Clinical Studies

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Last updated: 13th August 2010

Clinical Studies
Altered expression of innate immunity genes in different intestinal sites of children with ulcerative colitis.
Dig Liver Dis. 2010 May 6. [Epub ahead of print]
BACKGROUND: Innate immunity has been very rarely investigated in ulcerative colitis and never in paediatrics. The present study was aimed at describing expression of innate immunity genes (NOD2, RIP2, alpha-defensins HD5 and HD6) in inflamed colon and in ileum of children with ulcerative colitis. Expression of TNFalpha and IL-1beta was also analyzed. METHODS: 15 children with ulcerative colitis (9 pancolitis, 6 left-sided colitis) and 10 control children were enrolled. mRNA and protein expressions were detected by real time PCR and western blot assays. RESULTS: NOD2, RIP2, IL-1beta, TNFalpha expression levels were significantly increased in colonic mucosa of patients compared to controls (p<0.01). These genes were also upregulated (p<0.01) in the ileum of both pancolitis and left-sided colitis children. HD5 and HD6 were significantly upregulated (p<0.01) in the inflamed colon of patients as well as in the ileum of those with pancolitis. CONCLUSIONS: An increased mucosal expression of innate immunity genes was found in the inflamed colon of children with ulcerative colitis, outlining the role of the innate immune response in disease pathogenesis. Involvement of the ileum in ulcerative colitis suggests that an immune activation can also be established in intestinal sites classically uninvolved by the inflammation, carrying implications for the treatment and course of the disease. Copyright 2010 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

The Putative Role of Human Peritoneal Adipocytes in the Fight against Bacteria: Synthesis of the Antimicrobial Active Peptide DEFA1-3.
Nephron Exp Nephrol. 2010 Apr 28;115(4):e96-e100.
Background: Spontaneous peritonitis is a rather rare event, even in peritoneal dialysis (PD). As defensins are natural antimicrobial peptides, we hypothesized that adipocytes as the major constituents of the omentum could play an important role in protecting against infection by generating defensin (DEFA1-3). Methods: We isolated adipocytes from the omentum majus and conducted qualitative and quantitative RT-PCR and immunohistochemical analyses. Results: For the first time described, we were able to detect DEFA1-3 mRNA in highly purified isolated omental adipocytes. The expression of DEFA1-3 in adipocytes was confirmed on the protein level by immunohistochemistry. Conclusion: Our report of DEFA1-3 expression by human omental adipocytes adds to the role of adipocytes in the primary defense against bacterial infection. This may include PD, where the presence of the catheter as a foreign body and the nonphysiological dialysis solution may require constant defense measures to prevent peritonitis, a hypothesis that will require further testing.

Severity of Staphylococcus aureus infection of the skin is associated with inducibility of human beta-defensin 3 but not human beta-defensin 2.
Infect Immun. 2010 Jul;78(7):3112-7.
Gram-positive bacteria are the predominant cause of skin infections. Antimicrobial peptides (AMPs) are believed to be of major importance in skin's innate defense against these pathogens. This study aimed at providing clinical evidence for the contribution of AMP inducibility in determining the severity of gram-positive skin infection. Using real-time-PCR we determined the induction of human beta-defensin (HBD) -2, HBD-3 and RNase 7 by comparing healthy and lesional mRNA levels in 32 patients with gram-positive skin infection. We then examined whether AMP induction differed by disease severity, as measured by number of recurrences and need for surgical drainage in patients with S. aureus-positive lesions. We found that HBD-2 and -3, but not RNase 7 mRNA expression was highly induced by gram-positive infection in otherwise healthy skin. Lower induction of HBD-3, but not HBD-2, was associated with more severe S. aureus skin infection: HBD-3 mRNA levels were 11.4 times lower in patients with more than 6 recurrences (P=0.01) and 8.8 times lower in patients reporting surgical drainage (P=0.01) as compared to respective baseline groups. This suggests that inducibility of HBD-3 influences the severity of gram-positive skin infection in vivo. The physiological function of HBD-2 induction in this context remains unclear.

Defensin-mRNA expression in the upper gastrointestinal tract is modulated in children with celiac disease and Helicobacter pylori-positive gastritis.
J Pediatr Gastroenterol Nutr. 2010 Jun;50(6):596-600.
OBJECTIVES: Defensins are expressed in epithelial cells as cationic peptides with antimicrobial properties. Because of their role as immunologically important effector molecules, their contribution in maintaining a stable microenvironment in the gastrointestinal tract has recently received much attention. The present study was designed to further characterize expression patterns of defensins in diseases of the upper gastrointestinal tract in children, particularly in Helicobacter pylori (Hp)-associated gastritis or celiac disease (CD). PATIENTS AND METHODS: Semiquantitative real-time reverse transcriptase-polymerase chain reaction (PCR) was carried out with gene-specific primers for human beta-defensin 1 to 6 (hBD1 to 6) and human alpha-defensin 5 and 6 (hD5 and 6) in mucosal biopsies of children diagnosed as having CD (n = 11; 4.2-16.2 years) or Hp gastritis (n = 18; 3.2-16.7 years). Levels of expression were compared with those of healthy individuals (n = 21; 2.8-14.6 years). Expression levels in Hp-infected specimens were furthermore compared with those with histologic inflammation not associated with Hp infection (n = 30; 3.6-15.7 years). RESULTS: Expression of hBD2 was upregulated in the antrum and corpus of patients with Hp gastritis, whereas inflammation without detection of Hp was not associated with any change in defensin gene expression. In patients with CD, expression of hBD2 was upregulated in the antrum, whereas hBD1 and 4 were downregulated in duodenal biopsies. CONCLUSIONS: Different pathological conditions of the upper gastrointestinal tract lead to specific modulations of defensin gene expression in children. Especially the pathophysiological role of hBD2 in Hp infection and hBD1 and 4 in CD warrant further attention.

The Decrease of Serum Levels of Human Neutrophil Alpha-Defensins Parallels with the Surgery-Induced Amelioration of NASH in Obesity.
Obes Surg. 2010 Apr 9. [Epub ahead of print]
BACKGROUND: Innate immune system participates actively into inflammatory processes, with immune cells and liver secreting a number of immune peptides. Among them, both soluble CD14 receptor (sCD14) and human neutrophil alpha-defensins (HNDs) may represent serum markers of necro-inflammation in obese patients with non-alcoholic fatty liver disease. METHODS: To verify this hypothesis, we investigated changes in circulating levels of sCD14 and HNDs in 11 severely obese young women following surgery-induced weight loss (bilio-pancreatic diversion). Patients were evaluated before surgery and 2 years later, with NAS score evaluated on liver biopsies and whole body glucose uptake (M value) by euglycemic hyperinsulinemic clamp. RESULTS: NAS score improved in nine patients [median NAS score in the whole sample, 6 (5-6) vs. 3 (3-4), p = 0.016]. Serum concentrations of HNDs decreased significantly in all (p = 0.016), whilst sCD14 increased only in the nine women who showed the amelioration of the NAS score [2.4 (1.7-2.6) vs. 2.6 (2.3-3.3) mug/ml, p = 0.001]. NAS score and HNDs correlated significantly both before (r (o) = 0.671, p = 0.02) and after weight loss (r (o) = 0.683, p = 0.029), NAS score and sCD14 only before surgery (r (o) = 0.605, p = 0.04). The M value increased in all patients [2.67 (1.99-3.01) vs. 6.89 (6.35-7.32) mg kg (FFM) (-1) min(-1); p = 0.01], but independently of NAS score changes. CONCLUSIONS: Changes in levels of HNDs and sCD14 accompany those in hepatic necro-inflammation due to surgical-induced weight loss. Further studies are needed to verify any causative role of these peptides in non-alcoholic steatohepatitis.

Genomic copy number determines functional expression of {beta}-defensin 2 in airway epithelial cells and associates with chronic obstructive pulmonary disease.
Am J Respir Crit Care Med. 2010 Jul 15;182(2):163-9.
RATIONALE: Copy number variations of the cluster of beta-defensin genes have been associated with psoriasis and inflammatory bowel disease. Controversy still exists on whether the beta-defensins genes determine susceptibility for COPD. Aims: We investigated whether genomic copy number variations of the beta-defensin gene cluster have a functional role in airway epithelial cells and associate with the presence of COPD. METHODS: Baseline and inflammatory induced transcript expression of DEFB4 was studied in nasal epithelial cell cultures and its effect on Pseudomonas aeruginosa inhibition was assessed. Subsequently, relevant functional cut-offs for copy numbers were used to explore associations with COPD in two independent case-control studies. RESULTS: Copy number variation in the beta-defensin encoding genes correlated with baseline mRNA DEFB4 expression levels (R(2)= 0.96, p= 0.02), with a plateau effect from five copies or more. Only when higher copy numbers of beta-defensin genes were present, transcription was significantly up-regulated by tumor necrosis factor alpha (p< 0.0001), which resulted in a better anti-microbial activity in vitro. When comparing healthy smokers with COPD patients, a copy number >/= 5 was associated with increased risk for COPD with an adjusted Odds ratio of 1.8 (CI: 1.1- 2.8; p= 0.02), which was confirmed by a second independent case-control study. CONCLUSION: Genomic copy number variation of beta-defensin encoding genes has a functional role in airway epithelial cells, which may contribute to the pathogenesis of COPD.

The antimicrobial peptide DEFB1 is associated with caries.
J Dent Res. 2010 Jun;89(6):631-6.
Genetics is an important component in the determination of individual susceptibility to caries and periodontal diseases. Since beta defensin 1 (DEFB1) localizes in the oral cavity, we tested if variation in DEFB1 is associated with caries and periodontitis. We analyzed 3 single-nucleotide polymorphisms in DEFB1 in DNA samples from unrelated individuals. Carrying a copy of the variant allele of the DEFB1 marker rs11362 (G-20A) increased the DMFT and DMFS scores more than five-fold. Also, carrying a copy of the variant allele of the DEFB1 marker rs179946 (G-52A) correlated with low DMFT scores. We found a high-caries-experience haplotype (GCA), which increased DMFT scores two-fold, and a low- caries-experience haplotype (ACG), which decreased DMFT scores two-fold, in the DEFB1 promoter. No association between DEFB1 genetic markers and periodontal disease was found. Our results suggest that functional polymorphisms of DEFB1 are potential markers for caries.

Validation of a quantitative assay for human neutrophil peptide-1, -2, and -3 in human plasma and serum by liquid chromatography coupled to tandem mass spectrometry.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 May 1;878(15-16):1085-92.
A quantitative assay for simultaneous measurement of individual human neutrophil peptide-1, -2 and -3 concentrations will aid in exploring the potential of these antimicrobial peptides as biomarkers for various diseases. Therefore, a liquid chromatography-tandem mass spectrometry method has been developed and validated to allow separate quantification of the three human neutrophil peptides in human plasma and serum. Plasma and serum samples (100 microl) were deproteinized by precipitation, followed by chromatographic separation on a Symmetry 300 C18 column (50 mm x 2.1mm I.D., particle size 3.5 microm), using a water-methanol gradient containing 0.25% (v/v) formic acid and human alpha-defensin 5 as internal standard. Tandem mass spectrometric detection was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization. Despite low fragmentation efficiency of the antimicrobial peptides, multiple reaction monitoring was used for detection, though selecting the quaternary charged ions as both precursor and product. The method was linear for concentrations between 5 and 1000 ng/ml with a limit of detection around 3 ng/ml for all peptides. Intra- and inter-assay precisions were 14.8 and 19.1%, respectively, at the lowest measured endogenous concentration (6.4 ng/ml of HNP-1 in plasma), representing the lower limit of quantification of the assay. Recoveries of HNP-1, -2 and -3 from plasma and serum ranged between 85 and 128%. Analysis of serum samples from intensive care patients showed average concentrations of 362, 570 and 143 ng/ml for HNP-1, -2 and -3, respectively.

Expression of antimicrobial peptides in cutaneous infections after skin surgery.
Br J Dermatol. 2010 Jul;163(1):121-7.
BACKGROUND: Increasing numbers of antibiotics have lost efficiency because of bacterial resistance. The consequences can be severe when surgical wounds become infected during postoperative care. Natural peptide antibiotics, the so-called host defence peptides (HDPs), have been investigated since the 1990s in a search for alternative treatment strategies. HDPs build up a protection shield against pathological microorganisms, especially in human epithelium. The use of HDPs is currently being discussed as a new antimicrobial therapeutic strategy. Accordingly, a profound knowledge of the quantitative relationships of the effectors is essential. OBJECTIVES: To evaluate differences in HDP expression between postoperatively inflamed and healthy epithelium. METHODS: Expression profiles of the genes encoding HDP human beta-defensin (hBD)-1 (DEFB1, previously known as HBD-1), hBD-2 (DEFB4A, previously known as HBD-2), hBD-3 (DEFB103A, previously known as HBD-3) and psoriasin (S100A7) were assessed in samples of surgical wound healing disorders (n = 27) and healthy epithelium (n = 16) by using real-time polymerase chain reaction. Immunohistochemical staining was performed in the same samples. RESULTS: A significant overexpression of DEFB4A (P < 0.001), DEFB103A (P = 0.001) and S100A7 (P < 0.001) was found in cutaneous surgical site infections. Immunohistochemistry revealed intensely elevated protein levels of psoriasin in infected wounds, and differences in distribution with respect to the epithelial layers. CONCLUSIONS: The study demonstrates upregulated mRNA expression and protein levels of HDPs in postoperatively inflamed epithelium. The results may be a starting point for novel pharmacological treatments.

Impact of low-frequency pulsed magnetic fields on defensin and CRP concentrations in patients with painful diabetic polyneuropathy and in healthy subjects.
Electromagn Biol Med. 2010 Jun;29(1-2):19-25.
AIM: The aim was to assess whether magnetic field influences defensin and CRP concentrations in patients with diabetic polyneuropathy and in healthy subjects. METHODS: 61 diabetic patients were randomly divided into 2 groups: study group-32 patients exposed to low-frequency magnetic field; and control group-29 patients with sham exposure. Additionally, 20 healthy subjects exposed to low-frequency magnetic field. Exposures were performed during 3 weeks, 5 days in a week. Defensin and CRP concentrations were measured at baseline, after 3 weeks and at the end of the study. RESULTS: There were no significant changes in defensin concentration in patients with diabetes in both the real and sham exposure group. We observed increased concentration of defensin in healthy subjects in week 5 vs. baseline value (P<0.02). CONCLUSIONS: Magnetic field has no impact on defensin concentration in diabetic patients but has positive influence on this parameter in healthy subjects.