Nicole M. A. Nagtzaam

Are Fecal Immunochemical Test Characteristics Influenced by Sample Return Time? A Population-Based Colorectal Cancer Screening Trial

OBJECTIVES:Fecal immunochemical tests (FIT) are preferred over guaiac-based fecal occult blood testing as colorectal cancer (CRC) screening tool. However, hemoglobin (Hb) degradation over time may influence FIT outcome. We therefore evaluated the effect of sample return time on FIT performance characteristics in a population-based CRC screening trial.METHODS:A representative random sample of the Dutch population (n=17,677), aged 50–74 years, was invited for FIT screening (OC-Sensor Micro; cutoff ≥ 50 ng Hb/ml). Sample return time was defined as the interval in days between fecal sampling and FIT laboratory delivery. Moreover, a random sample of positive FITs were selected to be stored at room temperature and re-tested every 3–4 days.RESULTS:In total, 8,958 screenees fulfilled our inclusion criteria. The mean sample return time was 3 days (± 3). Overall, 792 screenees (8.8%) had a positive test. Between the sample return time groups, the positivity rate (PR) varied between 7.7 and 9.0%. No statistically significant associations were found between PR or detection rate (DR) and the different sample return time groups (P value=0.84 and 0.76, respectively). For the laboratory experiment, 71 positive FITs were stored at room temperature and re-tested with standard intervals. The mean daily fecal Hb decrease was 5.88% per day (95% confidence interval 4.78–6.96%). None of the positive FITs became negative before 10 days after fecal sampling.CONCLUSIONS:This population-based CRC screening trial demonstrates that both the PR and DR of FITs do not decrease with prolonged sample return times up to 10 days. This means that a delay in sending the FIT back to the laboratory, of up to at least 1 week, does not necessitate repeat sampling in case of a negative test result. These data support the use of FIT-based screening as a reliable tool for nationwide CRC screening programs.

The use of clinical, histologic, and serologic parameters to predict the intragastric extent of intestinal metaplasia: a recommendation for routine practice.

BACKGROUND Surveillance of intestinal metaplasia (IM) of the gastric mucosa should be limited to patients at high risk of gastric cancer. Patients with extensive IM are at increased cancer risk; however, the intragastric extent of IM is usually unknown at the time of the initial diagnosis. OBJECTIVE To assess the predictive value of clinical, histologic, and serologic parameters for the intragastric extent of IM. DESIGN AND SETTING Prospective, multicenter study. PATIENTS Eighty-eight patients with a previous diagnosis of IM of the gastric mucosa. INTERVENTION Surveillance gastroscopy with extensive random biopsy sampling. MAIN OUTCOME MEASUREMENTS Biopsy specimens were evaluated according to the Sydney classification system. In addition, serologic testing of Helicobacter pylori and cagA status, pepsinogens I and II, gastrin, and intrinsic factor antibodies was performed. The association between the available parameters and extensive IM was evaluated with logistic regression analysis. RESULTS In 51 patients (58%), IM was present in the biopsy specimens from at least 2 intragastric locations. The most important predictors of extensive IM were a family history of gastric cancer, alcohol use > or = 1 unit/d (1 glass, approximately 10 mL or 8 g ethanol), moderate or marked IM of the index biopsy specimen, and a pepsinogen I to II ratio < 3.0. A simple risk score based on these factors could identify extensive IM in 24 of 25 patients (sensitivity 96%). LIMITATION A prospective cohort study should confirm the proposed risk stratification. CONCLUSIONS A risk score of clinical, histologic, and serologic parameters can predict extensive intragastric IM and may serve as a practical tool to select patients for surveillance endoscopy in routine clinical practice.

Dexamethasone transforms lipopolysaccharide-stimulated human blood myeloid dendritic cells into myeloid dendritic cells that prime interleukin-10 production in T cells

Myeloid dendritic cells (MDC) play an important role in antigen‐specific immunity and tolerance. In transplantation setting donor‐derived MDC are a promising tool to realize donor‐specific tolerance. Current protocols enable generation of tolerogenic donor MDC from human monocytes during 1‐week cultures. However, for clinical application in transplantation medicine, a rapidly available source of tolerogenic MDC is desired. In this study we investigated whether primary human blood MDC could be transformed into tolerogenic MDC using dexamethasone (dex) and lipopolysaccharide (LPS). Human blood MDC were cultured with dex and subsequently matured with LPS in the presence or absence of dex. Activation of MDC with LPS after pretreatment with dex did not prevent maturation into immunostimulatory MDC. In contrast, simultaneous treatment with dex and LPS yielded tolerogenic MDC, that had a reduced expression of CD86 and CD83, that poorly stimulated allogeneic T‐cell proliferation and production of T helper 1 (Th1) cytokines, and primed production of the immunoregulatory cytokine interleukin‐10 (IL‐10) in T cells. In vitro, however, these tolerogenic MDC did not induce permanent donor‐specific hyporesponsiveness in T cells. Importantly, tolerogenic MDC obtained by LPS stimulation in the presence of dex did not convert into immunostimulatory MDC after subsequent activation with different maturation stimuli. In conclusion, these findings demonstrate that combined treatment with dex and LPS transforms primary human blood MDC into tolerogenic MDC that are impaired to stimulate Th1 cytokines, but strongly prime the production of the immunoregulatory cytokine IL‐10 in T cells, and are resistant to maturation stimuli. This strategy enables rapid generation of tolerogenic donor‐derived MDC for immunotherapy in clinical transplantation.

Thrombin induces epithelial-mesenchymal transition and collagen production by retinal pigment epithelial cells via autocrine PDGF-receptor signaling.

PURPOSE De-differentiation of RPE cells into mesenchymal cells (epithelial-mesenchymal transition; EMT) and associated collagen production contributes to development of proliferative vitreoretinopathy (PVR). In patients with PVR, intraocular coagulation cascade activation occurs and may play an important initiating role. Therefore, we examined the effect of the coagulation proteins factor Xa and thrombin on EMT and collagen production by RPE cells. METHODS Retinal pigment epithelial cells were stimulated with factor Xa or thrombin and the effect on zonula occludens (ZO)-1, α-smooth muscle actin (α-SMA), collagen, and platelet-derived growth factor (PDGF)-B were determined by real-time quantitative-polymerase chain reaction (RQ-PCR), immunofluorescence microscopy, and HPLC and ELISA for collagen and PDGF-BB in culture supernatants, respectively. PDGF-receptor activation was determined by phosphorylation analysis and inhibition studies using the PDGF-receptor tyrosine kinase inhibitor AG1296. RESULTS Thrombin reduced ZO-1 gene expression (P < 0.05) and enhanced expression of the genes encoding α-SMA and the pro-alpha1 chain of collagen type-1 (P < 0.05), indicating EMT. Also, ZO-1 protein expression declined on thrombin stimulation, whereas production of α-SMA and collagen increased. In contrast to thrombin, factor Xa hardly stimulated EMT by RPE. Thrombin clearly induced PDGF-BB production and PDGF-Rβ chain phosphorylation in RPE. Moreover, AG1296 significantly blocked the effect of thrombin on EMT and collagen production. CONCLUSIONS Our findings demonstrate that thrombin is a potent inducer of EMT by RPE via autocrine activation of PDGF-receptor signaling. Coagulation cascade-induced EMT of RPE may thus contribute to the formation of fibrotic retinal membranes in PVR and should be considered as treatment target in PVR.

The Role of Thrombin in Proliferative Vitreoretinopathy

PURPOSE To determine the role of thrombin in the development of proliferative vitreoretinopathy (PVR). METHODS Vitreous was collected from patients undergoing a vitrectomy (macular holes and puckers, n = 11 [controls]; retinal detachment without PVR development following vitrectomy, n = 15 [RRD1]; retinal detachment with PVR development within 6 months after vitrectomy, n = 11 [RRD2]; and established PVR, n = 14 [PVR]). Thrombin activity in vitreous was determined using a thrombin-specific chromogenic substrate. ARPE-19 cells were stimulated with 8× diluted vitreous samples in the presence and absence of hirudin. The samples were analyzed at t = 0 and t = 24 hours for the presence of 27 cytokines/chemokines and growth factors using a multiplex approach. In comparable studies, ARPE-19 cells were stimulated for 2 hours, and mRNA expression levels for CCL2, CXCL8, GMCSF, IL6, and PDGFB were determined by real-time quantitative (RQ)-PCR. RESULTS Thrombin activity was significantly (P < 0.05) higher in vitreous of the PVR group compared to the other groups. Proliferative vitreoretinopathy vitreous stimulated the production of chemokine (C-C motif) ligand (CCL)2, chemokine (C-X-C motif) ligand (CXCL)8, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, and platelet-derived growth factor (PDGF)-BB by ARPE-19 to significantly (P < 0.05) higher levels than vitreous from the RRD1 and RRD2 groups. These effects of PVR vitreous were significantly (P < 0.05) reduced by hirudin. These data were confirmed by mRNA studies. CONCLUSIONS Thrombin activity is increased in vitreous of patients with established PVR and is involved in the activation of proinflammatory and profibrotic pathways in RPE cells. Inhibition of thrombin activity may therefore represent a potential treatment option for proliferative vitreoretinopathy.

Mild versus strong anti-inflammatory therapy during early sepsis in mice: a matter of life and death.

Objective: A recent literature-based study suggested that low-dose corticosteroid treatment has a beneficial effect on mortality in septic patients, whereas high-dose corticosteroid treatment has not. This suggests that mild down-regulation of the inflammatory response during early sepsis may be beneficial while extensive reduction of the inflammatory response is not. To investigate this hypothesis, we examined the effect of dexamethasone in varying doses on cecal ligation and puncture-induced inflammation and mortality. Design: Animal study. Setting: University research laboratory. Subjects: Male C57BL/6 mice. Interventions: Mice were subjected to cecal ligation and puncture, and dexamethasone was administered intravenously at a dosage of 0.05 (L/DEX), 0.25 (M/DEX), or 2.5 (H/DEX) mg/kg body weight 20 mins postoperatively. Mice receiving phosphate-buffered saline served as controls. Survival was recorded up to 21 days and inflammatory markers were determined in plasma, lungs, liver, and kidney at 6 hrs following cecal ligation and puncture as well as bacterial load in blood and peritoneal fluid. Measurements and Main Results: L/DEX treatment significantly improved survival compared with control mice, whereas treatment with higher concentrations of dexamethasone (M/DEX and H/DEX) did not. Treatment with either M/DEX or H/DEX was associated with significantly (p < .05) reduced cytokine plasma levels as compared with controls at 6 hrs after cecal ligation and puncture. In addition, M/DEX or H/DEX powerfully reduced cytokine messenger RNA expression in the lung, liver, and kidney. In contrast, treatment with L/DEX was associated with a mild, but nonsignificant, reduction of cytokine plasma levels. In addition, L/DEX moderately reduced cytokine messenger RNA expression in lung, liver, and kidney tissue and reduced the occurrence of bacteremia. Conclusions: A modest down-regulation of the early sepsis-associated inflammatory response improves survival in a murine cecal ligation and puncture model. We propose that the success of anti-inflammatory therapies in a septic setting fundamentally depends on finding a treatment balance that reduces the hyperinflammation-induced pathology but still allows adequate defense against pathogens.

Serum Levels of Leptin As Marker For Patients At High Risk of Gastric Cancer

Background:  Serological screening for gastric cancer (GC) may reduce mortality. However, optimal serum markers for advanced gastric precursor lesions are lacking.

Encapsulating peritoneal sclerosis is associated with T-cell activation

BACKGROUND Encapsulating peritoneal sclerosis (EPS) is an excessive fibrotic response of the peritoneum that may occur after long-term peritoneal dialysis (PD). The underlying pathophysiology is poorly understood, but involvement of peritoneal inflammatory T helper 1 cells may be pivotal. METHODS Soluble interleukin-2 receptor alpha (sCD25) concentration was measured as a marker for T-cell activation in serum and ascites from EPS patients and various control patient groups. Peritoneal biopsies were stained for the presence of T cells, and T cells isolated from ascites of EPS patients were characterized in detail for differentiation status and cytokine expression. RESULTS Serum sCD25 concentrations are significantly and specifically increased in EPS patients compared with haemodialysis, PD and predialysis patients. Peritoneal effluent of stable PD patients contains very low levels of sCD25, while sCD25 levels in ascites of EPS patients are high and indicative of local production. In the years preceding the diagnosis of EPS, the serum sCD25 concentrations increased while remaining at stable levels in control PD patients. The peritoneum and ascites of EPS patients showed a significant influx of T cells with relatively increased numbers of CD4(+) T cells. These T cells were fully differentiated and displayed a T helper 1 cell type with a pro-inflammatory cytokine profile. CONCLUSIONS Increased serum sCD25 concentrations and peritoneal lymphocytosis in EPS patients indicate the involvement of activated T cells in the pathophysiology of excessive fibrosis.

Factor Xa and thrombin induce epithelial-mesenchymal transition by retinal pigment epithelial cells via PDGF-Rβ signaling

Proliferative vitreoretinopathy (PVR) is an inflammatory fibrotic disorder of the retina. Retinal pigment epithelial (RPE) cells contribute to PVR development through uncontrolled proliferation and extracellular matrix production as well as cytokine secretion. Insight into factors that stimulate these processes by RPE in PVR is limited, which may explain the lack of satisfying treatment so far. Blood-retinal barrier breakdown and vascular damage are early pathobiological events of PVR. Vascular damage results in activation of the coagulation system and subretinal fluids from retinal detachment patients have been described to contain high pro-coagulant activity. The effect of coagulation proteases on RPE is however hardly studied so far. Of all proteins involved in the coagulation cascade only factor Xa and thrombin are able to induce various cellular responses via their receptors which are expressed at the cell membranes of various cell types. These receptors, the so-called protease activated receptors (PARs) consist of 4 members: PAR1, -2, -3 and -4. Cleavage of PARs results in the activation of signal transduction pathways that control multiple cellular functions like inflammation, fibrosis and tissue repair. Here we examine the effect of factor Xa and thrombin on epithelial-mesenchymal transition (EMT) by RPE. EMT is a biological process that allows epithelial cells to undergo multiple biochemical changes that enable them to assume a mesenchymal phenotype. Similar processes have also been described for RPE cells in PVR membranes. Differentiation from an epithelial phenotype into a mesenchymal phenotype results in the loss of epithelial markers like zona occludens (ZO)-1 and the enrichment of mesenchymal markers like α-smooth muscle actin (α-SMA). Another component of PVR development as a result of EMT consists of excessive production of extracellular matrix (ECM) proteins; mainly collagen subtype-I. In this study we show that factor Xa and thrombin are able to upregulate the expression of pro-fibrotic mediators like PDGF-A, -B, TGF-α and TIMP-1 by RPE cells and induce fibrotic responses known to be involved in the development of PVR. In line with these findings we also show that mainly thrombin but also factor Xa, can induce EMT by RPE via PDGF-B mediated PDGF-Rβ signaling.