Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease

Inflammatory bowel disease (IBD) arises from inappropriate activation of the mucosal immune system resulting in a state of chronic inflammation with causal links to colon cancer. Helicobacter hepaticus-infected Rag2^−/− mice emulate many aspects of human IBD, and our recent work using this experimental model highlights the importance of neutrophils in the pathology of colitis. To define molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in tissues of mice and human IBD patients. Analysis in inflamed mouse colons identified the neutrophil- and macrophage-derived damage products 3-chlorotyrosine (Cl-Tyr) and 3-nitrotyrosine, both of which increased with disease duration. Analysis also revealed higher Cl-Tyr levels in colon relative to serum in patients with ulcerative colitis and Crohn disease. The DNA chlorination damage product, 5-chloro-2′-deoxycytidine, was quantified in diseased human colon samples and found to be present at levels similar to those in inflamed mouse colons. Multivariate analysis of these markers, together with serum proteins and cytokines, revealed a general signature of activated innate immunity in human IBD. Signatures in ulcerative colitis sera were strongly suggestive of neutrophil activity, and those in Crohn disease and mouse sera were suggestive of both macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into IBD disease processes.Inflammatory bowel disease (IBD) is a chronic and relapsing intestinal inflammatory disease that arises through unknown genetic, environmental, and bacterial origins (1, 2). Ulcerative colitis (UC) and Crohn disease (CD) are the two main forms of IBD, and their incidence is increasing in industrialized countries (3). Furthermore, IBD is a risk factor for the development of colon cancer (4). Although the specific determinants remain elusive, persistent inflammation is believed to play a significant role in colon cancer development (5).Neutrophil recruitment and activation are key steps in the intestinal innate immune response observed in IBD (6–8), and studies with animal models of colitis highlight the relationship between neutrophil infiltration and disease severity (9–11). We recently reported results of a comprehensive analysis of histopathology, changes in gene expression, and nucleic acid damage occurring during progression of lower bowel disease in Rag2^−/− mice infected with Helicobacter hepaticus (Hh) (10). This mouse model emulates many aspects of human IBD, and infected mice develop severe colitis that progress into colon carcinoma, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages (12, 13).Phagocytes produce strong oxidants and radicals that damage cellular macromolecules and promote tissue damage at sites of inflammation (14–16). Myeloperoxidase (MPO) is an abundant enzyme in neutrophils that produces hypochlorous acid (HOCl) from hydrogen peroxide (H₂O₂) and chloride ion (17, 18). HOCl can oxidize and chlorinate DNA, proteins, and lipids (19, 20). A prominent target of HOCl is tyrosine, which leads to the formation of the stable aromatic residue, 3-chlorotyrosine (Cl-Tyr) (21, 22). MPO also produces chlorinating species that react with DNA to form chlorinated adducts such as 5-chloro-2′-deoxycytidine (5-Cl-dC) (23), the presence of which was identified in colon tissue of H. hepaticus-infected Rag2^−/− mice (10). This modification of DNA may provide a mechanistic link between neutrophil activity and colitis-associated carcinoma (10, 24, 25).Macrophages also contribute to the array of oxidants and radicals at sites of inflammation through release of nitric oxide (NO) generated by the inducible NO synthase (iNOS) enzyme. NO reacts with superoxide anion (O₂^−•) at diffusion-controlled rates to yield highly reactive peroxynitrite (ONOO⁻) (26, 27). MPO also reacts H₂O₂ with nitrite (NO₂⁻, the endpoint of cellular NO oxidation) to produce the strong nitrating agent, nitrogen dioxide radical (NO₂^•) (28). Both NO₂^• and ONOO⁻ can react with tyrosine residues to generate the stable tyrosine nitration product, 3-nitrotyrosine (Nitro-Tyr) (29, 30).Multiple MS methods have been applied for determination of Cl-Tyr and Nitro-Tyr levels in biological systems (10, 31–38), and both have been detected in inflamed tissues from animals and humans (11, 39). The presence of Nitro-Tyr has been demonstrated in colon tissue of IBD patients by immunohistochemistry, and levels were reported to correlate with disease activity (40, 41). We undertook the present study to test the null hypothesis that the H. hepaticus-infected mouse model of colitis and colitis-associated carcinoma represents a useful surrogate of human IBD. To examine this hypothesis, we first quantified levels of Nitro-Tyr and Cl-Tyr in proteins and 5-Cl-dC in DNA of colon tissues of IBD patients. Comparison of these data with our previous findings (10) further assessed the validity of this animal model. We then tested the hypothesis that inflammation-induced damage in the colon would be reflected in changes in serum constituents, and would therefore serve as a noninvasive measure of IBD activity. For this purpose, we determined levels of protein chlorination and nitration products, acute-phase proteins, cytokines, and chemokines in human and mouse sera. In addition, gene expression of several inflammatory signaling molecules was monitored in mice colons to determine whether colonic inflammation was directly associated with serum cytokine levels. We then used multivariate analysis to determine which systemic inflammatory markers in serum were most closely associated with disease activity and were also common to human IBD and H. hepaticus-associated colitis in Rag2^−/− mice.     

Structurally Specific Heparan Sulfates Support Primitive Human Hematopoiesis by Formation of a Multimolecular Stem Cell Niche

Stem cell localization, conservation, and differentiation isbelieved to occur in niches in the marrow stromal microenvironment. Ourrecent observation that long-term in vitro human hematopoiesis requiresa stromal heparan sulfate proteoglycan (HSPG) led us to hypothesizethat such HSPG may orchestrate the formation of the stem cell niche. Wecompared the structure and function of HS from M2-10B4, ahematopoiesis-supportive cell line, with HS from a nonsupportive cellline, FHS-173-We. Long-term culture-initiating cell (LTC-IC)maintenance was enhanced by PG from supportive cells but not by PG fromnonsupportive cells (P < .005). The supportive HS weresignificantly larger and more highly sulfated than the nonsupportiveHS. Specifically, supportive HS contained higher 6-O-sulfation on theglucosamine residues. In agreement with these observations, purified6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarlymaintained LTC-IC. In contrast, completely desulfated heparin,N-sulfated heparin, and unmodified heparin did not support LTC-ICmaintenance. Moreover, the supportive HS promoted LTC-IC maintenancebut not differentiation of CD34⁺/HLA-DRcells into colony-forming cells (CFCs) and mature bloodcells. The supportive HS but not the nonsupportive HS bound bothcytokines and matrix components critical for hematopoiesis, includinginterleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1),and thrombospondin (TSP). Significantly more CD34⁺ cellsadhered directly to immobilized O-sulfated heparin than to N-sulfatedor desulfated heparin. Thus, hematopoiesis-supportive stromal HSPGpossessing large, highly 6-O-sulfated HS mediate the juxtaposition ofhematopoietic progenitors with stromal cells, specific growth-promoting(IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4])cytokines, and extracellular matrix (ECM) proteins such as TSP. Weconclude that the structural specificity of stromal HSPG thatdetermines the selective colocalization of cytokines and ECM componentsleads to the formation of discrete niches, thereby orchestrating thecontrolled growth and differentiation of stem cells. These findings mayhave important implications for ex vivo expansion of and gene transferinto primitive hematopoietic progenitors.     

Assessment of cardiac arrhythmias in patients suffering from essential hypertension

Heart rate turbulence (HRT) analysis is a novel, non-invasive method enabling to estimate sudden cardiac death risk. Up till now it was used to determine cardiac death risk in patients suffering from congestive heart failure and especially after myocardial infarction. The aim of the study was to estimate heart rate turbulence parameters in patients with essential hypertension and to find correlations between those parameters and age, left ventricular mass and hypertension stages. Studies were performed in a group of 55 persons: 26 women (54.79 +/- 8.26 years old) and in 29 men (52.34 +/- 5.72 years old) with essential pharmacologically untreated hypertension. Patients were divided into two groups: increased (group I, n = 32) and normal left ventricle mass (group II, n = 23). In each patient 24-hour ECG Holter recording was performed and then turbulence onset (TO) (expressed in %) and turbulence slope (TS) (in ms/RR interval) were estimated. In the group of patients with increased left ventricle mass parameter TO was significantly higher (-0.74 +/- 0.24 vs -1.32 +/- 0.28; p < 0.05) and parameter TS significantly lower (5.26 +/- 1.17 vs 8.46 +/- 2.91; p < 0.01) in comparison to study subjects with normal left ventricle mass. Heart rate turbulence is decreased in people with essential hypertension and increased left ventricle mass in comparison to normal left ventricle mass group. Decreased heart rate turbulence in patients with essential hypertension and increased left ventricle mass may be a predictor of worse prognosis in that group.     

Serum aminoterminal peptide of type III procollagen (PIIINP) and transforming growth factor-beta1 (TGF-beta1) levels in patients with chronic hepatitis B and C

Fibrosis is the process accompanying majority of chronic diseases of liver, independent of etiological factor and leading to cirrhosis and hepatic failure. Monitoring fibrosis process by liver's biopsy is limited, so many attempts are undertaken to assess concentrations of definite proteins in blood, which could be easily accessible marker of intrahepatic process. It seems, that among others, determinations of blood concentration of aminoterminal propeptide of procollagen III--index of collagen's III synthesis and TGF-beta 1--cytokine of antiproliferative action and inhibiting hepatocytes' growth, yet inducing fibroblasts' growth and stimulating fibrosis process brings out such a possibility. The aim of the study was simultaneous determination of TGF-beta 1 and PIIINP concentration in blood of patients with chronic hepatitis B and C before interferone's therapy in comparison to healthy controls, assessment of the parameters in dependence on stage of liver fibrosis and determination of correlation between TGF-beta 1 and PIIINP. Studies were performed in 40 patients with chronic hepatitis B (CAH B) and 35 patients with chronic hepatitis C (CAH C). Significantly increased serum concentrations of TGF-beta 1 as PIIINP in both groups of patients (CAH B and CAH C; grading 2-3, staging 1-2) in comparison with control group was noted. Significant positive correlation of TGF-beta 1 and PIIINP serum concentrations in both groups of patients was observed. There was not significant changes in PIIINP serum levels in patients with hepatitis B and C in dependence on stage of liver fibrosis (staging 1 vs staging 2) but TGF-beta 1 serum levels was significantly increased in CAH B and C patients with higher stage of liver fibrosis process. On the base of obtained results, it seems that changes in TGF-beta 1 concentrations in blood reflect "grading" and "staging" and can be a marker of intensification of intrahepatic fibrosis process whereas PIIINP levels in blood have rather the relation with "grading".     

Influence of Pre-Storage Irradiation on the Oxidative Stress Markers,Membrane Integrity,Size and Shape of the Cold Stored Red Blood Cells

Background

To investigate the extent of oxidative damage and changes in morphology of manually isolated red blood cells (RBCs) from whole blood, cold stored (up to 20 days) in polystyrene tubes and subjected to pre-storage irradiation (50 Gy) and to compare the properties of SAGM-preserved RBCs stored under experimental conditions (polystyrene tubes) with RBCs from standard blood bag storage.

Methods

The percentage of hemolysis as well as the extracellular activity of LDH, thiobarbituric acid-reactive substances, reduced glutathione (GSH), and total antioxidant capacity (TAC) were measured. Changes in the topology of RBC membrane, shape, and size were evaluated by flow cytometry and judged against microscopy images.

Results

Irradiation caused significant LDH release as well as increased hemolysis and lipid peroxidation, GSH depletion, and reduction of TAC. Prolonged storage of irradiated RBCs resulted in phosphatidylserine exposure on the cell surface. By day 20, approximately 60% of RBCs displayed non-discoid shape. We did not notice significant differences in percentage of altered cells and cell volume between RBCs exposed to irradiation and those not exposed.

Conclusion

Irradiation of RBC transfusion units with a dose of 50 Gy should be avoided. For research purposes such as studying the role of antioxidants, storage of small volumes of RBCs derived from the same donor would be more useful, cheaper, and blood-saving.Key Words: Red blood cell, Gamma irradiation, Storage, Oxygen-free radical, Flow cytometry     

Analysis of sentinel lymph node biopsy results in colon cancer in regard of the anthropometric features of the population and body composition assessment formulas

Purpose

The aim of the study was to assess sentinel lymph node biopsy (SLNB) results in colon cancer (CC) regarding basic anthropometric features of the studied population and their derivatives calculated using mathematical formulas.

Methods

One hundred three SLNBs in CC have been analysed. Various indicators were calculated for every patient using mathematical formulas: BMI, Roher’s index, lean body weight, body fat percentage and body weight/ideal body weight for a given height ratios using the following formulas: Broca’s, Broca’s ideal weight, Broca–Brugsch, Lorenz’s, Potton’s, Devine’s, Robinson’s, Miller’s and Hamwi. The results were compared with accuracy, sensitivity and false negative results percentage by means of ROC curves and the test for structure indicators (for determined cut-off points).

Results

No statistically significant relationship between the results and patients' sex or age were found. ROC curve analysis did not reveal statistically significant relationships between the obtained results and indicators calculated on the basis of growth and weigh (all p?>?0.05). The analyses of sensitivity and accuracy with determined cut-off point, in spite of differences amounting to 19 % (analysis of lean body weight/weight ratio), showed no statistical significance for any of the relationships (all p?>?0.05).

Conclusions

No indicator with high diagnostic and prognostic value has been found. The problem of qualifying patients for SLNB in CC in regard of the anthropometric features of the population and body composition assessment formulas remains open and requires further analysis on larger populations.