Vitamin C, Hydrocortisone, and the Combination Thereof Significantly Inhibited Two of Nine Inflammatory Markers Induced by Escherichia Coli But Not by Staphylococcus Aureus - When Incubated in Human Whole Blood.

ABSTRACT: Vitamin C combined with hydrocortisone is increasingly being used to treat septic patients, even though this treatment regimen is based on questionable evidence. When used, a marked effect on key players of innate immunity would be expected, as sepsis is featured by a dysregulated immune response.Here, we explored the effect of vitamin C and hydrocortisone alone and combined, in an ex vivo human whole-blood model of Escherichia coli- or Staphylococcus aureus-induced inflammation. Inflammatory markers for activation of complement (terminal C5b-9 complement complex [TCC]), granulocytes (myeloperoxidase), platelets (ß-thromboglobulin), cytokines (tumor necrosis factor [TNF], IL-1ß, IL6, and IL-8), and leukocytes (CD11b and oxidative burst) were quantified, by enzyme-linked immunosorbent assay, multiplex technology, and flow cytometry.In E. coli- and S. aureus-stimulated whole blood, a broad dose-titration of vitamin C and hydrocortisone alone did not lead to dose-response effects for the central innate immune mediators TCC and IL-6. Hence, the clinically relevant doses were used further. Compared to the untreated control sample, two of the nine biomarkers induced by E. coli were reduced by hydrocortisone and/or vitamin C. TNF was reduced by hydrocortisone alone (19%, P = 0.01) and by the combination (31%, P = 0.01). The oxidative burst of monocytes and granulocytes was reduced for both drugs alone and their combination, (ranging 8-19%, P < 0.05). Using S. aureus, neither of the drugs, alone nor in combination, had any effects on the nine biomarkers.In conclusion, despite the limitation of the ex vivo model, the effect of vitamin C and hydrocortisone on bacteria-induced inflammatory response in human whole blood is limited and following the clinical data.

Body Composition, Inflammation, and 5-Year Outcomes in Colon Cancer.

Importance: Obesity, particularly visceral obesity and sarcopenia, are poor prognostic indicators in colon cancer. Objectives: To explore the association between body composition profiles and 5-year colon cancer outcomes and delineate the associated underlying inflammatory processes. Design, Setting, and Participants: This multicenter translational cohort study included patients with nonmetastatic colon cancer who did not have underlying chronic inflammatory disorders and were not receiving anti-inflammatory drugs referred to tertiary cancer centers from 2009 to 2015. Preoperative acute phase proteins (white cell count, C-reactive protein, and albumin), cytokines (interleukin [IL]-1b, IL-2, IL-6, IL-10, interferon γ, and tumor necrosis factor α), vascular endothelial growth factor (VEGF), and cell surface receptor expression levels (CD11b and CD14) were measured. All patients underwent follow-up for at least 5 years. Data were analyzed in December 2020. Exposure: Nonmetastatic colon cancer. Main Outcomes and Measures: The associations of body composition profiles with 5-year cancer recurrence and disease-specific mortality were analyzed using Mantel Cox log-rank test and Kaplan-Meier curves. Results: A total of 28 patients were included (median [interquartile range] age, 67 [58-72] years; 22 [78.6%] men). Low skeletal muscle area (SMA) and high visceral to total fat ratio were associated with poor clinical and oncological outcomes, including increased 5-year recurrence (low SMA: hazard ratio [HR], 2.30 [95% CI, 1.41-2.89]; P = .04; high visceral to total fat ratio: HR, 5.78 [95% CI, 3.66-7.95]; P = .02). High visceral to total fat ratio was associated with increased 5-year disease-specific mortality (HR, 5.92 [95% CI, 4.04-8.00]; P = .02). Patients with low SMA who developed a cancer recurrence, compared with those who did not, had higher C-reactive protein (mean [SD], 31.24 [6.95] mg/dL vs 8.11 [0.58] mg/dL; P = .003), IL-6 (mean [SD], 1.93 [1.16] ng/mL vs 0.88 [0.14] ng/mL; P = .004), VEGF (mean [SD], 310.03 [122.66] ng/mL vs 176.12 [22.94] ng/mL; P = .007), and CD14 (mean [SD], 521.23 [302.02] ng/mL vs 322.07 [98.35] ng/mL; P = .03) expression and lower albumin (mean [SD], 3.8 [0.6] g/dL vs 43.50 [3.69] g/dL; P = .01), IL-2 (mean [SD], 0.45 [0.25] ng/mL vs 0.94 [0.43] ng/mL; P < .001), IL-10 (mean [SD], 8.15 [1.09] ng/mL vs 16.32 [4.43] ng/mL; P = .004), and interferon γ (mean [SD], 2.61 [1.36] ng/mL vs 14.87 [3.43] ng/mL; P = .02) levels. Patients with high visceral to total fat ratio who developed recurrence had higher levels of IL-6 (mean [SD], 5.26 [7.05] ng/mL vs 2.76 [3.11] ng/mL; P = .03) and tumor necrosis factor α (mean [SD], 5.74 [4.53] ng/mL vs 4.50 [1.99] ng/mL; P = .03). Conclusions and Relevance: These findings suggest that low SMA and high visceral to total fat ratio were associated with worse colon cancer outcomes and with increased expression of proinflammatory cytokines and VEGF and inhibition of anti-inflammatory cytokines.

Report of a new six-panel flow cytometry marker for early differential diagnosis of APL from HLA-DR negative Non-APL leukemia.

Although AML-M3 (APL) and HLA-DR negative non-APL are characterized by negative HLA-DR antigen, they are different entities with similar morphology in some cases. The aim of this study is the precise, differential diagnosis of APL from HLA-DR negative non-APL by flow cytometry to narrow the diagnosis window. Bone marrow or blood samples of 580 AML patients were analyzed, and flow cytometry and molecular analysis were performed for the diagnosis of blood disorders. In 105 HLA-DR negative AML patients, expression of HLA-DR, CD33, CD117, CD11b, CD64, CD34, CD9 and myeloperoxidase staining pattern were evaluated. Fifty-six patients were diagnosed with APL, and 49 patients were diagnosed with HLA-DR negative non-APL. The APL blasts expressed CD33, CD117, CD64, and CD9 in 100%, 80.3%, 94.6%, and 100% of the cases, respectively. HLA-DR negative non-APL blasts expressed CD33, CD117, CD64 and CD9 in 75.5%, 59.1%, 32.6%, and 73.4% of the cases, respectively. APL cells were negative for HLA-DR, CD11b, and CD34 in 96.4%, 94.6%, and 91.0%, respectively. Blasts in HLA-DR negative non M3-AML were negative for CD11b, CD117, and CD34 in 77.5%, 40.9%, and 22.4%, respectively. We also investigated myeloperoxidase (MPO) staining pattern and found strong diffuse reaction in APL cells while HLA-DR negative non-APL cells showed focal positive reaction. In all of the APL patients, except for one, PML/RARA translocation was positive, and in another case with HLA-DR negative non-APL, PML/RARA and other translocations were not detected. The six-panel combination profile rapidly and specifically identifies APL from other HLA-DR negative AML.

Automated flow cytometry enables high performance point-of-care analysis of leukocyte phenotypes.

INTRODUCTION: Phagocytes such as granulocytes and monocytes are fundamental players in the innate immune system. Activation of these cells can be quantified by the measurement of activation marker expression using flow cytometry. Analysis of receptor expression on inflammatory cells facilitates the diagnosis of inflammatory diseases and can be used to determine the extent of inflammation. However, several major limitations of this analysis precludes application of inflammation monitoring in clinical practice. Fast and automated analysis would minimalize ex vivo manipulation and allow reproducible processing. The aim of this study was to evaluate a fully automated "load & go" flow cytometer for analyzing activation of granulocytes and monocytes in a clinically applicable setting. METHODS: Blood samples were obtained from 10 anonymous and healthy volunteers between the age of 18 and 65 years. Granulocyte and monocyte activation was determined by the use of the markers CD35, CD11b and CD10 measured in the automated AQUIOS CL® "load & go" flow cytometer. This machine is able to pierce the tube caps, add antibodies, lyse and measure the sample within 20 min after vena puncture. Reproducibility tests were performed to test the stability of activation marker expression on phagocytes. The expression of activation markers was measured at different time points after blood drawing to analyze the effect of bench time on granulocyte and monocyte activation. RESULTS: The duplicate experiments demonstrate a high reproducibility of the measurements of the activation state of phagocytes. Healthy controls showed a very homogenous expression of activation markers at T = 0 (immediately after vena puncture). Activation markers on neutrophils were already significantly increased after 1 h (T = 1) depicted as means (95%Cl) CD35: 2.2× (1.5×-2.5×) p = .028, CD11b: 2.5× (1.7×-3.1×) p = .023, CD10: 2.5× (2.1×-2.7×) p = .009) and a further increase in activation markers was observed after 2 and 3 h. Monocytes also showed a increase in activation markers in 1 h (mean (95%Cl) CD35: 1.8× (1.3×-2.2×) p = .058, CD11b: 2.13× (1.6×-2.4×) p = .025) and also a further significant increase in 2 and 3 h was observed. CONCLUSION: This study showed that bench time of one hour already leads to a significant upregulation and bigger variance in activation markers of granulocytes and monocytes. In addition, it is likely that automated flow cytometry reduces intra-assay variability in the analysis of activation markers on inflammatory cells. Therefore, we found that it is of utmost importance to perform immune activation analysis as fast as possible to prevent drawing wrong conclusions. Automated flow cytometry is able to reduce this analysis from 2 h to only 15-20 min without the need of dedicated personnel and in a point-of-care context. This now allows fast and automated inflammation monitoring in blood samples obtained from a variety of patient groups. FUND: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Cyclophosphamide treatment for hypertension and renal injury in an experimental model of systemic lupus erythematosus.

Cardiovascular disease is the major cause of mortality among patients with the autoimmune disorder systemic lupus erythematosus (SLE). Our laboratory previously reported that immunosuppression with mycophenolate mofetil, a common therapy in patients with SLE, attenuates the development of hypertension in an experimental model of SLE. Cyclophosphamide (CYC) is another common therapy for patients with SLE that has contributed to improved disease management; however, its impact on the development of hypertension associated with SLE is not clear. We tested whether treatment with CYC (25 mg/kg, once/week, IP injection) for 4 weeks would attenuate hypertension in an established female mouse model of SLE with hypertension (30-week-old NZBWF1 females). Plasma anti-dsDNA IgG levels, pathogenic for the disease, were lower in CYC-treated SLE mice compared to vehicle-treated SLE mice, suggesting efficacy of the therapy to suppress aberrant immune system function. Mean arterial pressure (MAP) was assessed by carotid artery catheters in conscious mice. Treatment did not attenuate the development of hypertension when compared to vehicle-treated SLE mice; however, urinary albumin excretion was lower in CYC-treated animals. Corresponding with the reduction in autoantibodies, data suggest that CYC treatment lowered circulating CD45R+ B cells. Paradoxically, circulating CD11b+ Ly6G+ neutrophils were increased in CYC-treated SLE mice compared to vehicle treated. Estrus cycling data also suggest that CYC treatment had an impact on ovarian function that may be consistent with reduced circulating estrogen levels. Taken together, these data suggest that CYC treatment attenuates autoantibody production and renal disease during SLE, but that the potential to affect MAP may be blunted by the increase in circulating neutrophils and CYC's impact on ovarian function.

Microcirculatory consequences of limb ischemia/reperfusion in ovariectomized rats treated with zoledronic acid.

BACKGROUND: Nitrogen-containing bisphosphonates (BIS) are potent therapeutics in osteoporosis, but their use may result in osteonecrotic side-effects in the maxillofacial region. Periosteal microcirculatory reactions may contribute to the development of bone-healing complications, particularly in osteoporotic bones, where ischemia-reperfusion (IR) events often develop during orthopaedic/trauma interventions. The effect of BIS on the inflammatory reactions of appendicular long bones has not yet been evaluated; thus, we aimed to examine the influence of chronic zoledronate (ZOL) administration on the periosteal microcirculatory consequences of hindlimb IR in osteopenic rats. MATERIALS AND METHODS: Twelve-week-old female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated, and ZOL (80 µg/kg iv, weekly) or a vehicle was administered for 8 weeks, 4 weeks after the operation. At the end of the pre-treatment protocols, 60-min limb ischemia was induced, followed by 180-min reperfusion. Leukocyte-endothelial interactions were quantitated in tibial periosteal postcapillary venules by intravital fluorescence videomicroscopy. CD11b expression of circulating polymorphonuclear leukocytes (PMN, flow cytometry) and plasma TNF-alpha levels (ELISA) were also determined. Two-way RM ANOVA followed by the Holm-Sidak and Dunn tests was used to assess differences within and between groups, respectively. RESULTS: Limb IR induced significant increases in PMN rolling and firm adhesion in sham-operated and OVX rats, which were exacerbated temporarily in the first 60 min of reperfusion by a ZOL treatment regimen. Postischemic TNF-alpha values showed a similar level of postischemic elevations in all groups, whereas CD11b expression only increased in rats not treated with ZOL. CONCLUSIONS: The present data do not show substantial postischemic periosteal microcirculatory complications after chronic ZOL treatment either in sham-operated or OVX rats. The unaltered extent of limb IR-induced local periosteal microcirculatory reactions in the presence of reduced CD11b adhesion molecule expression on circulating PMNs, however, may be attributable to local endothelial injury/activation caused by ZOL.

The CD9+ CD11b- HLA-DR- immunophenotype can be used to diagnose acute promyelocytic leukemia.

OBJECTIVE: To investigate the immunophenotypic characteristics of acute promyelocytic leukemia (APL) and explore the sensitivity and specificity of various antibody combinations for the timely and accurate diagnosis APL. METHODS: A retrospective analysis was performed using morphological, immunological, genetic, and molecular biological data from 92 patients diagnosed with APL and 190 controls diagnosed with non-APL acute myeloid leukemia. RESULTS: For APL diagnosis, the CD9/CD11b/human leukocyte antigen (HLA)-DR antibody combination had 85% sensitivity and 95% specificity, AUC = 0.85. However, the sensitivity and specificity were 39% and 92%, AUC = 0.65, respectively, for the HLA-DR/CD34/CD117 combination, and 80% and 80%, AUC = 0.80, respectively for the CD11b/HLA-DR combination. Significant differences were observed between the different antibody combinations. CONCLUSIONS: The CD9/CD11b/HLA-DR antibody combination displays high sensitivity and specificity and can be used to diagnose APL.

Resistance Exercise Selectively Mobilizes Monocyte Subsets: Role of Polyphenols.

PURPOSE: To examine the impact of polyphenol supplementation on the recruitment, mobilization, and activation of monocyte subsets after resistance exercise. METHODS: Thirty-eight recreationally active males (22.1 ± 3.1 yr; 173.9 ± 7.9 cm; 77.8 ± 14.5 kg) were assigned to 28 d of polyphenol blend (PPB) supplementation, placebo (PL), or control (CON). Blood samples were obtained before (PRE) postresistance exercise, immediately (IP) postresistance exercise, 1 h (1H) postresistance exercise, 5 h (5H) postresistance exercise, 24 h (24H) postresistance exercise, and 48 h (48H) postresistance exercise (PPB/PL) or rest (CON). Fine-needle biopsies were obtained from the vastus lateralis at PRE, 1H, 5H, and 48H. Circulating concentrations of macrophage chemoattractant protein-1 (MCP-1) and fractalkine, as well as intramuscular MCP-1 were analyzed via multiplex assay. Changes in the proportions and expression of CD11b on monocyte subsets were assessed via flow cytometry. RESULTS: Circulating MCP-1 increased in PPB and PL at IP with further increases at 5H. Intramuscular MCP-1 was increased at 1H, 5H, and 48H in all groups. Classical monocyte proportions were reduced in PPB and PL at IP, and increased at 1H. Nonclassical monocytes were increased in PPB and PL at IP, whereas intermediate monocytes were increased at IP, and reduced at 1H. Intermediate monocytes were increased in PPB at 24H and 48H. CD11b expression was reduced on PPB compared with PL and CON at PRE on intermediate and nonclassical monocytes. CONCLUSIONS: Resistance exercise may elicit selective mobilization of intermediate monocytes at 24H and 48H, which may be mediated by tissue damage. Additionally, polyphenol supplementation may suppress CD11b expression on monocyte subsets at rest.

Nbeal2 Deficiency Increases Organ Damage but Does Not Affect Host Defense During Gram-Negative Pneumonia-Derived Sepsis.

Objective- Nbeal2-/- mice, a model of human gray platelet syndrome, have reduced neutrophil granularity and impaired host defense against systemic Staphylococcus aureus infection. We here aimed to study the role of Nbeal2 deficiency in both leukocytes and platelets during gram-negative pneumonia and sepsis. Approach and Results- We studied the role of Nbeal2 in platelets and leukocytes during murine pneumonia and sepsis by Klebsiella pneumoniae. Apart from platelet α-granule deficiency and reduced neutrophil granularity, also monocyte granularity was reduced in Nbeal2-/- mice, whereas plasma levels of MPO (myeloperoxidase), elastase, NGAL (neutrophil gelatinase-associated lipocalin), and MMP-9 (matrix metalloproteinase 9), and leukocyte CD11b expression were increased. Nbeal2-/- leukocytes showed unaltered in vitro antibacterial response and phagocytosis capacity against Klebsiella, and unchanged reactive nitrogen species and cytokine production. Also during Klebsiella pneumonia and sepsis, Nbeal2-/- mice had similar bacterial growth in lung and distant body sites, with enhanced leukocyte migration to the bronchoalveolar space. Despite similar infection-induced inflammation, organ damage was increased in Nbeal2-/- mice, which was also seen during endotoxemia. Platelet-specific Nbeal2 deficiency did not influence leukocyte functions, indicating that Nbeal2 directly modifies leukocytes. Transfusion of Nbeal2-/- but not of Nbeal2+/+ platelets into thrombocytopenic mice was associated with bleeding in the lung but similar host defense, pointing at a role for platelet α-granules in maintaining vascular integrity but not host defense during Klebsiella pneumosepsis. Conclusions- These data show that Nbeal2 deficiency-resulting in gray platelet syndrome-affects platelets, neutrophils, and monocytes, with intact host defense but increased organ damage during gram-negative pneumosepsis.

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.

Cre/lox Studies Identify Resident Macrophages as the Major Source of Circulating Coagulation Factor XIII-A.

OBJECTIVE: To establish the cellular source of plasma factor (F)XIII-A. APPROACH AND RESULTS: A novel mouse floxed for the F13a1 gene, FXIII-Aflox/flox (Flox), was crossed with myeloid- and platelet-cre-expressing mice, and cellular FXIII-A mRNA expression and plasma and platelet FXIII-A levels were measured. The platelet factor 4-cre.Flox cross abolished platelet FXIII-A and reduced plasma FXIII-A to 23±3% (P<0.001). However, the effect of platelet factor 4-cre on plasma FXIII-A was exerted outside of the megakaryocyte lineage because plasma FXIII-A was not reduced in the Mpl-/- mouse, despite marked thrombocytopenia. In support of this, platelet factor 4-cre depleted FXIII-A mRNA in brain, aorta, and heart of floxed mice, where FXIII-Apos cells were identified as macrophages as they costained with CD163. In the integrin αM-cre.Flox and the double copy lysozyme 2-cre.cre.Flox crosses, plasma FXIII-A was reduced to, respectively, 75±5% (P=0.003) and 30±7% (P<0.001), with no change in FXIII-A content per platelet, further consistent with a macrophage origin of plasma FXIII-A. The change in plasma FXIII-A levels across the various mouse genotypes mirrored the change in FXIII-A mRNA expression in aorta. Bone marrow transplantation of FXIII-A+/+ bone marrow into FXIII-A-/- mice both restored plasma FXIII-A to normal levels and replaced aortic and cardiac FXIII-A mRNA, while its transplantation into FXIII-A+/+ mice did not increase plasma FXIII-A levels, suggesting that a limited population of niches exists that support FXIII-A-releasing cells. CONCLUSIONS: This work suggests that resident macrophages maintain plasma FXIII-A and exclude the platelet lineage as a major contributor.

Angiopoietin-1 promotes atherosclerosis by increasing the proportion of circulating Gr1+ monocytes.

AIMS: Atherosclerosis is a chronic inflammatory disease occurring within the artery wall. A crucial step in atherogenesis is the infiltration and retention of monocytes into the subendothelial space of large arteries induced by chemokines and growth factors. Angiopoietin-1 (Ang-1) regulates angiogenesis and reduces vascular permeability and has also been reported to promote monocyte migration in vitro. We investigated the role of Ang-1 in atherosclerosis-prone apolipoprotein-E (Apo-E) knockout mouse. METHODS AND RESULTS: Apo-E knockout (Apo-E-/-) mice fed a western or normal chow diet received a single iv injection of adenovirus encoding Ang-1 or control vector. Adenovirus-mediated systemic expression of Ang-1 induced a significant increase in early atherosclerotic lesion size and monocyte/macrophage accumulation compared with control animals receiving empty vector. Ang-1 significantly increased plasma MCP-1 and VEGF levels as measured by ELISA. FACS analysis showed that Ang-1 selectively increased inflammatory Gr1+ monocytes in the circulation, while the cell-surface expression of CD11b, which mediates monocyte emigration, was significantly reduced. CONCLUSIONS: Ang-1 specifically increases circulating Gr1+ inflammatory monocytes and increases monocyte/macrophage retention in atherosclerotic plaques, thereby contributing to development of atherosclerosis.

Increased Activity and Apoptosis of Eosinophils in Blister Fluids, Skin and Peripheral Blood of Patients with Bullous Pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering skin disease that is more common in elderly individuals. The aim of this study was to determine the functional activity of eosinophils in patients with BP compared with healthy donors. Blood, skin and blister-derived eosinophils were strongly activated in patients with BP, seen by increased surface expression of CD69 compared with controls. CD11b was also increased in BP blood eosinophils, which may explain the striking accumulation of eosinophils in BP (1×106 per ml blister fluid). Furthermore, CCL26 was expressed by activated eosinophils in BP skin and in blister fluid. BP eosinophils also released IL-6, IL-8 and IL-1α in BP blister fluids. Apoptosis in cultivated BP eosinophils was increased and accompanied by enhanced surface externalization of CD95. Caspase 3 positive eosinophils in lesional BP skin and blister fluid also showed the initiation of apoptosis. These results reveal novel pathophysiological aspects of BP, with a strong activation pattern and increased apoptosis of eosinophils in the peripheral blood, skin and blister fluids.

Downregulation of Blood Monocyte HLA-DR in ICU Patients Is Also Present in Bone Marrow Cells.

BACKGROUND: The downregulation of blood monocyte HLA-DR expression also occurs in tissue infiltrative cells in a context of acute clinical inflammation, especially sepsis. This context favors the development of secondary infections and results from various mechanisms. Little is known about HLA-DR expression on bone marrow (BM) cells of the monocyte lineage, the source of circulating monocytes. This study analyzed the BM HLA-DR expression in ICU patients compared to BM monocytes from non-ICU patients and to blood monocytes of control healthy donors. A potential dysfunction of myeloid differentiation was investigated in a sub-population of these ICU patients to characterize the phenotype of the immature forms of monocytes and granulocytes in BM. METHODS AND FINDINGS: BM and blood were drawn from 33 ICU and 9 non-ICU patients having a BM analysis to precise the etiology of abnormal low count in blood cells. The data were compared with blood cells of 28 control donors. Flow cytometry was used for both HLA-DR expression and phenotyping of immature forms of monocytes and granulocytes. HLA-DR expression was downregulated in both blood and BM monocyte in ICU patients compared to BM of non-ICU patients and blood of control donors. Amplitude of HLA-DR downregulation was comparable in septic and non-septic ICU patients. The phenotype of immature forms of monocytes and granulocytes in BM (n = 11) did not show abnormal myeloid (monocyte + granulocyte) differentiation. CONCLUSION: The downregulation of HLA-DR in BM monocyte lineage is present in ICU patients without major changes in myeloid cells. It may result from a regulation mediated by soluble and/or neuro-endocrine factors present in BM cell microenvironment.

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.

Clinical Significance of Circulating CD33+CD11b+HLA-DR- Myeloid Cells in Patients with Stage IV Melanoma Treated with Ipilimumab.

PURPOSE: High levels of circulating myeloid-derived suppressor cells (MDSCs) in various cancer types, including melanoma, were shown to correlate with poor survival. We investigated whether frequencies of circulating CD33+CD11b+HLA-DR- MDSCs could be used as immune system monitoring biomarkers to predict response and survival of patients with stage IV melanoma treated with anti-CTLA4 (ipilimumab) therapy. EXPERIMENTAL DESIGN: Peripheral blood samples from 56 patients and 50 healthy donors (HDs) were analyzed for CD33+CD11b+HLA-DR- MDSC percentage, NO-, and hROS levels by flow cytometry. We determined whether MDSC levels and suppressive features detected before anti-CTLA4 therapy correlate with the patients' response and overall survival (OS). RESULTS: Patients with melanoma had significantly higher levels of circulating CD33+CD11b+HLA-DR- MDSCs with suppressive phenotype when compared with HDs. Low levels of MDSCs before CTLA-4 therapy correlated with an objective clinical response, long-term survival, increased CD247 expression in T cells, and an improved clinical status. No predictive impact was observed for lactate dehydrogenase (LDH). Kaplan-Meier and log-rank tests performed on the 56 patients showed that the presence of more than 55.5% of circulating CD33+CD11b+ out of the HLA-DR- cells, were associated with significant short OS (P < 0.003), a median of 6.5 months, in comparison with the group showing lower MDSC frequencies, with a median survival of 15.6 months. CONCLUSIONS: Our study suggests the use of CD33+CD11b+HLA-DR- cells as a predictive and prognostic biomarker in patients with stage IV melanoma treated with anti-CTLA4 therapy. This monitoring system may aid in the development of combinatorial modalities, targeting the suppressive environment in conjunction with iplimumab, toward facilitating better disease outcomes. Clin Cancer Res; 22(23); 5661-72. ©2016 AACR.

Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis.

OBJECTIVE: Reactive oxygen species (ROS) are known to regulate platelet activation; however, the mechanisms of ROS production during platelet activation remain unclear. Platelets express different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs). Here, we investigated the role of NOX1 and NOX2 in ROS generation and platelet activation using NOX1 and NOX2 knockout mice. APPROACH AND RESULTS: NOX1(-/Y) platelets showed selective defects in G-protein-coupled receptor-mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide, a glycoprotein VI agonist. In contrast, NOX2(-/-) platelets showed potent inhibition of collagen-related peptide-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet activation. Consistently, production of ROS was inhibited in NOX1(-/Y) platelets stimulated with thrombin, but not collagen-related peptide, whereas NOX2(-/-) platelets showed reduced ROS generation induced by collagen-related peptide or thrombin. Reduced ROS generation in NOX1/2-deficient platelets is associated with impaired activation of Syk and phospholipase Cγ2, but minimally affected mitogen-activated protein kinase pathways. Interestingly, laser-induced arterial thrombosis was impaired but the bleeding time was not affected in NOX2(-/-) mice. Wild-type thrombocytopenic mice injected with NOX2(-/-) platelets also showed defective arterial thrombosis, suggesting an important role for platelet NOX2 in thrombosis in vivo but not hemostasis. CONCLUSIONS: NOX1 and NOX2 play differential roles in different platelet activation pathways and in thrombosis. ROS generated by these enzymes promotes platelet activation via the Syk/phospholipase Cγ2/calcium signaling pathway.

[Identification and significance of myeloid-derived suppressor cells in peripheral blood of breast cancer patients].

OBJECTIVE: To investigate the presence, biological features, and clinical significance of myeloid-derived suppressor cells (MDSCs) in breast cancer patients. METHODS: Eighty-four cases of breast cancer, 37 cases of benign breast tumor and 21 cases of healthy individuals were included in this study. Samples of peripheral blood (2 ml) were collected, and in the breast cancer patients, blood samples were taken both before and after treatment. Flow cytometry using anti-CD11b, CD33, CD14 and HLA-DR antibody was conducted to identify the unique membrane markers of MDSCs, and statistical analysis was performed to explore the relationship between MDSCs and clinical factors. Cell isolation and in vitro assay were used to test T cell function. RESULTS: CD11b(+) CD33(+) CD14(-) MDSCs were present in the blood of breast cancer patients, and these MDSCs were histologically of mononuclear cells. Cell proliferation assay confirmed that MDSCs inhibited proliferation of homologous T cells in vitro. MDSCs levels in patients with breast cancer, benign disease and the health control were (15.93±3.17)%, (8.92±4.42)% and (5.02±2.75)%, respectively, with a statistically significant difference (P<0.001) between breast cancer patients and the other subjects (patients with benign lesions and healthy controls). The expression level of MDSCs in patients with breast cancer was associated with surgical treatment, but not with age, disease stage, lymph node metastasis, ER or PR expression. MDSCs levels were significantly lower in post-operative patients[(7.83±3.78) %] than the (15.37±2.49) % in patients before surgery (P<0.001). CONCLUSIONS: The results of this study demonstrate that MDSCs are present in the peripheral blood of breast cancer patients and the level of MDSCs is associated with surgical treatment. Our findings suggest that CD11b(+) CD33(+) CD14(-) MDSCs are likely involved in breast cancer initiation and development, and may become a novel biomarker to facilitate diagnosis and to predict clinical outcomes of breast cancer.

Circulating myeloid-derived suppressor cells in patients with pancreatic cancer.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are heterogeneous cell types that suppress T-cell responses in cancer patients and animal models, some MDSC subpopulations are increased in patients with pancreatic cancer. The present study was to investigate a specific subset of MDSCs in patients with pancreatic cancer and the mechanism of MDSCs increase in these patients. METHODS: Myeloid cells from whole blood were collected from 37 patients with pancreatic cancer, 17 with cholangiocarcinoma, and 47 healthy controls. Four pancreatic cancer cell lines were co-cultured with normal peripheral blood mononuclear cells (PBMCs) to test the effect of tumor cells on the conversion of PBMCs to MDSCs. Levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and arginase activity in the plasma of cancer patients were analyzed by enzyme-linked immunosorbent assay. RESULTS: CD14+/CD11b+/HLA-DR- MDSCs were increased in patients with pancreatic or bile duct cancer compared with those in healthy controls, and this increase was correlated with clinical cancer stage. Pancreatic cancer cell lines induced PBMCs to MDSCs in a dose-dependent manner. GM-CSF and arginase activity levels were significantly increased in the serum of patients with pancreatic cancer. CONCLUSIONS: MDSCs were tumor related: tumor cells induced PBMCs to MDSCs in a dose-dependent manner and circulating CD14+/CD11b+/HLA-DR- MDSCs in pancreatic cancer patients were positively correlated with tumor burden. MDSCs might be useful markers for pancreatic cancer detection and progression.

Monocyte Recruitment after High-Intensity and High-Volume Resistance Exercise.

UNLABELLED: The innate immune response is generally considered to have an important role in tissue remodeling after resistance exercise. PURPOSE: The purpose of this study was to compare changes in markers of monocyte recruitment after an acute bout of high-intensity (HVY) versus high-volume (VOL) lower-body resistance exercise. METHODS: Ten resistance-trained men (24.7 ± 3.4 yr, 90.1 ± 11.3 kg, 176.0 ± 4.9 cm) performed each protocol in a randomized, counterbalanced order. Blood samples were collected at baseline, immediately (IP), 30 min (30P), 1 h (1H), 2 h (2H), and 5 h (5H) postexercise. Plasma concentrations of monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α), myoglobin, and cortisol were measured via assay. Tumor necrosis factor receptor 1 (TNFr1), macrophage-1 antigen (cluster of differentiation 11b [CD11b]), and C-C chemokine receptor 2 (CCR2) expression levels were measured using flow cytometry. TNFr1 and CD11b were assessed on CD14CD16 monocytes, whereas CCR2 was assessed on CD14 monocytes. RESULTS: Plasma myoglobin concentrations were significantly greater after HVY compared with VOL (P < 0.001). Changes in plasma TNF-α, MCP-1, and expression levels of CCR2 and CD11b were similar between HVY and VOL. When collapsed across groups, TNF-α was significantly increased at IP, 30P, 1H, and 2H (P values < 0.05), whereas MCP-1 was significantly elevated at all postexercise time points (P values < 0.05). CCR2 expression on CD14 monocytes was significantly lower at IP, 1H, 2H, and 5H (P values < 0.05). CD11b expression on CD14 CD16 was significantly greater at IP (P < 0.014) and 1H (P = 0.009). TNFr1 expression did not differ from baseline at any time point. Plasma cortisol concentrations did not seem to be related to receptor expression. CONCLUSIONS: Results indicate that both HVY and VOL protocols stimulate a robust proinflammatory response. However, no differences were noted between resistance exercise training paradigms.