Sequential engagement of adhesion molecules and cytokine receptors impacts both piecemeal and anaphylactic degranulation of human basophils.

Basophils are rare granulocytes in circulation which home to tissues in a process depending on rolling, adhesion and cytokine exposure. However, it is still unclear how these steps affect basophil degranulation. Our aim was to imitate these processes associated with homing by sequential crosslinking of adhesion molecules and cytokine exposure and evaluate the effect on basophil piecemeal (PMD) and anaphylactic degranulation (AND). Blood donors with or without allergic asthma were recruited from an ongoing cohort study. Basophils were subjected to CD62L-, CD49d- or CD11b crosslinking and IL-3 or IL-33 stimulation in different orders followed by anti-IgE and fMLP stimulation. Basophil CD203c and CD63 expression were analysed by flow cytometry to determine PMD and AND, respectively. IL-3 induced PMD in basophils and combined with CD62L- or CD11b crosslinking, IL-3 potentiated the degranulation regardless of sequential order. IL-3 priming followed by adhesion molecule crosslinking induced AND and potentiated the effect of anti-IgE. CD62L- and CD11b crosslinking did not further potentiate this effect. CD49d crosslinking followed by IL-3 increased CD63 expression following anti-IgE. IL-3 potentiated the effect of fMLP on AND while adhesion molecule crosslinking did not. IL-33 had impact on PMD only when followed by adhesion molecule crosslinking but did not potentiate neither IgE-dependent nor IgE-independent degranulation. Our data indicate that sequential interactions between basophils, cytokines and adhesion molecule ligands have a decisive effect on basophil degranulation and that these interactions are operational for fine-tuning the activity of tissue dwelling basophils. These data should be considered when the effect of different pharmaceutical on basophil function is studied.

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.

"Role of kidney function and concentrations of BAFF, sPD-L1 and sCD25 on mortality in hospitalized patients with COVID-19".

BACKGROUND: Chronic kidney disease (CKD) is a recognized risk factor for severe complications in COVID-19. Our objective was to analyze the association between kidney function / T and B lymphocyte modulatory factors and risk of mortality in COVID-19 patients. METHODS: In-hospital and 30-day mortality were analyzed in COVID-19 patients (n = 110). Plasma levels of selected T and B cell modulators were analyzed and correlated to mortality risk. A subgroup of sex- and eGFR-matched COVID-19 patients was compared to CKD patients without infection and healthy subjects. RESULTS: COVID-19 patients who died in hospital and within 30 days had significantly higher BAFF and sCD25 plasma levels than survivors. In logistic regression models patients with high BAFF, sCD25 and sPD-L1 levels had significantly higher risk of both in-hospital and 30-day mortality while there was no association to eGFR. In the subgroup analysis, a higher level of BAFF, IFN-α, sCD25, sPD-L1 and a lower level of sCD40L was observed in COVID-19 patients compared to the CKD group with corresponding kidney function. CONCLUSIONS: We demonstrate that kidney function and concentrations of BAFF, sCD25 and PD-L1, independent of previously recognized risk factors; age, male gender, and leukocytosis are associated with risk of in-hospital and 30-day mortality in patients with COVID-19. These data indicate the significance of adaptive immune system modulators in COVID-19 and motivate further analysis to identify new potential prognostic and therapeutic approaches.

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.

B cell and monocyte phenotyping: A quick asset to investigate the immune status in patients with IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) advances from multiple pathogenic "hits" resulting in poorly O-galactosylated IgA1 glycoforms (Gd-IgA1), production of antibodies and glomerular deposition of immune complexes. A sequence of immune responses arising from plasma cells, T cells and antigen presenting cells (APCs), causes glomerular injury. This study was designed to phenotype subsets of B cells, monocytes and T cells in the peripheral circulation and their association with inflammatory cytokines and kidney function in patients with IgAN, healthy controls (HC) and disease controls with autosomal dominant polycystic kidney disease (ADPKD). METHODS: Patients with IgAN (n = 13), median estimated glomerular filtration rate (eGFR) of 57 ml/min/1.73m2 (IQR 42-84), patients with ADPKD (n = 13) matched for kidney function, gender and age and gender and age-matched HC (n = 13) were recruited. CD3+ and CD3- peripheral blood mononuclear cells were isolated and profiled based on their specific surface markers for different subsets of monocytes, B and T cells and analyzed by flow cytometry. Cytokines were analyzed by ELISA. RESULTS: We observed a significant decrease in the proportion of pre-switched B cells and plasmablasts, but an increase in long-lived plasma cells in the peripheral circulation of IgAN patients compared to HC. The proportion of non-classical monocytes was significantly higher in IgAN patients compared to both HC and ADPKD. We also report an association between sCD40L levels and the proportion of pre-switched B cells, as well as sCD40L and MCP-1 levels and albuminuria in IgAN patients. CONCLUSIONS: We applied an easy-access method to analyze subsets of immune cells as well as relevant inflammatory mediators in IgAN patients. Our data demonstrate an altered B cell profile that indicates a pathophysiological role of the B cell lineage and an increased proportion of non-classical monocytes that suggests their role in the disease process.

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.

Vitamin D receptor activation reduces inflammatory cytokines and plasma MicroRNAs in moderate chronic kidney disease - a randomized trial.

BACKGROUND: Chronic kidney disease (CKD) is a major risk factor for cardiovascular disease (CVD), partly due to endothelial dysfunction and chronic inflammation. Vitamin D treatment in end stage renal disease is suggested to modulate the immune system and lead to improved outcomes. We and others have demonstrated that treatment with vitamin D or activated vitamin D analogues protects the endothelial function in less severe renal disease as well. Since the endothelial protection might be mediated by vitamin D effects on inflammation, we assessed levels of pro-inflammatory cytokines and micro RNAs (miRs) in patients with moderate CKD, treated with an active vitamin D analogue (paricalcitol). METHODS: Thirty-six patients with moderate CKD were randomized to 12 weeks treatment with placebo, 1 µg, or 2 µg paricalcitol daily. Cytokines were measured by Milliplex 26-plex. Total RNA was isolated from plasma and miRs were determined by quantitative reverse transcription PCR analysis. RESULTS: Selected pro-inflammatory cytokines decreased significantly following treatment, while no change was observed in the placebo group. The micro RNAs; miR 432-5p, miR 495-3p, and miR 576-5p were significantly downregulated in the active treated groups, compared to the placebo group. CONCLUSION: Paricalcitol treatment for 12 weeks in patients with moderate CKD reduces cytokines and micro RNAs involved in atherosclerosis and inflammation. The potentially protective role of vitamin D receptor activation in the inflammatory processes regarding the long-term outcomes in CKD patients warrants further studies. TRIAL REGISTRATION: SOLID study; NCT01204528 , April 27, 2010.

Hemodialysis Patients Display a Declined Proportion of Th2 and Regulatory T Cells in Parallel with a High Interferon-γ Profile.

BACKGROUND: A high prevalence of cardiovascular diseases (CVDs) and infections in patients with chronic kidney disease (CKD) arises partly due to a high inflammatory state and aberrations in immune cells function. Following in vitro stimulation of leukocytes with different T-cell mitogens, we observed a lower level of interleukin (IL)-2 and IL-10 production in CKD patients. To gain more knowledge as to whether this is the result of an alteration in T-cell function, we investigated the T-cell subsets profile and cytokine production in hemodialysis patients. METHODS: CD4+ cells were isolated from whole blood of 10 hemodialysis patients and 10 age- and gender-matched healthy controls. Following in vitro stimulation with an antigen-independent T-cell mitogen, Th1, Th2, and regulatory T (Treg) cell subsets were analyzed by flow cytometry through the expression of specific transcription factors. The levels of cytokines, interferon (IFN)-γ, IL-4, and IL-10 were analyzed by enzyme-linked immunosorbent assay in the supernatants. RESULTS: The proportion of CD4+CD25+FOXP3+ (Treg) and CD4+GATA3+ (Th2) cells was significantly lower in patients compared to healthy controls, while the proportion of CD4+T-bet+ (Th1) cells was similar. Moreover, levels of IL-4 were significantly lower in supernatants from patients, while IFN-γ levels were higher. IL-10 levels did not differ compared to those of the healthy controls. CONCLUSIONS: Our findings indicate a diminished anti-inflammatory Treg, and Th2 cell profile in hemodialysis patients, accompanied by a high pro-inflammatory IFN-γ profile. Since this profile is characterized in CVDs, we propose that an imbalance between the inflammatory and anti-inflammatory responses may contribute to the pathogenesis of CVD in advanced 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.

Leukocyte proliferation and immune modulator production in patients with chronic kidney disease.

INTRODUCTION: In Chronic Kidney Disease (CKD), immune cells are affected by uremic retention toxins. Given this effect, we analyzed lymphocyte proliferative response and immune modulators production following in vitro stimulation. METHODS: Whole blood was drawn from healthy controls, patients with eGFR <20 ml/min/1.73 m(2) (Pre-dialysis, CKD stages 4 and 5) and hemodialysis patients (stage 5D). Peripheral cells were incubated for six days with pokeweed mitogen, concanavalin A, Staphylococcus enterotoxin A or influenza A vaccine. Peripheral lymphocyte proliferation was then analyzed by the "Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood" (FASCIA) method, and cytokine profile in the cell supernatants was analyzed by the Milliplex multi-array method. RESULTS: The absolute number of lymphoblasts in response to mitogenic stimulation and the number of cells in each CD4+ and CD8+ subpopulation were similar comparing the three groups, except for a single decline in number of lymphoblasts after stimulation with Staphylococcus enterotoxin A, comparing dialysis patients with healthy controls. Levels of interleukin (IL)-2 (p=0.026), -10 (p=0.019) and -15 (p=0.027) in the Staphylococcus enterotoxin A-stimulated supernatant were lower in hemodialysis patients compared to healthy controls. Levels of IL-15 (p=0.017) from pre-dialysis patients and levels of IL-5 (p=0.019) from hemodialysis patients in influenza A vaccine-stimulated supernatants were also lower compared to controls. In pokeweed mitogen-stimulated supernatant, IL-2 levels (p=0.013) were lower in hemodialysis patients compared to pre-dialysis patients. TNF-α, IL-10, IL-12, IL-15, IL-8, MCP-1, IP-10, IFN-α2, IL-1α and eotaxin levels were all significantly higher in plasma obtained from CKD patients. CONCLUSION: Our results suggest that T-cells from CKD patients have similar proliferative response to stimulation compared with healthy individuals. Moreover, however the immune cells show inability to produce selected cytokines, most likely due to the uremic milieu or dialysis procedure.

Adaptive and Pathogenic Responses to Stress by Stem Cells during Development.

Cellular stress is the basis of a dose-dependent continuum of responses leading to adaptive health or pathogenesis. For all cells, stress leads to reduction in macromolecular synthesis by shared pathways and tissue and stress-specific homeostatic mechanisms. For stem cells during embryonic, fetal, and placental development, higher exposures of stress lead to decreased anabolism, macromolecular synthesis and cell proliferation. Coupled with diminished stem cell proliferation is a stress-induced differentiation which generates minimal necessary function by producing more differentiated product/cell. This compensatory differentiation is accompanied by a second strategy to insure organismal survival as multipotent and pluripotent stem cells differentiate into the lineages in their repertoire. During stressed differentiation, the first lineage in the repertoire is increased and later lineages are suppressed, thus prioritized differentiation occurs. Compensatory and prioritized differentiation is regulated by at least two types of stress enzymes. AMP-activated protein kinase (AMPK) which mediates loss of nuclear potency factors and stress-activated protein kinase (SAPK) that does not. SAPK mediates an increase in the first essential lineage and decreases in later lineages in placental stem cells. The clinical significance of compensatory and prioritized differentiation is that stem cell pools are depleted and imbalanced differentiation leads to gestational diseases and long term postnatal pathologies.