Soluble CD14 and Osteoprotegerin Associate with Ankle-Brachial Index as a Measure of Arterial Stiffness in Patients with Mild-to-Moderate Chronic Kidney Disease in a Five-Year Prospective Study.

INTRODUCTION: Vascular lesions and arterial stiffness appear at early stages of chronic kidney disease (CKD) and follow an accelerated course with disease progression, contributing to high cardiovascular mortality. There are limited prospective data on mechanisms contributing to progression of arterial stiffness in mild-to-moderate CKD (stages 2-3). METHODS: We applied an affinity proteomics approach to identify candidates of circulating biomarkers with potential impact on vascular lesions in CKD and selected soluble cluster of differentiation 14 (sCD14), angiogenin (ANG), and osteoprotegerin (OPG) for further analysis. We studied their association with ankle-brachial index (ABI) and carotid intima-media thickness, as measures of arteriosclerosis and atherosclerosis, respectively, in 48 patients with CKD stages 2-3, who were prospectively followed and intensively treated for 5 years, and 44 healthy controls. RESULTS: Concentrations of sCD14 (p < 0.001), ANG (p < 0.001), and OPG (p < 0.05) were higher in patients with CKD 2-3 at baseline, and sCD14 (p < 0.001) and ANG (p < 0.001) remained elevated in CKD patients at follow-up. There were positive correlations between ABI and sCD14 levels (r = 0.36, p = 0.01) and between ABI and OPG (r = 0.31, p = 0.03) at 5 years. The changes in sCD14 during follow-up correlated to changes in ABI from baseline to 5 years (r = 0.41, p = 0.004). CONCLUSION: Elevated levels of circulating sCD14 and OPG in patients with CKD 2-3 were significantly associated with ABI, a measure of arterial stiffness. An increase in sCD14 over time in CKD 2-3 patients was associated with a corresponding increase in ABI. Further studies are needed to examine if early intensive multifactorial medication to align with international treatment targets may influence cardiovascular outcomes.

Soluble PD-L1 Expression After Intravenous Treatment of Cancer Patients With Selenite in Phase I Clinical Trial.

A high expression level of programmed death-ligand 1 (PD-L1) is observed in different types of cancers (particularly lung cancer). Soluble (s)PD-L1 may be used as a prognostic marker and a target for anti-cancer immunity, as well as, predicting gene therapy or systemic immunotherapy in blocking the PD-1 and PD-L1 checkpoint. Studies that evaluate the effects of the immune regulator selenium on PD-L1 expression show ambiguous results. Thus, we aimed to analyze sPD-L1 levels in samples from patients who underwent different dosages of selenite treatment in phase I clinical trial. We hypothesized that selenite modulates the sPD-L1 levels in the plasma as a consequence of the suggested mode of action of selenotherapy in cancer patients. In conclusion, our results support the view that selenotherapy does not substantially affect the PD-1/PD-L1 axis judged by sPD-L1 analysis. Furthermore, no significant correlation was observed between the survival and sPD-L1 expression nor sPD-L1 changes. However, due to a dynamic individual sPD-L1 profile and a high variation in survival, we suggest that further studies are needed to identify whether individual patients can be benefited from combinational seleno- and anti-PD-L1 therapy.

Impact of monocyte-related modulators and kidney function on mortality in hospitalized patients with COVID-19.

Patients with chronic kidney disease (CKD) are at high risk of severe complications from COVID-19 and functional monocyte disturbances have been implicated to play a role. Our objective was to analyse the association between kidney function and monocyte modulatory factors, with risk of mortality in patients with COVID-19. Hospitalized patients with COVID-19 (n = 110) were included and in-hospital mortality was analysed with unadjusted and adjusted multiple logistic regression analysis. Plasma levels of monocyte chemoattractant factors (MIP-1α, MCP-1, IL-6) and a monocyte immune modulator (sCD14) were analysed and correlated to kidney function and risk of mortality. Monocyte modulatory factors were also determined in CKD patients without infection (disease controls) and in healthy subjects. Patients who died in hospital were more often in CKD stages 3-5, with lower estimated glomerular filtration rate (eGFR) and had significantly higher MIP-1α and IL-6 levels than survivors. In multiple regression analyses adjusted for age, sex and eGFR, both high MCP-1 and high MIP-1α were significantly associated with risk of in-hospital mortality. Apart from impaired kidney function, also the concentrations of MCP-1 and MIP-1α add important prognostic information in hospitalized patients with COVID-19. These data provide an increased understanding of the impact of monocyte modulators in patients with COVID-19 and normal or impaired kidney function, and warrant consideration in the pursuit of new effective therapies.

Adhesion molecule cross-linking and cytokine exposure modulate IgE- and non-IgE-dependent basophil activation.

Basophils are known for their role in allergic inflammation, which makes them suitable targets in allergy diagnostics such as the basophil activation test (BAT) and the microfluidic immunoaffinity basophil activation test (miBAT). Beside their role in allergy, basophils have an immune modulatory role in both innate immunity and adaptive immunity. To accomplish this mission, basophils depend on the capability to migrate from blood to extravascular tissues, which includes interactions with endothelial cells, extracellular matrix and soluble mediators. Their receptor repertoire is well known, but less is known how these receptor-ligand interactions impact the degranulation process and the responsiveness to subsequent activation. As the consequences of these interactions are crucial to fully appreciate the role of basophils in immune modulation and to enable optimization of the miBAT, we explored how basophil activation status is regulated by cytokines and cross-linking of adhesion molecules. The expression of adhesion molecules and activation markers on basophils from healthy blood donors was analysed by flow cytometry. Cross-linking of CD203c, CD62L, CD11b and CD49d induced a significant upregulation of CD63 and CD203c. To mimic in vivo conditions, valid also for miBAT, CD62L and CD49d were cross-linked followed by IgE-dependent activation (anti-IgE), which caused a reduced CD63 expression compared with anti-IgE activation only. IL-3 and IL-33 priming caused increased CD63 expression after IgE-independent activation (fMLP). Together, our data suggest that mechanisms operational both in the microfluidic chip and in vivo during basophil adhesion may impact basophil anaphylactic and piecemeal degranulation procedures and hence their immune regulatory function.

Associations of Fibroblast Growth Factor 23 with Markers of Inflammation and Leukocyte Transmigration in Chronic Kidney Disease.

BACKGROUND: Patients with chronic kidney disease (CKD) show elevated levels of inflammatory markers and have an increased risk of infections as well as cardiovascular morbidity. Recent studies have implied effects of fibroblast growth factor 23 (FGF23) on inflammation in CKD. We analyzed potential correlations between levels of FGF23 with pro-inflammatory chemokines and markers of leukocyte transmigration in CKD patients. METHODS: One hundred three patients with CKD 2-5ND and 54 healthy controls, had biochemical markers in blood and urine analyzed according to routine protocol. Pro-inflammatory cytokines were analyzed by Milliplex technique and leukocyte CD11b adhesion molecule expression was measured by flow cytometry. FGF23 levels were measured with ELISA technique. Treatment of leukocytes from healthy blood donors with FGF23 was performed in vitro and effects analyzed by flow cytometry. RESULTS: Tumor necrosis factor-alpha, RANTES and interleukin (IL)-12 levels were significantly higher (p = 0.001, p < 0.001, and p < 0.001) in patients with CKD. Elevated FGF23 levels in the CKD group correlated to glomerular filtration rate, parathyroid hormone, urinary albumin excretion and phosphate as well as to IL-12 and RANTES. CD11b expression on resting granulocytes and monocytes, and on activated monocytes, was associated with FGF23. In vitro treatment of leukocytes with FGF23 reduced CD11b expression in resting as well as in formyl-methyinoyl-leucyl-phenylalanine-stimulated granulocytes (p = 0.03). CONCLUSION: FGF23 levels are associated with various inflammatory markers such as pro-inflammatory cytokines and adhesion molecules on innate immune cells. However, further studies are warranted to define the direct role of FGF23 in modulation of the innate immune system in CKD.

Early Changes in Monocyte Adhesion Molecule Expression and Tumor Necrosis Factor-α Levels in Chronic Kidney Disease - A 5-Year Prospective Study.

BACKGROUND: Despite the absence of clinical symptoms, patients with chronic kidney disease (CKD) exhibit elevated levels of pro-inflammatory markers. To investigate whether it is possible to detect inflammatory activity and altered monocyte function at an early stage of renal disease, we studied patients with CKD stages 2-3 over 5 years. METHODS: The expression of adhesion molecules on monocytes at resting state and after stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP), as well as oxidative metabolism capacity was measured with flow cytometry in 108 CKD patients and healthy controls. Soluble markers of inflammation, such as cytokines, were analyzed using the Milliplex technique. RESULTS: Patients showed significantly lower CD11b expression after stimulation during the 3rd (p = 0.002) and the 5th year (p < 0.001), together with a lower oxidative burst in response to fMLP over time (p = 0.02). The expression of CD62L on resting monocytes was lower during the 3rd (p = 0.001) and the 5th (p = 0.001) year in patients. Levels of tumor necrosis factor-α and RANTES were significantly increased (p = 0.001, p = 0.006) and interleukin-12 levels were also higher in CKD patients during the 5th year (p = 0.007). CONCLUSION: Monocytes in CKD stages 2-3 show emerging functional abrasions, with altered adhesion molecule expression and impaired fMLP response. These findings suggest that a transformation of monocyte function occurs at an early phase of renal impairment and may together with increased plasma levels of pro-inflammatory cytokines contribute to the higher vulnerability of CKD patients to comorbidities, such as infections and cardiovascular disease.

Activation of basophils is a new and sensitive marker of biocompatibility in hemodialysis.

The hemodialysis procedure involves contact between peripheral blood and the surface of dialyzer membranes, which may lead to alterations in the pathways of innate and adaptive immunity. We aimed to study the effect of blood-membrane interaction on human peripheral basophils and neutrophils in hemodialysis with high- and low-permeability polysulfone dialyzers. The surface expression of CD203c (basophil selection marker) and CD63 (activation marker) after activation by the bacterial peptide formyl-methionyl-leucyl-phenylalanine (fMLP) or anti-Fcε receptor I (FcεRI) antibody and the absolute number of basophils was investigated before and after hemodialysis with each of the dialyzers. Moreover, the expression on neutrophils of CD11b, the CD11b active epitope, and CD88 was analyzed in the same groups of individuals. The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared with that in healthy controls, but no differences were observed after activation by anti-FcεRI. During the hemodialysis procedure, the low-flux membrane induced up-regulation of CD63 expression on basophils, while passage through the high-flux membrane did not significantly alter the responsiveness. In addition, the absolute number of basophils was unchanged after hemodialysis with either of the dialyzers and compared with healthy controls. We found no significant differences in the expression of the neutrophil activation markers (CD11b, the active epitope of CD11b, and CD88) comparing the two different dialyzers before and after dialysis and healthy controls. Together, these findings suggest that alterations in basophil activity may be a useful marker of membrane bioincompatibility in hemodialysis.

The tyrosine kinase receptor ROR1 is constitutively phosphorylated in chronic lymphocytic leukemia (CLL) cells.

Phosphorylation of receptor tyrosine kinases (RTKs) has a key role in cellular functions contributing to the malignant phenotype of tumor cells. We and others have previously demonstrated that RTK ROR1 is overexpressed in chronic lymphocytic leukemia (CLL). Silencing siRNA downregulated ROR1 and induced apoptosis of CLL cells. In the present study we analysed ROR1 isoforms and the phosphorylation pattern in CLL cells (n=38) applying western blot and flow-cytometry using anti-ROR1 antibodies and an anti-phospho-ROR1 antibody against the TK domain. Two major ROR1 bands with the size of 105 and 130 kDa respectively were identified, presumably representing unglycosylated (immature) and glycosylated (mature) ROR1 respectively as well as a 260 kDa band which may represent dimerized ROR1. A ROR1 band of 64 kDa that may correspond to a C-terminal fragment was also noted, present only in the nucleus. The 105 kDa ROR1 isoform was more frequently expressed in non-progressive as compared to progressive CLL patients (p=0.03). The 64, 105, 130 and 260 kDa bands were constitutively phosphorylated both at tyrosine and serine residues. Phosphorylation intensity of the mature (130 kDa) isoform was significantly higher in progressive than in non-progressive disease (p<0.001). Incubation of CLL cells with a mouse anti-ROR1 KNG or an anti-ROR1 CRD mAb respectively induced dephosphorylation of ROR1 before entering apoptosis. In conclusion CLL cells expressed different isoforms of ROR1 which were constitutively phosphorylated. The mature, phosphorylated ROR1 isoform was associated with a progressive disease stage. Targeting ROR1 by mAbs induced specific dephosphorylation and leukemic cell death. ROR1 might be an interesting therapeutic target.