Immunological profiling in long COVID: overall low grade inflammation and T-lymphocyte senescence and increased monocyte activation correlating with increasing fatigue severity.

Background: Many patients with SARS-CoV-2 infection develop long COVID with fatigue as one of the most disabling symptoms. We performed clinical and immune profiling of fatigued and non-fatigued long COVID patients and age- and sex-matched healthy controls (HCs). Methods: Long COVID symptoms were assessed using patient-reported outcome measures, including the fatigue assessment scale (FAS, scores ≥22 denote fatigue), and followed up to one year after hospital discharge. We assessed inflammation-related genes in circulating monocytes, serum levels of inflammation-regulating cytokines, and leukocyte and lymphocyte subsets, including major monocyte subsets and senescent T-lymphocytes, at 3-6 months post-discharge. Results: We included 37 fatigued and 36 non-fatigued long COVID patients and 42 HCs. Fatigued long COVID patients represented a more severe clinical profile than non-fatigued patients, with many concurrent symptoms (median 9 [IQR 5.0-10.0] vs 3 [1.0-5.0] symptoms, p<0.001), and signs of cognitive failure (41%) and depression (>24%). Immune abnormalities that were found in the entire group of long COVID patients were low grade inflammation (increased inflammatory gene expression in monocytes, increased serum pro-inflammatory cytokines) and signs of T-lymphocyte senescence (increased exhausted CD8+ TEMRA-lymphocytes). Immune profiles did not significantly differ between fatigued and non-fatigued long COVID groups. However, the severity of fatigue (total FAS score) significantly correlated with increases of intermediate and non-classical monocytes, upregulated gene levels of CCL2, CCL7, and SERPINB2 in monocytes, increases in serum Galectin-9, and higher CD8+ T-lymphocyte counts. Conclusion: Long COVID with fatigue is associated with many concurrent and persistent symptoms lasting up to one year after hospitalization. Increased fatigue severity associated with stronger signs of monocyte activation in long COVID patients and potentially point in the direction of monocyte-endothelial interaction. These abnormalities were present against a background of immune abnormalities common to the entire group of long COVID patients.

Altered Functionality of Lipoprotein(a) Impacts on Angiogenesis in Diabetic Retinopathy.

Purpose: Diabetic retinopathy (DR) is a complication of type 2 diabetes mellitus (T2DM). Lipoprotein(a) (Lp(a)) contributes to the progression of DR, but how is unclear. In homeostasis of the retinal microvasculature, myeloid-derived pro-angiogenic cells (PACs) also play a pivotal role, and fail to function properly in diabetic conditions. Here, we explored the putative contribution of Lp(a) from patients with T2DM with/without DR and healthy controls on inflammation and angiogenesis of retinal endothelial cells (RECs), and on PAC differentiation. Subsequently, we compared the lipid composition of Lp(a) from patients to that from healthy controls. Methods: Lp(a)/LDL obtained from patients and healthy controls were added to TNF-alpha-activated RECs. Expression of VCAM-1/ICAM-1 was measured using flowcytometry. Angiogenesis was determined in REC-pericyte co-cultures stimulated by pro-angiogenic growth factors. PAC differentiation from peripheral blood mononuclear cells was determined by measuring expression of PAC markers. The lipoprotein lipid composition was quantified using detailed lipidomics analysis. Results: Lp(a) from patients with DR (DR-Lp(a)) failed to block TNF-alpha-induced expression of VCAM-1/ICAM-1 in REC whereas Lp(a) from healthy controls (healthy control [HC]-Lp(a)) did. DR-Lp(a) increased REC angiogenesis more than HC-Lp(a) did. Lp(a) from patients without DR showed intermediate profiles. HC-Lp(a) reduced the expression of CD16 and CD105 in PAC, but T2DM-Lp(a) did not. Phosphatidylethanolamine content was lower in T2DM-Lp(a) than in HC-Lp(a). Conclusions: DR-Lp(a) does not show the anti-inflammatory capacity seen with HC-Lp(a), but increases REC angiogenesis, and affects PAC differentiation less than HC-Lp(a). These functional differences in Lp(a) in T2DM-related retinopathy are associated with alterations in the lipid composition as compared to healthy conditions.

Monocyte adaptations in patients with obesity during a 1.5 year lifestyle intervention.

Background: Obesity is associated with chronic, low-grade inflammation, which is reflected in altered peripheral blood monocyte characteristics. The aim of this study was to analyze the monocyte subset composition (classical (CM), intermediate (IM) and non-classical monocytes (NCM)), and their inflammatory marker profile (CD14, CD16, CD36, CD45, CD64, CD300e, HLA-DR) in individuals with obesity during a 1.5 year combined lifestyle intervention (CLI), comprising healthy nutrition, increased exercise and behavioral changes. Methods: We analyzed monocyte subset counts and immunophenotypes in 73 individuals with obesity, and associated these to baseline body mass index (BMI) and waist circumference (WC). The measurements were repeated after 10 weeks and at the end of the intervention (1.5 years). Results: Generally, monocyte subset counts were not associated to BMI or WC at baseline, neither did monocyte counts change during the 1.5 year CLI. Immunophenotypically, higher baseline BMI and WC were associated to lower CD14 and higher CD300e expression by all subsets. During CLI there were remarkable changes in marker profiles: expression of CD14, CD36, CD45 and CD64 significantly decreased in CM and IM, as did CD16 (IM and NCM) (p<0.05). CD300e initially decreased after 10 weeks, but increased sharply at 1.5 years (all subsets). We observed no consistent associations between changes in monocyte characteristics and anthropometric changes. Conclusion: A 1.5 year CLI in individuals with obesity mediates persistent immunophenotypic adaptations related to cellular activation in blood monocytes, whereas changes in subset distribution are limited. Lifestyle-induced changes in the inflammatory profile of monocytes differ from the 'less-severe-obesity'-phenotype, suggesting a novel, 'post-weight-loss' monocyte setpoint.

Monocyte response after colorectal surgery: A prospective cohort study.

Background: Tumor resection is the common approach in patients with colorectal malignancy. Profound insight into inflammatory changes that accompany the normal post-operative stress response will establish reference parameters useful for identification of putative complications. Alterations in circulating monocytes might be indicative as these cells are considered to be the most responsive leukocytes to trauma. Therefore, the aim of this study is to assess the monocyte subset kinetic and phenotypic changes in response to surgery. Methods: Fifty patients undergoing colorectal tumor resection were included in a multicenter prospective cohort study. Blood samples were collected early in the morning prior to surgery and the next days through postoperative day three for flowcytometric analysis. Leukocyte subtypes were identified and expression of activation stage-related markers by monocyte subsets was quantified. Results: Changes in leukocyte subset composition and monocyte subset phenotypes were most prominent at the first day postoperatively, after which these parameters typically returned to normal or near-normal preoperative values. The immunophenotypic alterations after surgery were most notable in classical and intermediate monocytes. These included up-regulation of activation markers CD64 and CD62L, but down-regulation of HLA-DR and CD54. Markers of de-activation, CD163 and CD206, were consistently increasingly expressed. Discussion/conclusion: The current study gives detailed insight into the peripheral blood leukocyte response after colorectal cancer surgery. This form of short-term stress induces a rapid and significant redistribution of immune cells. Immunophenotypic alterations in monocytes as a response to surgery suggest a mixed profile of cellular activation and de-activation.

Minimal Development of Liver Fibrosis in Adult Tolerant Liver Transplant Recipients Late After Immunosuppressive Drug Weaning and Transplantation.

BACKGROUND: Operationally tolerant liver transplant (LTx)-recipients can be weaned off immunosuppressive (IS) drugs without development of graft rejection. However, it is feared that liver fibrosis might develop after complete IS weaning. The purpose of this small single-center study was to assess liver fibrosis in adult tolerant LTx recipients long after LTx and IS weaning. METHODS: Liver fibrosis was assessed in adult tolerant LTx-recipients (n = 9) using noninvasive transient elastography and measurements of multiple pro- and antifibrotic serum markers associated with liver fibrosis. The data was collected for 2 subsequent years; 8 and 9 years after IS weaning and 19 and 20 years after transplantation. Healthy individuals (n = 9) matched for age and sex were included as a reference for fibrosis-related serum markers. This study was conducted in accordance with the Declaration of Helsinki and approved by the medical ethics committee of our institution. RESULTS: Transient elastography indicated that 7 of 9 tolerant LTx recipients had no or minimal liver fibrosis (F0-F1), whereas 2 recipients had moderate or severe liver fibrosis (F2-F3). Most fibrosis-related serum markers in tolerant LTx recipients were within or close to the range obtained for healthy individuals. CONCLUSIONS: The results from this small, single-center study indicated that most adult tolerant LTx recipients have no or minimal liver graft fibrosis long after transplantation and IS weaning, and their fibrosis-related serum marker profile indicates an absence of a profibrotic status.

Soluble guanylate cyclase activator BAY 54-6544 improves vasomotor function and survival in an accelerated ageing mouse model.

DNA damage is a causative factor in ageing of the vasculature and other organs. One of the most important vascular ageing features is reduced nitric oxide (NO)soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) signaling. We hypothesized that the restoration of NO-sGC-cGMP signaling with an sGC activator (BAY 54-6544) may have beneficial effects on vascular ageing and premature death in DNA repair-defective mice undergoing accelerated ageing. Eight weeks of treatment with a non-pressor dosage of BAY 54-6544 restored the decreased in vivo microvascular cutaneous perfusion in progeroid Ercc1∆/- mice to the level of wild-type mice. In addition, BAY 54-6544 increased survival of Ercc1∆/- mice. In isolated Ercc1∆/- aorta, the decreased endothelium-independent vasodilation was restored after chronic BAY 54-6544 treatment. Senescence markers p16 and p21, and markers of inflammation, including Ccl2, Il6 in aorta and liver, and circulating IL-6 and TNF-α were increased in Ercc1∆/- , which was lowered by the treatment. Expression of antioxidant genes, including Cyb5r3 and Nqo1, was favorably changed by chronic BAY 54-6544 treatment. In summary, BAY 54-6544 treatment improved the vascular function and survival rates in mice with accelerated ageing, which may have implication in prolonging health span in progeria and normal ageing.

Vitreous proteomics, a gateway to improved understanding and stratification of diverse uveitis aetiologies.

PURPOSE: The vitreous proteome might provide an attractive gateway to discriminate between various uveitis aetiologies and gain novel insights into the underlying pathophysiological processes. Here, we investigated 180 vitreous proteins to discover novel biomarkers and broaden disease insights by comparing (1). primary vitreoretinal lymphoma ((P)VRL) versus other aetiologies, (2). sarcoid uveitis versus tuberculosis (TB)-associated uveitis and (3). granulomatous (sarcoid and TB) uveitis versus other aetiologies. METHODS: Vitreous protein levels were determined by proximity extension assay in 47 patients with intraocular inflammation and a prestudy diagnosis (cohort 1; training) and 22 patients with a blinded diagnosis (cohort 2; validation). Differentially expressed proteins identified by t-tests on cohort 1 were used to calculate Youden's indices. Pathway and network analysis was performed by ingenuity pathway analysis. A random forest classifier was trained to predict the diagnosis of blinded patients. RESULTS: For (P)VRL stratification, the previously reported combined diagnostic value of IL-10 and IL-6 was confirmed. Additionally, CD70 was identified as potential novel marker for (P)VRL. However, the classifier trained on the entire cohort (cohort 1 and 2) relied primarily on the interleukin score for intraocular lymphoma diagnosis (ISOLD) or IL-10/IL-6 ratio and only showed a supportive role for CD70. Furthermore, sarcoid uveitis displayed increased levels of vitreous CCL17 as compared to TB-associated uveitis. CONCLUSION: We underline the previously reported value of the ISOLD and the IL-10/IL-6 ratio for (P)VRL identification and present CD70 as a potentially valuable target for (P)VRL stratification. Finally, we also show that increased CCL17 levels might help to distinguish sarcoid uveitis from TB-associated uveitis.

Association of monocyte HLA-DR expression over time with secondary infection in critically ill children: a prospective observational study.

An impaired immune response could play a role in the acquisition of secondary infections in critically ill children. Human leukocyte antigen-DR expression on monocytes (mHLA-DR) has been proposed as marker to detect immunosuppression, but its potential to predict secondary infections in critically ill children is unclear. We aimed to assess the association between mHLA-DR expression at several timepoints and the change of mHLA-DR expression over time with the acquisition of secondary infections in critically ill children. In this prospective observational study, children < 18 years with fever and/or suspected infection (community-acquired or hospital-acquired) were included at a paediatric intensive care unit in the Netherlands. mHLA-DR expression was determined by flow cytometry on day 1, day 2-3 and day 4-7. The association between delta-mHLA-DR expression (difference between last and first measurement) and secondary infection was assessed by multivariable regression analysis, adjusted for age and Paediatric Logistic Organ Dysfunction-2 score. We included 104 patients at the PICU (median age 1.2 years [IQR 0.3-4.2]), of whom 28 patients (27%) developed a secondary infection. Compared to 93 healthy controls, mHLA-DR expression of critically ill children was significantly lower at all timepoints. mHLA-DR expression did not differ at any of the time points between patients with and without secondary infection. In addition, delta-mHLA-DR expression was not associated with secondary infection (aOR 1.00 [95% CI 0.96-1.04]).Conclusions: Our results confirm that infectious critically ill children have significantly lower mHLA-DR expression than controls. mHLA-DR expression was not associated with the acquisition of secondary infections. What is Known: • An impaired immune response, estimated by mHLA-DR expression, could play an essential role in the acquisition of secondary infections in critically ill children. • In critically ill children, large studies on the association of mHLA-DR expression with secondary infections are scarce. What is New: • Our study confirms that critically ill children have lower mHLA-DR expression than healthy controls. • mHLA-DR expression and change in mHLA-DR was not associated with the acquisition of secondary infection.

Leukocyte toll-like receptor expression in pathergy positive and negative Behçet's disease patients.

OBJECTIVES: To investigate whether the auto-inflammatory nature and the pathergic reaction in Behçet's disease (BD) are driven by a disturbed toll-like receptor (TLR) response. METHODS: We compared both TLR expression by flow-cytometry and TLR response by stimulation assay in 18 BD patients (both pathergy positive and pathergy negative) with 15 healthy controls. RESULTS: Expression of TLR1 and 2 was significantly elevated in B-lymphocytes of BD patients compared with healthy controls. TLR1, 2 and 4 were significantly more highly expressed in both CD4+ and CD8+ T-lymphocytes of BD patients. Granulocytes of BD patients displayed significantly higher expression of TLR1, 2, 4 and 6. TLR2, 4 and 5 expression was significantly increased on classical monocytes of BD patients. Intermediate monocytes of BD patients showed an increase in expression of TLR2. Furthermore, TLR2 and 5 were significantly more highly expressed in non-classical monocytes of BD patients. In pathergy positive patients, TLR5 was even more highly expressed compared with pathergy negative patients on B- and T-lymphocytes and granulocytes. Furthermore, TLR2 and 5 showed an elevated TNF-α response to stimulation with their cognate ligands. CONCLUSION: Immune cells of BD patients overexpress TLR1, 2, 4, 5 and 6. Furthermore, after stimulation of TLR2 and 5, BD patients demonstrate a more potent TNF-α response. Although this is a small cohort, in the pathergy positive patients, TLR5 expression is even further augmented, suggesting that a microbial (flagellin) or damage (HMGB1) associated signal may trigger the exaggerated immune response that is characteristic for the pathergy phenomenon in BD. In conclusion, these results point to an exaggerated TLR response in the auto-inflammatory nature of BD.

HLA-DR Expression on Monocyte Subsets in Critically Ill Children.

BACKGROUND: To longitudinally study blood monocyte subset distribution and human leukocyte antigen-DR (HLA-DR) expression on monocyte subsets in children with sepsis, post-surgery and trauma in relation to nosocomial infections and mortality. METHODS: In 37 healthy children and 37 critically ill children (12 sepsis, 11 post-surgery, 10 trauma and 4 admitted for other reasons)-participating in a randomized controlled trial on early versus late initiation of parenteral nutrition-monocyte subset distribution and HLA-DR expression on monocyte subsets were measured by flow cytometry upon admission and on days 2, 3 and 4 of pediatric intensive care unit (PICU) stay. RESULTS: Upon PICU admission, critically ill children had a higher proportion of classical monocytes (CD14++CD16-) than healthy children [PICU 95% (interquartile range [IQR] 88%-98%); controls, 87% (IQR 85%-90%), P < 0.001]. HLA-DR expression was significantly decreased within all monocyte subsets and at all time points, being most manifest on classical monocytes and in patients with sepsis. Percentage of HLA-DR expressing classical monocytes [upon PICU admission 67% (IQR 44%-88%); controls 95% (IQR 92%-98%), P < 0.001], as well as the HLA-DR mean fluorescence intensity [upon PICU admission 3219 (IQR 2650-4211); controls 6545 (IQR 5558-7647), P < 0.001], decreased during PICU stay. Patients who developed nosocomial infections (n = 13) or who died (n = 6) had lower HLA-DR expression on classical monocytes at day 2 (P = 0.002) and day 3 (P = 0.04), respectively. CONCLUSIONS: Monocytic HLA-DR expression decreased during PICU stay and was lower compared with controls on all examined time points, especially on classical monocytes and in children admitted for sepsis. Low HLA-DR expression on classical monocytes was associated with nosocomial infections and death.

A Novel Heterozygous Mutation in the STAT1 SH2 Domain Causes Chronic Mucocutaneous Candidiasis, Atypically Diverse Infections, Autoimmunity, and Impaired Cytokine Regulation.

Chronic mucocutaneous candidiasis (CMC) is a primary immunodeficiency characterized by persistent or recurrent skin and mucosal surface infections with Candida species. Different gene mutations leading to CMC have been identified. These include various heterozygous gain-of-function (GOF) mutations in signal transducer and activator of transcription 1 (STAT1) that are not only associated with infections but also with autoimmune manifestations. Recently, two STAT1 GOF mutations involving the Src homology 2 (SH2) domain have been reported, while so far, over 50 mutations have been described mainly in the coiled coil and the DNA-binding domains. Here, we present two members of a Dutch family with a novel STAT1 mutation located in the SH2 domain. T lymphocytes of these patients revealed STAT1 hyperphosphorylation and higher expression of STAT1 target genes. The clinical picture of CMC in our patients could be explained by diminished production of interleukin (IL)-17 and IL-22, cytokines important in the protection against fungal infections.

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.

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.

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-Rß 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.

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.

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 hyper-inflammation-induced pathology but still allows adequate defense against pathogens.