Toni Karhu

Stage-dependent alterations of the serum cytokine pattern in colorectal carcinoma

Background:Inflammation contributes to the pathogenesis of colorectal cancer (CRC), and cytokine levels are altered during colorectal carcinogenesis.Methods:The serum levels of 13 cytokines and their relation to clinical and pathological parameters, and systemic inflammatory response (mGPS, CRP and neutrophil–lymphocyte ratio), were analysed from a prospective series of 148 CRC patients and 86 healthy age- and sex-matched controls.Results:CRC patients had higher serum platelet-derived growth factor, interleukin (IL)-6, IL-7, and IL-8 levels and lower monocyte chemotactic protein-1 (MCP-1) levels than the controls. A logistic regression model for discriminating the patients from the controls – including the five most predictive cytokines (high IL-8, high IL-6, low MCP-1, low IL-1ra, and low IP-10) – yielded an area under curve value of 0.890 in receiver operating characteristics analysis. Serum cytokines showed distinct correlation with other markers of systemic inflammatory response, and advanced CRCs were associated with higher levels of IL-8, IL-1ra, and IL-6. A metastasised disease was accompanied by an orientation towards Th2 cytokine milieu.Conclusion:CRC is associated with extensive alterations in serum cytokine environment, highlighting the importance of studying relative cytokine level alterations. Serum cytokine profile shows promise in separating CRC patients from healthy controls but its clinical value is yet to be confirmed.

Inhibition of cytokine secretion from adipocytes by 1,25-dihydroxyvitamin D3 via the NF-κB pathway

Adipose tissue inflammation is an important pathological process in obese people, associated with diabetes and cardiovascular disease. We hypothesized that 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] inhibits cytokine secretion from adipocytes via direct inhibition of transcription factor nuclear factor‐κB (NF‐κB). We utilized two different human models. Bone marrow‐derived human mesenchymal stromal cells (hMSCs) differentiated into adipocytes, and adipocytes isolated from biopsies stimulated with lipopolysaccharide (LPS) were treated with or without 1,25(OH)2D3. Expression and secretion of interleukin‐6 (IL‐6) were measured by quantitative RT‐PCR analysis and ELISA. Assessment of NF‐κB nuclear translocation, DNA binding activity was performed by immunofluorescence (IF) and electrophoretic mobility assay (EMSA). Inhibitor κB (IκB) and its phosphorylation were detected by Western blot (WB) analysis. Simultaneous 1,25(OH)2D3 cotreatment significantly reduced LPS‐stimulated (10 ng/ml) IL‐6 secretion dose dependently by 15% at 10–10 M and 26% at 10–7 M (P<0.05) in hMSCs, while preincubation with 1,25(OH)2D3 (10–7 M) for 24 h reduced IL‐6 secretion by 24 and 35% (P<0.001) and mRNA levels by 34 and 30% (P<0.05) in hMSCs and isolated adipocytes, respectively. 1,25(OH)2D3 suppressed LPS‐stimulated IκB phosphorylation‐mediated NF‐κB translocation into the nucleus were evident from WB, IF, and EMSA. 1,25(OH)2D3 inhibits LPS‐stimulated IL‐6 secretion in two human adipocyte models via interference with NF‐κB signaling.—Mutt, S. J., Karhu, T., Lehtonen, S., Lehenkari, P., Carlberg, C., Saarnio, J., Sebert, S., Hyppönen, E., Järvelin, M.‐R., Herzig, K.‐H. Inhibition of cytokine secretion from adipocytes by 1,25‐dihydroxyvitamin D3 via the NF‐κB pathway. FASEB J. 26, 4400–4407 (2012). www.fasebj.org

Differential effects of dietary protein sources on postprandial low-grade inflammation after a single high fat meal in obese non-diabetic subjects

BackgroundObesity is a state of chronic low-grade inflammation. Chronic low-grade inflammation is associated with the pathophysiology of both type-2 diabetes and atherosclerosis. Prevention or reduction of chronic low-grade inflammation may be advantageous in relation to obesity related co-morbidity. In this study we investigated the acute effect of dietary protein sources on postprandial low-grade inflammatory markers after a high-fat meal in obese non-diabetic subjects.MethodsWe conducted a randomized, acute clinical intervention study in a crossover design. We supplemented a fat rich mixed meal with one of four dietary proteins - cod protein, whey isolate, gluten or casein. 11 obese non-diabetic subjects (age: 40-68, BMI: 30.3-42.0 kg/m2) participated and blood samples were drawn in the 4 h postprandial period. Adiponectin was estimated by ELISA methods and cytokines were analyzed by multiplex assay.ResultsMCP-1 and CCL5/RANTES displayed significant postprandial dynamics. CCL5/RANTES initially increased after all meals, but overall CCL5/RANTES incremental area under the curve (iAUC) was significantly lower after the whey meal compared with the cod and casein meals (P = 0.0053). MCP-1 was initially suppressed after all protein meals. However, the iAUC was significantly higher after whey meal compared to the cod and gluten meals (P = 0.04).ConclusionWe have demonstrated acute differential effects on postprandial low grade inflammation of four dietary proteins in obese non-diabetic subjects. CCL5/RANTES initially increased after all meals but the smallest overall postprandial increase was observed after the whey meal. MCP-1 was initially suppressed after all 4 protein meals and the whey meal caused the smallest overall postprandial suppression.Trial RegistrationClinicalTrials.gov ID: NCT00863564

The relationships between serum cytokine levels and tumor infiltrating immune cells and their clinical significance in colorectal cancer

Increased inflammatory cell infiltration correlates to improved survival in colorectal cancer (CRC). Development and progression of CRC is associated with alterations in serum cytokine levels but their significance is not well defined. In this study, we investigated the relationships between the serum levels of 13 cytokines and the densities of eight types of tumor infiltrating inflammatory cells and their impact on disease‐free survival (DFS), cancer‐specific survival (CSS) and overall survival (OS) in a prospectively recruited group of 147 CRC patients. There were strong positive correlations between the serum concentrations of different cytokines, as well as between the different types of tumor infiltrating immune cells, whereas the associations between serum cytokines and tumor infiltrating immune cells were generally weak. High serum IL‐12 levels associated with increased densities of peritumoral CD8+ T cells, intraepithelial CD3+ T cells and intratumoral neutrophils, while high serum CCL4 levels associated with increased densities of peritumoral CD68+ cells. In multivariate survival models, increased infiltration of intraepithelial CD3+ T cells and increased serum CCL4 associated with improved DFS, whereas higher intratumoral CD83+ dendritic cell density and increased serum interferon gamma levels associated with improved CSS and OS. Also high density of peritumoral CD3+ T cells associated with improved CSS. In conclusion, serum cytokines and tumor infiltrating immune cells in CRC represent entities with high intragroup correlations but relatively weak intergroup correlations. The results suggest that tumor infiltrating CD3+ T cells, CD83+ dendritic cells, serum CCL4 and serum interferon gamma represent relevant markers of disease outcome.

Neurogenic pulmonary edema in patients with nontraumatic intracerebral hemorrhage: predictors and association with outcome.

BACKGROUND:Neurogenic pulmonary edema (NPE) is a well-recognized phenomenon after intracranial insult. In this study, we evaluated the predictors for NPE and its association with outcome in patients with intensive care unit–treated nontraumatic intracranial hemorrhage. METHODS:This was a prospective, observational clinical study in a university-level intensive care unit. Clinical characteristics, level of consciousness, and Acute Physiology and Chronic Health Evaluation (APACHE) II score were recorded on admission and the findings of primary head computed tomography were reviewed. A chest radiograph and arterial blood gas analysis were taken serially and NPE was determined as acute bilateral infiltrates in chest radiograph and hypoxemia. Echocardiography and cardiac and inflammatory markers were recorded. The 1-year outcome was assessed using the Glasgow Outcome Scale. RESULTS:NPE developed in 38 (35%) of the 108 patients included. Predictors for NPE were higher APACHE II score (≥20, odds ratio 6.17, P = 0.003) and higher interleukin-6 plasma concentration (>40 pg/mL, odds ratio 5.62, P = 0.003). Of patients with 0, 1, or 2 predictors mentioned above, 4%, 37%, and 65% had NPE, respectively. NPE was associated with a higher 1-year mortality (37% vs 14%, P = 0.007, respectively), but with an unchanged functional outcome after 1 year (Glasgow Outcome Scale score 1–3, 53% vs 51%, P > 0.9). CONCLUSIONS:Predictors for NPE are the severity of disease defined by APACHE II scores and higher levels of systemic inflammatory mediators. NPE is associated with a higher 1-year mortality, but not with a poorer 1-year functional outcome.

Mast cell degranulation via MRGPRX2 by isolated human albumin fragments

BACKGROUND Mast cells are important modulators of the human immune system via their release of several inflammatory mediators and proteases. The release can be activated by different pathways: the classical immunoglobulin E-dependent pathway and by the non-immunological immunoglobulin E-independent pathway. MAS-related G protein-coupled receptor X2 (MRGPRX2) is expressed in mast cells and it is one of the endogenous receptor responsible for the IgE-independent activation of human mast cell. The MRGPRX2 is classified as orphan receptor and unlike most GPCRs, the MRGPRX2 recognizes a wide range of basic molecules. Thus, there still might be several unknown ligands for the receptor. METHODS MRGPRX2 activating peptides were isolated from human plasma using consecutive HPLC purification steps. The isolation process was monitored with MRGPRX2 transfected HEK 293 cells. The isolated peptides were sequenced by MS and synthetized. The synthetic peptides were used to determine degranulation of the human LAD 2 mast cell line by measuring β-hexosaminidase release. RESULTS Three endogenous MRGPRX2 activating peptides were isolated from human plasma. These peptides are identified as fragments of albumin. The isolated fragments activate MRGPRX2 and degranulate MRGPRX2 expressing LAD 2 cells in dose-dependent manner. CONCLUSIONS The isolated basic peptides generated from human albumin are able to degranulate mast cells via the MRGPRX2. GENERAL SIGNIFICANCE These endogenous albumin fragments, cleaved from albumin by mast cell secreted proteases, provide a possible pathway for self-perpetuating mast cell dependent inflammation.

Preoperative anemia in colorectal cancer: relationships with tumor characteristics, systemic inflammation, and survival

Anemia is common in colorectal cancer (CRC) but its relationships with tumor characteristics, systemic inflammation, and survival have not been well characterized. In this study, blood hemoglobin levels and erythrocyte mean corpuscular volume (MCV) levels were measured in two independent cohorts of 148 CRC patients and 208 CRC patients, and their correlation with patient and tumor characteristics, systemic inflammatory markers (modified Glasgow Prognostic Score: mGPS; serum levels of thirteen cytokines, C-reactive protein, albumin), and survival were analyzed. We found that anemia, most frequently normocytic, followed by microcytic, was present in 43% of the patients. Microcytic anemia was most commonly associated with proximal colon tumor location. Average MCV and blood hemoglobin levels were lower in tumors with high T-class. Low blood hemoglobin associated with systemic inflammation, including high mGPS and high serum levels of C-reactive protein and IL-8. Particularly, normocytic anemia associated with higher mGPS. Normocytic anemia associated with a tendency towards worse overall survival (multivariate hazard ratio 1.61, 95% confidence interval 1.07–2.42, p = 0.023; borderline statistical significance considering multiple hypothesis testing). In conclusion, anemia in CRC patients is most frequently normocytic. Proximal tumor location is associated with predominantly microcytic anemia and systemic inflammation is associated with normocytic anemia.

Portal vein cytokines in the early phase of acute experimental oedematous and necrotizing porcine pancreatitis.

Abstract Objective. Cytokines initiate and modify systemic inflammatory response in early acute pancreatitis. The aim of this study was to analyze which cytokines are released from the pancreas to portal venous blood in the early phase of acute experimental necrotizing and oedematous pancreatitis and which of those cytokines are correlated with the more severe form of the disease. Material and methods. Fifteen pigs were randomized to develop mild oedematous pancreatitis (n = 5, saline infusion to pancreatic duct), severe necrotizing pancreatitis (n = 5, taurocholic acid infusion) along with a control group (n = 5). Arterial and venous blood samples were drawn and cytokine levels were measured from portal vein blood at 0, 120, 240 and 360 min after the induction of pancreatitis. Tissue samples from the pancreas were harvested at 0 and 360 min. Results. White blood cell count increased in necrotizing pancreatitis and the control group. The amount of neutrophils increased (p < 0.001) and the lymphocyte and eosinophil counts decreased in all groups (p < 0.001, p < 0.001). The monocyte count, as well as PDGF and IL-6 concentrations, increased only in necrotizing pancreatitis. IL-8 and eotaxin increased both in oedematous and necrotizing pancreatitis. MCP-1 increased in all groups. IL-9, IL-4, MIP-1α, IFN- γ concentrations did not change. Eotaxin and MCP-1 plasma levels from a previous series between portal venous and pulmonary arterial blood were not significantly different. Conclusions. The initial inflammatory process was diverse in oedematous and necrotizing pancreatitis. Increased monocyte count in combination with elevated PDGF and IL-6 are characteric of necrotizing pancreatitis in our model.

Blister fluid and serum cytokine levels in severe sepsis in humans reflect skin dysfunction.

Knowledge of sepsis‐related end‐organ inflammation in vivo is limited. We investigated the cytokine response in skin and in serum in sepsis and its relation to multiorgan failure (MOF) and survival.

Decreased preoperative serum 25-Hydroxyvitamin D levels in colorectal cancer are associated with systemic inflammation and serrated morphology.

Deficiency of vitamin D is associated with increased risk of several types of cancer including colorectal cancer (CRC). However, factors contributing to low levels of 25-hydroxyvitamin D [25(OH)D] in CRC are not clear. Therefore, in this study serum 25(OH)D levels in 117 CRC patients and 86 controls were analyzed and correlated with the clinicopathological data including morphological subtype (serrated or conventional), quantity of tumor infiltrating immune cells, levels of systemic inflammatory markers, and disease outcome. We found that the patients had lower serum 25(OH)D levels compared to the controls. Interestingly, among the patients mismatch repair deficiency, serrated morphology, and high body mass index associated with lowest serum 25(OH)D levels. In addition, patients operated in summer or autumn had higher serum 25(OH)D levels. Furthermore, serum 25(OH)D levels inversely correlated with several systemic inflammatory markers, e.g. serum C reactive protein, but did not associate with prognosis. Mechanism leading to vitamin D deficiency in these patients are not clear but could be related to the effects of systemic inflammation. Longitudinal studies are warranted to assess vitamin D deficiency as a potential risk factor for serrated colorectal polyps and adenocarcinoma.