rat anti-glomerular basement membrane GN model, we established bone marrow (BM) chimeras between GN-resistant Lewis (LEW) and GN-susceptible Wistar Kyoto (WKY) rats. Glomerular inflammation and fibrosis were compared between chimeras. <b><i>Results:</i></b> LEW's BM to WKY chimeras with or without co-transfer of host WKY's T cells were GN-resistant. On the other hand, WKY's BM to LEW (LEW^WKY) chimeras developed glomerular inflammation and albuminuria upon immunization. Quantitative analysis showed that the number and composition of inflammatory cells in glomeruli of immunized LEW^WKY chimeras were similar to those in immunized WKY rats at their inflammatory peak. Thus, glomerular inflammation was controlled by BM-derived non-T cell populations. However, unlike WKY rats, LEW^WKY rats did not develop fibrosis until the end of experiments (84 days) in spite of persistent inflammation and albuminuria. <b><i>Conclusion:</i></b> Inflammation alone was not sufficient to trigger fibrosis, suggesting a critical role of glomerular cells in the fibrotic process. As LEW^WKY chimera allows us to separate glomerular inflammation from fibrosis, this model provides a useful tool to study how fibrosis is initiated following inflammation