Clinical Studies

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Last updated: 13th August 2010

Clinical Studies
Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis
Clin Exp Immunol. 2003 Jan;131(1):90-101
The impact of chronic inflammation on the expression of human alpha-defensins 5 and 6 (HD-5, HD-6), beta-defensins 1 and 2 (hBD-1, hBD-2) and lysozyme in epithelial cells of small and large intestine was investigated. Intestinal specimens from 16 patients with ulcerative colitis (UC), 14 patients with Crohn's disease (CD) and 40 controls with no history of inflammatory bowel disease were studied. mRNA expression levels of the five defence molecules were determined in freshly isolated epithelial cells by real-time quantitative RT-PCR. Specific copy standards were used allowing comparison between the expression levels of the different defensins. HD-5 and lysozyme protein expression was also studied by immunohistochemistry. Colonic epithelial cells from patients with UC displayed a significant increase of hBD-2, HD-5, HD-6 and lysozyme mRNA as compared to epithelial cells in controls. Lysozyme mRNA was expressed at very high average copy numbers followed by HD-5, HD-6, hBD-1 and hBD-2 mRNA. HD-5 and lysozyme protein was demonstrated in metaplastic Paneth-like cells in UC colon. There was no correlation between hBD-2 mRNA levels and HD-5 or HD-6 mRNA levels in colon epithelial cells of UC patients. Colonic epithelial cells of Crohn's colitis patients showed increased mRNA levels of HD-5 and lysozyme mRNA whereas ileal epithelial cells of Crohn's patients with ileo-caecal inflammation did not. Chronic inflammation in colon results in induction of hBD-2 and alpha-defensins and increased lysozyme expression. hBD-1 expression levels in colon remain unchanged in colitis. The high antimicrobial activity of epithelial cells in chronic colitis may be a consequence of changes in the epithelial lining, permitting adherence of both pathogenic bacteria and commensals directly to the epithelial cell surface.

Single-nucleotide polymorphisms (SNPs) in human beta-defensin 1: high-throughput SNP assays and association with Candida carriage in type I diabetics and nondiabetic controls.
J Clin Microbiol. 2003 Jan;41(1):90-6.
beta-Defensins are cationic antimicrobial peptides expressed in epithelia. They exhibit antibacterial, antifungal, and antiviral properties. Defensins are a component of the innate immune response, and it has been proposed that they have a protective role in the oral cavity. Previous studies have shown that human beta-defensin 1 (hBD-1) is constitutively expressed in oral epithelial cells but that expression varies between individuals. We tested the hypothesis that genetic variations in defensin peptide expression may be associated with opportunistic infections. This may be critical in the immunocompromised patient population, in which innate immune responses may have a relatively more important role. Oral Candida carriage status and the presence of six single-nucleotide polymorphisms (SNPs) in the DEFB1 gene encoding hBD-1 were evaluated in type I diabetic patients (n = 43) and nondiabetic controls (n = 50). Genomic DNA was obtained from buccal swabs. Portions of the DEFB1 gene were amplified, and each SNP was analyzed by a TaqMan assay, standardized with control DNA of known genotype. Candida carriage status was determined from unstimulated saliva on CHROMagar plating medium. A low level of Candida carriage was defined as < or = 350 CFU/ml. A high level of Candida carriage was seen in 44% of the diabetic subjects but only in 28% of the nondiabetic controls (P < 0.05). C. albicans predominated; however, diabetic subjects, especially those with high levels of carriage, showed an increased proportion of Candida glabrata and C. tropicalis. There was a strong association between an SNP in the 5' untranslated region (C-->G at position -44) and Candida carriage in both groups. Among individuals in the diabetic population who had the SNP allele 2 (G), 58% had low CFU, while 6% had high CFU. The C-->G SNP at position -44 is associated with low levels of Candida carriage. The resultant odd ratios are statistically significant for a protective effect (odd ratios, 25 for diabetic subjects and 8.5 for nondiabetic subjects). These results indicate that genetic variations in the DEFB1 gene encoding hBD-1 may have a major role in mediating and/or contributing to susceptibility to oral infection.

Differential expression of alpha- and beta-defensins in human peripheral blood.
Eur J Clin Invest. 2003 Jan;33(1):82-7.
BACKGROUND: Human defensin peptides with broad-spectrum antimicrobial activity have been implicated in the human defence response towards microbial invasion. Two families of defensins designated alpha- and beta-defensins, respectively, have been identified. Little is known about the expression of both defensin families in human peripheral blood. The purpose of this study was to examine the expression of alpha- and beta-defensin genes in human peripheral blood. MATERIAL AND METHODS: Fifty-one healthy blood donors were screened for defensin expression. Blood from defensin responders was stimulated by lipopolysaccharide or heat-inactivated Pseudomonas aeruginosa ex vivo. Levels of mRNA were assessed by semiquantitative RT-PCR. Southern blot analysis and sequencing were used to confirm the identity of defensin gene transcripts. Western blotting analysis was used to detect the expression of defensin peptides. RESULTS: beta-defensin was undetected in human peripheral blood without stimulation. Following stimulation by lipopolysaccharide or heat-inactivated bacterial cells, the majority (88.2%) of healthy individuals had a detectable expression for beta-defensin-1 gene and 39.2% for beta-defensin-2 gene, compared with none for beta-defensin-3. beta-defensin-1 and -2 mRNAs in the stimulated human peripheral blood of responders became detectable at 3 h and showed a maximum at 6 h following induction by 100 ng mL-1 of lipopolysaccharide or bacterial cells. In contrast, human alpha-defensins 1-3 mRNA are constitutively expressed in peripheral leukocytes but not up-regulated by lipopolysaccharide or bacterial cells. CONCLUSIONS: In human peripheral blood, beta-defensin-1 and -2 genes were transiently transcribed and translated following the induction of lipopolysaccharide or heat-inactivated bacterial cells, whereas alpha-defensins 1-3 genes were constitutively transcribed, and beta-defensin-3 gene was not expressed. The inducible expression of beta-defensin-1 and -2 genes showed interindividual variability.

Bacterial vaginosis and vaginal fluid defensins during pregnancy
Am J Obstet Gynecol. 2002 Nov;187(5):1267-71
OBJECTIVE: The purpose of this study was to examine the association between Gram stain findings of vaginal fluid and the concentration of vaginal fluid neutrophil defensins. STUDY DESIGN: Vaginal fluid specimens obtained from 749 women at 24 to 29 weeks of gestation were tested for bacterial vaginosis and assayed for neutrophil defensins. Bacterial vaginosis was studied as a categoric variable (negative, intermediate, and positive), whereas defensins were examined as a continuous measure and dichotomized on the basis of presence versus absence and at the 90th percentile. Multiple linear and logistic regression models were used to assess the relationship between bacterial vaginosis and defensins. RESULTS: Women with intermediate bacterial vaginosis were more likely to have elevated vaginal fluid neutrophil defensins (>90th percentile) than women with normal vaginal flora (adjusted odds ratio, 2.3; 95% CI, 1.3, 4.2), whereas women with frank bacterial vaginosis were not (adjusted odds ratio, 1.3; 95% CI, 0.7, 2.6). Among women with any detectable defensin (69.5% of the study population), intermediate bacterial vaginosis was associated positively with defensin concentrations in multiple linear regression models (P =.007). Women with intermediate and frank bacterial vaginosis had 5.9 microg/mL and 2.2 microg/mL higher defensin concentrations, respectively, than women who did not have bacterial vaginosis. The presence of leukocytes in vaginal fluids was associated positively with defensin concentrations (P <.0001). CONCLUSION: Changes in vaginal microflora during mid pregnancy are associated with an increased concentration of vaginal fluid neutrophil defensins.

Association between elevated neutrophil defensin levels and endometritis.
J Infect Dis. 2002 Sep 15;186(6):792-7.
The role of host defenses in the pathogenesis of pelvic inflammatory disease (PID) remains largely uncharacterized. The antimicrobial peptides defensins are important components of innate host defense. To explore the relationship between neutrophil defensins and upper genital tract infection, 377 women who were at risk for PID were enrolled in a study. Women infected with Neisseria gonorrhoeae, Trichomonas vaginalis, or Chlamydia trachomatis had higher median levels of neutrophil defensins (human neutrophil peptides 1-3) in the vagina than did uninfected women. Neutrophil defensins were strongly associated with the presence of endometritis after the analysis was controlled for the presence of sexually transmitted diseases. Vaginal neutrophils were associated with endometritis only in the presence of elevated defensin levels, which highlights the importance of neutrophil activation, rather than the presence of neutrophils alone, in this inflammatory process. Neutrophil defensins appear to participate in the host defense in ascending pelvic infection and the pathogenesis of PID.

Human beta-defensin 2 but not beta-defensin 1 is expressed preferentially in colonic mucosa of inflammatory bowel disease.
Eur J Gastroenterol Hepatol. 2002 Jul;14(7):745-52.
OBJECTIVE : Various antimicrobial peptides such as defensins are part of innate immunity and contribute to the intestinal barrier that may be defective in inflammatory bowel disease (IBD). This study investigated beta-defensin mRNA and peptide expression in the colon from controls and patients with Crohn's disease, ulcerative colitis or unspecific colitis as inflammatory controls. METHODS : Mucosal mRNA expression was measured by multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) with primers for human beta-defensin 1 (HBD-1) and human beta-defensin 2 (HBD-2) in CaCo-2 cells and in biopsies from 103 patients (33 controls, 24 Crohn's disease patients, 36 ulcerative colitis patients, 10 unspecific colitis patients). Paraffin-embedded tissue from colonic resections was tested for HBD-1 and HBD-2 peptides by immunohistochemistry. RESULTS : HBD-1 mRNA was expressed constitutively whereas HBD-2 was induced by pro-inflammatory cytokines in CaCo-2 cells. HBD-1 mRNA was detectable in 61% of control and Crohn's disease biopsies and 53% of ulcerative colitis biopsies. HBD-2 transcript was expressed differentially, with 18% of control biopsies positive as opposed to 34% in Crohn's disease and 53% in ulcerative colitis. HBD-2 mRNA but not HBD-1 mRNA was expressed preferentially in inflamed areas. Immunohistochemical investigation demonstrated the presence of defensin peptides in colonic epithelium as well as the differential induction in IBD. CONCLUSIONS : HBD-1 is expressed constitutively in colonic tissue irrespective of inflammation. HBD-2 is barely present in uninflamed colon but it is induced in inflammation. The lower expression of HBD-2 in Crohn's disease compared with ulcerative colitis indicates different responses of the mucosal innate defence. Defensins may play a crucial role in controlling pathogen invasion in IBD, although the functional significance remains to be established.

Raised plasma concentrations of alpha-defensins in patients with idiopathic pulmonary fibrosis.
Thorax. 2002 Jul;57(7):623-8.
BACKGROUND: Neutrophils are thought to play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Human neutrophils contain antimicrobial and cytotoxic peptides in the azurophil granules which belong to a family of mammalian neutrophil peptides named alpha-defensins. A study was undertaken to investigate the role of alpha-defensins in the pathogenesis of IPF. METHODS: The concentrations of alpha-defensins (human neutrophil peptides (HNPs) 1, 2, and 3) in plasma and bronchoalveolar lavage (BAL) fluid of 30 patients with IPF and 15 healthy subjects were measured by radioimmunoassay. RESULTS: The concentrations of alpha-defensins in plasma, but not in BAL fluid, were significantly higher in IPF patients than in controls. BAL fluid concentrations of interleukin (IL)-8 in patients with IPF, which were significantly higher than in controls, correlated with those of alpha-defensins. An inverse relationship was seen between plasma alpha-defensin levels and the arterial oxygen tension (PaO(2)) and pulmonary function (vital capacity (%VC), forced expiratory volume in 1 second (FEV(1)), and carbon monoxide transfer factor (%TLCO)) in patients with IPF. Plasma levels of alpha-defensins also correlated with the clinical course in IPF patients with an acute exacerbation. Immunohistochemically, positive staining was observed inside and outside neutrophils in the alveolar septa, especially in dense fibrotic areas. CONCLUSION: These findings suggest that alpha-defensins play an important role in the pathogenesis of IPF, and that the plasma alpha-defensin level may be a useful marker of disease severity and activity.

Neutrophil defense in patients undergoing bone marrow transplantation: bactericidal/permeability-increasing protein (BPI) and defensins in graft-derived neutrophils.
Transplantation. 2002 May 15;73(9):1522-6.
BACKGROUND: Even after neutrophil counts return to near normal levels, patients undergoing myeloablative chemotherapy and bone marrow transplantation (BMT) are at risk for invasive bacterial infections, raising the possibility that their neutrophil function might be impaired. To assess potential qualitative defects in neutrophil function in patients undergoing BMT, we measured neutrophil content of the antimicrobial (poly)peptides BPI and defensins. METHODS: Neutrophil extracts were analyzed for content of BPI by Western blotting and ELISA and for defensin peptides by acid-urea polyacrylamide gel electrophoresis (PAGE). Antibacterial activity of neutrophil extracts was measured against Escherichia coli K1/r, a clinical isolate sensitive to synergistic killing by BPI and defensins. RESULTS: Neutrophil extract BPI content on post-BMT days +20, +30, and +100 (169+/-35, 232+/-57, and 160+/-55 ng per 106 neutrophils, respectively) was similar to the neutrophil BPI content of normal controls (163+/-35 ng per 106 neutrophils). Neutrophil defensin content also did not vary during this time-span. Activity of neutrophil extracts against E. coli K1/r did not differ between BMT patients and controls. CONCLUSION: At post-BMT days +20 to +100, neutrophils derived from engrafted marrow contain normal quantities of BPI and defensins. Any deficiencies of neutrophil function during this phase are not due to inadequate expression of these antimicrobial (poly)peptides but could reflect abnormalities in other aspects of neutrophil function.

The novel human beta-defensin-3 is widely expressed in oral tissues.
Eur J Oral Sci. 2002 Apr;110(2):121-4.
The purpose of this study was to investigate the expression of human beta-defensins (hBD), especially of the recently discovered hBD-3, in oral tissues by reverse-transcription polymerase chain reaction (RT-PCR). Primary oral keratinocytes (n = 3) and fibroblasts (n = 3), 64 non-inflamed and 40 inflamed oral tissue samples, and 10 samples of salivary glands, were examined. The transcripts for hBD-3 (61/64), as well as for hBD-1 (64/64) and hBD-2 (54/64), were found to be widely expressed in non-inflamed oral tissues. In contrast, only 23, 22 and 24 of the 40 inflamed tissues showed detectable hBD-1, -2 and -3 transcripts, respectively. In salivary glands, mRNA expression was constitutive for hBD-1, frequent for hBD-2 (9/10), and infrequent for hBD-3 (4/10). Oral keratinocytes, but not fibroblasts, contained transcripts for all beta-defensins, suggesting that the novel hBD-3 is also produced in the epithelial compartment of oral tissues. The results indicate an important role for the novel hBD-3, as well as for hBD-1 and hBD-2, in the innate oral epithelial host defense.

Increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection.
Am J Respir Crit Care Med. 2002 Apr 1;165(7):992-5.
Pneumonia and systemic infection are common in premature infants. The antimicrobial peptides human beta-defensin 1 and 2 (hBD-1 and hBD-2) and the cathelicidin LL-37/hCAP-18 are effector molecules of the innate respiratory immune system. It is unknown whether these host defense substances are produced in the respiratory tract of newborns. Concentrations of these peptides were determined in tracheal aspirates of mechanically ventilated newborn infants. All three antimicrobial peptides could be detected in airway lining fluid with equivalent levels in term and preterm newborns. Concentrations of antimicrobial peptides correlated with each other and with levels of interleukin-8 and tumor necrosis factor-alpha in the bronchoalveolar lavage fluid. Pulmonary or systemic infections were associated with significantly increased concentrations of LL-37, hBD-1, and hBD-2. Western blotting detected mature peptides in the lavage fluid. In conclusion, mucosal antimicrobial peptides are present in lung secretions of premature and mature newborns. The molecules are upregulated in response to infection and inflammation and probably represent effector molecules of the respiratory defense system.