Clinical Studies

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Last updated: 13th August 2010

Clinical Studies
Differential expression of human beta defensin 2 and human beta defensin 3 in human middle ear cholesteatoma.
Ann Otol Rhinol Laryngol. 2007 Mar;116(3):235-40.
OBJECTIVES: The purpose of this study was to investigate the differential expressions of human beta defensin (hBD) 2 and hBD-3 in human middle ear cholesteatoma epithelium. METHODS: The expressions of hBD-2 and hBD-3 were analyzed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical staining. Samples were obtained from 10 patients who underwent middle ear surgery for middle ear cholesteatoma. RESULTS: Real-time RT-PCR and Western blot analysis showed that the messenger RNAs and proteins of hBD-2 and hBD-3 were higher in the cholesteatoma epithelium than in normal external auditory canal skin. In cholesteatoma epithelium, hBD-2 and hBD-3 activities were present in the upper granular layer and in the prickle cell layer, but in the normal skin they were poorly expressed in all layers. CONCLUSIONS: Increased expressions of hBD-2 and hBD-3 in cholesteatoma epithelium suggest that cholesteatoma, a chronic inflammatory state of middle ear keratinocytes, may induce an innate immune response. That the induction of hBD-2 was found to be more intense than that of hBD-3 in cholesteatoma epithelium implies that hBD-2 is the major effector in terms of chronic epithelial inflammatory responses.

Expression of human beta-defensins in patients with mycosis fungoides.
Arch Dermatol Res. 2007 Jul;299(4):221-4.
The human beta-defensins (hBDs) are peptides with a strong antimicrobial activity. Patients with atopic dermatitis (AD) and mycosis fungoides (MF) are prone to skin infections. We aimed to investigate the mRNA expression of hBDs in lesional and non-lesional skin of MF patients, and to compare the data with hBD levels found in AD patients and healthy controls. In this prospective pilot study, 13 patients with MF were recruited. Punch biopsies were harvested from the centre of the tumour (lesional) as well as a healthy skin site (non-lesional controls). In addition to the specimens of MF patients, skin samples (healthy controls) were obtained from healthy subjects (n = 15) and patients with acute AD (n = 14). In order to detect mRNA of hBDs, we performed quantitative real-time reverse transcriptase polymerase chain reaction. As compared to healthy controls, skin of patients with MF (non-lesional and lesional) and AD patients showed significantly lower hBD-1 mRNA expression and significantly higher hBD-2 and hBD-3 mRNA expression. HBD-1 mRNA levels of lesional skin were significantly lower than those of non-lesional skin. By contrast, significantly increased hBD-2 and hBD-3 mRNA expression was found in lesional skin of MF patients when compared to non-lesional skin. HBD mRNA expression in lesional skin of MF patients did not significantly differ from hBD expression that was observed in AD lesions. We observed an identical pattern of hBD expression in MD and AD suggesting a common regulatory mechanism that might mainly be driven by T helper 2 lymphocytes.

Alteration in human defensin-5 expression following gastric bypass surgery.
J Clin Pathol. 2007 Sep;60(9):1029-34.
BACKGROUND: Roux-en-Y gastric bypass surgery provides a novel human model to investigate small bowel mucosal innate immunity, in which there is loss of gastric acid-mediated protection against orally-acquired microorganisms. AIM: To study changes in jejunal mucosal immunoreactivity of human defensin (HD)-5, an antimicrobial peptide normally produced by Paneth cells. METHODS: Mucosal samples were obtained from 18 female patients (24-54 years), from the same segment of jejunum during and after gastric bypass surgery. Samples were used for bacterial culture and immunohistochemistry using anti-HD-5 antibody. The number of immunoreactive cells per crypt and villus were determined and expressed as mean (SD). RESULTS: No bacteria were cultured from any of the perioperative jejunal samples but colonies of bacteria normally present in the pharynx were identified during culture of all postoperative jejunal biopsy specimens (1->100 colonies). Paneth cell numbers per crypt were unchanged after gastric bypass (4.16 (0.71) vs 4.24 (0.78)). However, following surgery, there was an increase in HD-5-positive intermediate cells per crypt (0.25 (0.41) vs 1.12 (0.66), p<0.01), HD-5 staining enterocytes per crypt (0.03 (0.09) vs 1.38 (1.10), p<0.01), HD-5 staining material in the crypt lumen (crypt lumens: 5.0% (10.9%) vs 68.1% (27.9%), p<0.01) and HD-5 immunoreactivity coating the luminal surface of villus enterocytes (villi sampled: 15.0% (31.0%) vs 67.5% (42.0%), p<0.01). CONCLUSIONS: Bacteria normally resident in the pharynx were present in the proximal jejunal mucosa following Roux-en-Y gastric bypass surgery. After gastric bypass, there was increased secretion of HD-5 and an increase in HD-5 expressing intermediate cells and enterocytes in the crypt. The increase in HD-5 expression in the jejunal mucosa following gastric bypass surgery is likely to be secondary to exposure to orally-acquired microorganisms.

Changes in vaginal morphology, steroid receptor and natural antimicrobial content following treatment with low-dose mifepristone
Contraception. 2007 Apr;75(4):271-80
BACKGROUND: We have previously shown that the antigestagen mifepristone is contraceptive when given in a daily dose of 5 mg, po. Epidemiological studies suggest that gestagen-only contraceptives may increase the risk of transmission of human immunodeficiency virus (HIV) due to effects on the vaginal defenses to infection. We investigate the effects of mifepristone on vaginal thickness, steroid receptor and natural antimicrobial content and pharmacokinetics of mifepristone. METHODS: In a pilot study, eight women were given mifepristone 5 mg/day for an average of 33 days. Ovarian function was assessed by measurement of estradiol and progesterone in blood and their metabolites in urine and by serial ultrasound of their ovaries. Vaginal biopsies were collected before (late proliferative) and after taking mifepristone. RESULTS: All subjects showed a similar pattern of descending serum concentrations of mifepristone. The elimination phase half-life was 18+/-5.1 h (mean+/-SD). Mean Cmax measured at 1 h was 641.7 nmol/L (range, 502-740 nmol/L). All eight women reported amenorrhea for the duration of treatment and seven of eight women showed biochemical and ultrasound evidence of anovulation. There was no significant change in vaginal thickness following treatment [342+/-40 microm pretreatment, 303+/-69 microm posttreatment (mean+/-SEM); p>.05]. Estrogen (ERalpha, ERbeta) and androgen receptor were expressed in both vaginal epithelium and subepithelial stroma, whereas progesterone receptor was expressed predominantly in the subepithelial stroma. There was no change in receptor content and distribution following mifepristone treatment. Natural antimicrobial mRNA [secretory leukocyte protease inhibitor, human beta defensins mRNA (HBD1, HBD2, HBD3, HBD5), granulysin and elafin] was extracted from the vaginal tissues, and the content was unaffected by mifepristone treatment. CONCLUSION: The absence of changes in vaginal thickness, steroid receptor and natural antimicrobial content and its distribution in this preliminary study suggests that in contrast to other estrogen-free contraceptives, mifepristone is unlikely to be associated with the increased risk of transmission of HIV and other sexually transmitted infections.

Expression and immunolocalisation of antimicrobial peptides within human palatine tonsils
J Laryngol Otol. 2007 Oct;121(10):973-8
BACKGROUND: Recurrent acute tonsillitis is one of the most frequent ENT referrals, yet its pathogenesis remains poorly understood, and tonsillectomy still costs the National Health Service more than pound 60,000000 annually. Antimicrobial cationic peptides are components of the innate immune system. They are generally small, highly positively charged peptides with broad spectrum antimicrobial activity which function as the body's 'natural antibiotics'. The role of antimicrobial cationic peptides in the susceptibility of patients to recurrent acute tonsillitis is unknown. AIMS: To characterise and compare antimicrobial cationic peptide expression and localisation in human palatine tonsils from control subjects and recurrent acute tonsillitis patients, and to assess the potential role of these peptides in the pathogenesis of tonsillitis. METHODS: Palatine tonsils were harvested with informed consent from 19 recurrent acute tonsillitis patients and from five control subjects undergoing tonsillectomy for sleep disorders. Total ribonucleic acid was isolated and antimicrobial cationic peptide expression was characterised using reverse transcription polymerase chain reaction. Fluorescent immunohistochemical techniques were used to localise antimicrobial cationic peptides within fresh frozen tonsil sections. RESULTS: Using molecular analyses, the palatine tonsils from control and recurrent acute tonsillitis subjects were confirmed as a site of expression of the antimicrobial cationic peptides human beta-defensin 1-3, LL-37 (cathelicidin) and Liver expressed antimicrobial peptide-1 (LEAP-1). We also demonstrated for the first time the expression of Liver expressed antimicrobial peptide-2 (LEAP-2). Our analyses indicated that all six antimicrobial cationic peptides were expressed in all 26 tonsil samples. Immunohistochemical staining indicated that the antimicrobial cationic peptides were localised to the tonsil surface and crypt epithelium. However, the surface epithelium of tonsils from recurrent acute tonsillitis patients showed reduced amounts of antimicrobial peptides human beta-defensins 1 and 3, and LL-37, compared with healthy controls. CONCLUSION: The tonsil epithelium synthesizes an array of antimicrobial cationic peptides which function as host defence. Preliminary immunohistochemical data suggest that the surface epithelium of tonsils from recurrent acute tonsillitis patients contains reduced amounts of such peptides, which may increase these patients' susceptibility to infection.

Inducibility of the endogenous antibiotic peptide beta-defensin 2 is impaired in patients with severe sepsis.
Crit Care. 2007;11(1):R19.
INTRODUCTION: The potent endogenous antimicrobial peptide human beta-defensin 2 (hBD2) is a crucial mediator of innate immunity. In addition to direct antimicrobial properties, different effects on immune cells have been described. In contrast to the well-documented epithelial beta-defensin actions in local infections, little is known about the leukocyte-released hBD2 in systemic infectious disorders. This study investigated the basic expression levels and the ex vivo inducibility of hBD2 mRNA in peripheral whole blood cells from patients with severe sepsis in comparison to non-septic critically ill patients and healthy individuals. METHODS: This investigation was a prospective case-control study performed at a surgical intensive care unit at a university hospital. A total of 34 individuals were tested: 16 patients with severe sepsis, 9 critically ill but non-septic patients, and 9 healthy individuals. Serial blood samples were drawn from septic patients, and singular samples were obtained from critically ill non-septic patients and healthy controls. hBD2 mRNA levels in peripheral white blood cells were quantified by real-time polymerase chain reaction in native peripheral blood cells and following ex vivo endotoxin stimulation. Defensin plasma levels were quantified by enzyme-linked immunosorbent assay. RESULTS: Endotoxin-inducible hBD2 mRNA expression was significantly decreased in patients with severe sepsis compared to healthy controls and non-septic critically ill patients (0.02 versus 0.95 versus 0.52, p < 0.05, arbitrary units). hBD2 plasma levels in septic patients were significantly higher compared to healthy controls and critically ill non-septic patients (541 versus 339 versus 295 pg/ml, p < 0.05). CONCLUSION: In contrast to healthy individuals and critically ill non-septic patients, ex vivo inducibility of hBD2 in peripheral blood cells from septic patients is reduced. Impaired hBD2 inducibility may contribute to the complex immunological dysfunction in patients with severe sepsis.

Association of plasma neutrophil elastase levels with other inflammatory mediators and clinical features in adult patients with moderate and severe pneumonia
Respir Med. 2007 Jul;101(7):1521-8
BACKGROUND: Plasma levels of neutrophil elastase (NE) are elevated in several inflammatory diseases and thus this enzyme might be a critical inflammatory marker. However, the role of NE in the pathogenesis of pneumonia has not been determined. The association between the severity of pneumonia and blood levels of inflammatory markers could be relevant to developing a useful indicator of severity and new therapeutic strategies for pneumonia. METHODS: We searched for a useful predictive marker and a new therapeutic strategy against pneumonia, using a prospective, multicenter, population-based investigation. Several inflammatory markers in the circulation including NE, cytokines, defensins, C-reactive protein (CRP) and white blood cell (WBC) counts as well as clinical features were prospectively monitored in 28 adult patients with moderate (n=11) and severe pneumonia (n=17) over a period of 14 days. RESULTS: The value of plasma NE was the highest at entry and significantly declined 2 days later. Trends of cytokines, defensins, CRP and WBC counts were similar but blunter. Microorganisms and the outcome of initial treatment did not significantly affect plasma NE levels. Baseline values of plasma NE were significantly higher in severe, than in moderate pneumonia and this difference between the two types of pneumonia persisted longer than those of any other markers. CONCLUSIONS: Neutrophil elastase appears to play a critical role in severe pneumonia and determination of its concentration in blood could be a useful indicator of severity. Furthermore, clinical trials of anti-NE drugs in patients with severe pneumonia should be promising.

Significance of human beta-defensins in the epithelial lining fluid of patients with chronic lower respiratory tract infections.
Clin Microbiol Infect. 2007 Jan;13(1):63-9.
Human beta-defensins (hBDs) are the most abundant antimicrobial peptides in epithelial cells, and function in the host immune system. Respiratory epithelial cells express hBDs to inhibit bacterial proliferation during respiratory tract infections. The aim of this study was to investigate the release of hBDs into the respiratory tract and their benefit as a host defence system in chronic Pseudomonas aeruginosa infections. The levels of four hBD peptides (hBD-1-hBD-4) were measured in the bronchial epithelial lining fluid (ELF) of nine patients with chronic lower respiratory tract infection caused by P. aeruginosa. Eight patients with idiopathic pulmonary fibrosis and eight volunteers free of pulmonary disease were recruited as controls. ELF was obtained by bronchoscopic microsampling and hBD levels were measured by radioimmunoassays. The antimicrobial effects of hBDs were studied individually and in combination using an in-vitro colony count assay for P. aeruginosa. Concentrations of hBD-1 and hBD-3 tended to be higher in patients with chronic lower respiratory tract infection than in the controls. hBD-2 and hBD-4 were detected in ELF from five and four of nine patients, respectively, but the hBD levels in controls were all below the limits of detection. All patients with infection caused by mucoid P. aeruginosa had detectable hBD-2 and hBD-4 levels in ELF. In-vitro colony count assays showed a potential synergism between hBD-2 and hBD-4 in inhibiting bacterial proliferation. The findings indicate that hBDs, especially hBD-2 and hBD-4, are pathophysiologically important in infections caused by mucoid strains of P. aeruginosa.

Human beta-defensin-2, an antimicrobial peptide, is elevated in scales collected from tinea pedis patients.
Int J Dermatol. 2006 Nov;45(11):1389-90
Human beta-defensin-2 (hBD-2) is a cysteine-rich, cationic, low-molecular-weight, antimicrobial peptide occurring in psoriatic skin scales,1 and belongs to the defensin family of peptides, known for their broad spectrum of antimicrobial activity.2 In order to demonstrate the hypothesis that hBD-2 is elevated in the lesional skin of patients with tinea pedis, relative to normal plantar skin, we measured and compared the level of hBD-2 in tinea pedis, psoriasis, and normal skin using enzyme-linked immunosorbent assay (ELISA).

SNPs in human beta-defensin 1 gene (DEFB1): frequencies in a Mexican population and new PCR-RFLPs assays
Int J Immunogenet. 2006 Oct;33(5):339-42
Polymerase chain reaction-restriction fragment length polymorphism assays for two single nucleotide polymorphisms in the human beta-defensin 1 gene have been validated with real-time PCR in 101 healthy individuals from western Mexico. Allele frequencies were 52.5% (692-A) and 98.5% (1836-A). These assays can be confidently used as a cheaper alternative genotyping method for these sites.