Expression and function of human ribonuclease 4 in the kidney and urinary tract.

Antimicrobial peptides are essential host defense mechanisms that prevent urinary tract infections. Recent studies have demonstrated that peptides in the ribonuclease A superfamily have antimicrobial activity against uropathogens and protect the urinary tract from uropathogenic Escherichia coli (UPEC). Little is known about the antibacterial function or expression of ribonuclease 4 (RNase 4) in the human urinary tract. Here, we show that full-length recombinant RNase 4 peptide and synthetic amino-terminal RNase 4 peptide fragment have antibacterial activity against UPEC and multidrug-resistant (MDR)-UPEC. RNASE4 transcript expression was detected in human kidney and bladder tissue using quantitative real-time PCR. Immunostaining or in situ hybridization localized RNase 4 expression to proximal tubules, principal and intercalated cells in the kidney's collecting duct, and the bladder urothelium. Urinary RNase 4 concentrations were quantified in healthy controls and females with a history of urinary tract infection. Compared with controls, urinary RNase 4 concentrations were significantly lower in females with a history of urinary tract infection. When RNase 4 was neutralized in human urine or silenced in vitro using siRNA, urinary UPEC replication or attachment to and invasion of urothelial and kidney medullary cells increased. These data show that RNase 4 has antibacterial activity against UPEC, is expressed in the human urinary tract, and can contribute to host defense against urinary tract infections.NEW & NOTEWORTHY Ribonuclease 4 (RNase 4) is a newly identified host defense peptide in the human kidney and bladder. RNase 4 kills uropathogenic Escherichia coli (UPEC) and multidrug-resistant UPEC. RNase 4 prevents invasive UPEC infection and suppressed RNase 4 expression may be a risk factor for more severe or recurrent urinary tract infection.

Urine concentrations of human beta-defensins and ribonuclease 7 in urinary tract infection and asymptomatic bacteriuria.

Antimicrobial peptides (AMPs) provide a first line of defense against bacterial infections. Here we report that urine levels of AMPs, locally produced in the urinary tract, are lower in individuals with asymptomatic bacteriuria (ABU) compared to patients with urinary tract infection (UTI).

Insulin and the phosphatidylinositol 3-kinase signaling pathway regulate Ribonuclease 7 expression in the human urinary tract.

Diabetes mellitus is a systemic disease associated with a deficiency of insulin production or action. Diabetic patients have an increased susceptibility to infection with the urinary tract being the most common site. Recent studies suggest that Ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that plays an important role in protecting the urinary tract from bacterial insult. Because the impact of diabetes on RNase 7 expression and function are unknown, we investigated the effects of insulin on RNase 7 using human urine specimens. The urinary RNase 7 concentrations were measured in healthy control patients and insulin-deficient type 1 diabetics before and after starting insulin therapy. Compared with controls, diabetic patients had suppressed urinary RNase 7 concentrations, which increased with insulin. Using primary human urothelial cells, the mechanisms by which insulin stimulates RNase 7 synthesis were next explored. Insulin induced RNase 7 production via the phosphatidylinositide 3-kinase signaling pathway (PI3K/AKT) to shield urothelial cells from uropathogenic E. coli. In contrast, uropathogenic E. coli suppressed PI3K/AKT activity and RNase 7 production. Thus, insulin and PI3K/AKT signaling are essential for RNase 7 expression and increased infection risks in diabetic patients may be secondary to suppressed RNase 7 production. Our data may provide unique insight into novel urinary tract infection therapeutic strategies in at-risk populations.

Novel Method for the Analysis of Ribonuclease Based on Fluorescence Recovery of a Cationic Aluminum Phthalocyanine-RNA Association Complex as a Red-emitting Fluorogenic Substrate.

The conventional spectrophotometric method that is often applied to determine ribonuclease (RNase) has disadvantages that include cumbersome manipulation, time-consuming processing and a lack of linear range. We had found that a low concentration of RNA could induce cationic aluminum phthalocyanine (tetra(trimethylammonio)aluminum phthalocyanine (TTMAAlPc)), which emitted strong red fluorescence to aggregate in neutral media, resulting in an almost complete quenching of fluorescence from the cationic aluminum phthalocyanine. The RNA is degraded through hydrolysis by RNase, which destroys the induced aggregation of TTMAAlPc on RNA and releases free TTMAAlPc, leading to a significant fluorescence recovery of the reaction system. Based on this new finding, a method to detect RNase by enhanced fluorescence was established using the TTMAAlPc-RNA association complex as a new fluorogenic substrate of RNase. The optimal conditions were determined, and the interfering foreign substances were investigated. Under optimal conditions, the linear range was 0.05 - 50 µg/L, and the detection limit was 0.02 µg/L. This method was applied for the analysis of ribonuclease in urine specimens from normal adults, and the results were consistent with those determined by conventional spectrophotometric methods. The developed method is easy to operate and highly sensitive, and has a wide linear range, thus solving issues with conventional methods. This study applied, for the first time, cationic phthalocyanine as a fluorescent probe in the detection of nuclease, which provides new applications of phthalocyanine as a fluorescent probe emitting at the red wavelength region.

An endogenous ribonuclease inhibitor regulates the antimicrobial activity of ribonuclease 7 in the human urinary tract.

Recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Previously, we have shown that ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that has a broad-spectrum antimicrobial activity against uropathogenic bacteria. The urothelium of the lower urinary tract and intercalated cells of the kidney produce RNase 7, but regulation of its antimicrobial activity has not been well defined. Here, we characterize the expression of an endogenous inhibitor, ribonuclease inhibitor (RI), in the urinary tract and evaluate its effect on the antimicrobial activity of RNase 7. Using RNA isolated from non-infected human bladder and kidney tissue, quantitative real-time polymerase chain reaction showed that RNH1, the gene encoding RI, is constitutively expressed throughout the urinary tract. With pyelonephritis, RNH1 expression and RI peptide production significantly decrease. Immunostaining localized RI production to the umbrella cells of the bladder and intercalated cells of the renal collecting tubule. In vitro assays showed that RI bound to RNase 7 and suppressed its antimicrobial activity by blocking its ability to bind the cell wall of uropathogenic bacteria. Thus, these results demonstrate a new immunomodulatory role for RI and identified a unique regulatory pathway that may affect how RNase 7 maintains urinary tract sterility.

Ribonuclease 7, an antimicrobial peptide upregulated during infection, contributes to microbial defense of the human urinary tract.

The mechanisms that maintain sterility in the urinary tract are incompletely understood; however, recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Ribonuclease 7 (RNase 7), a potent antimicrobial peptide contributing to urinary tract sterility, is expressed by intercalated cells in the renal collecting tubules and is present in the urine at levels sufficient to kill bacteria at baseline. Here, we characterize the expression and function of RNase 7 in the human urinary tract during infection. Both quantitative real-time PCR and enzyme-linked immunosorbant assays demonstrated increases in RNASE7 expression in the kidney along with kidney and urinary RNase 7 peptide concentrations with infection. While immunostaining localized RNase 7 production to the intercalated cells of the collecting tubule during sterility, its expression during pyelonephritis was found to increase throughout the nephron but not in glomeruli or the interstitium. Recombinant RNase 7 exhibited antimicrobial activity against uropathogens at low micromolar concentrations by disrupting the microbial membrane as determined by atomic force microscopy. Thus, RNase 7 expression is increased in the urinary tract with infection and has antibacterial activity against uropathogens at micromolar concentrations.

[Effect of two doses of budesonide on exhaled nitric oxide and urinary EPX excretion in asthmatic children]. / Einfluss unterschiedlicher Dosen von Budesonid auf die exhalierte NO-Konzentration und die EPX-Konzentration im Urin bei Kindern mit Asthma bronchiale.

The use of objective outcome measures that assess airway inflammation in pediatric asthma can provide a good evaluation of asthma severity and treatment response. In this double-blind and randomized study the effects of 200 micro g of budesonide and 800 micro g of budesonide on markers of inflammation (exhaled nitric oxide (eNO), eosinophil protein X (EPX) excretion in urine) and on lung function (FEV (1)) were prospectively investigated in 24 ICS-naive children with mild persistent to moderate persistent asthma over a period of eight weeks. After eight weeks of treatment 200 micro g and 800 micro g of budesonide led to a significant decrease (p < 0.025) in eNO [median (90 % interval): 200 micro g: - 17.2 ppb (- 54.6 to 0.9); 800 micro g: - 13.2 ppb (- 44.6 to - 1.7)]. A significant change in urinary EPX excretion was only observed in the high dose group [200 micro g: - 10.3 micro g/mmol creatinine (- 116.2 to 50.5), p = 0.9; 800 micro g: - 49.2 micro g/mmol creatinine (- 231.0 to 48.7), p = 0.02]. However, a significant difference between the change from baseline after 8 weeks of either group was found neither for eNO (p = 0.66) nor for EPX excretion (p = 0.04). In conclusion, our data demonstrate that 800 micro g budesonide per day did not show any advantage in reduction of airway inflammation, measured by eNO and urinary EPX excretion, in children with mild persistent to moderate persistent asthma.

Urinary eosinophilic protein X, atopy, and symptoms suggestive of allergic disease at 3 years of age.

BACKGROUND: Urinary eosinophilic protein X (U-EPX) measurement is easy to perform in children. However, its use for prediction, diagnosis, and monitoring of asthma and atopy is unclear. OBJECTIVE: We sought to investigate the relationship between U-EPX and clinical phenotypes suggestive of allergic diseases. METHODS: U-EPX measurement (RIA), respiratory questionnaires, and skin testing were completed at age 3 years in 903 children followed prospectively from birth. Specific airway resistance was measured in 503 currently asymptomatic children by using whole-body plethysmography during tidal breathing. RESULTS: Nonatopic children with wheezing or eczema had slightly increased U-EPX levels compared with nonatopic asymptomatic children. U-EPX levels (geometric mean EPX/creatinine ratio) were as follows: nonatopic asymptomatic children (n = 313), 61.3 microg/mmol (95% CI, 56.4-66.6 microg/mmol); nonatopic children with wheezing (n = 148), 71.2 microg/mmol (95% CI, 63.2-80.1 microg/mmol); nonatopic children with eczema (n = 90), 65.7 microg/mmol (95% CI, 56.7-76.2 microg/mmol); and nonatopic children with wheezing and eczema (n= 86), 79.7 microg/mmol (95% CI, 67.4-94.3 microg/mmol). Children who had persistent atopy early in life had significantly higher U-EPX levels at age 3 years (nonatopic at 1 and 3 years [n = 263], 63.4 microg/mmol [95% CI, 58.4-69.0 microg/mmol]; atopic at 1 but not 3 years [n = 24], 65.1 microg/mmol [95% CI, 43.8-96.7 microg/mmol]; nonatopic at 1 year and atopic at 3 years [n = 62], 90.0 microg/mmol [95% CI, 74.6-108.4 microg/mmol]; atopic at 1 and 3 years [n = 35], 111.5 microg/mmol [95% CI, 89.2-139.3 microg/mmol]; P <.002). Atopy alone and with wheezing, eczema, or both was associated with significantly increased U-EPX levels (P <.0001). Wheezing appeared to be associated with higher U-EPX levels compared with eczema in both atopic and nonatopic children. The highest U-EPX level was found in atopic children with a history of wheezing and eczema (P <.0001). There was no relationship between U-EPX level and lung function. CONCLUSION: U-EPX level reflects the presence of atopy and associated symptoms and might be useful for monitoring the progression of allergic disease.

Eosinophil activation and preschool viral wheeze.

BACKGROUND: A study was undertaken to ascertain whether systemic eosinophil activation is associated with preschool viral wheeze (PVW). METHODS: Urinary eosinophil protein X (uEPX) and serum total IgE (IgE) levels were measured in children admitted to hospital with PVW, and uEPX was measured 6 weeks after discharge. Two years after admission, current wheeze in children aged > or =5 years was determined by questionnaire. Controls were recruited from children undergoing elective surgery (normal controls) and from those with skin prick test reactivity to foods (atopic controls). RESULTS: There was no difference in uEPX levels between normal controls (n=15) and atopic controls (n=8). uEPX levels were increased in children with acute PVW (n=84; p<0.001 v normal controls, p<0.01 v atopic controls) and fell on convalescence (n=20, 95% CI -217 to -31 microg/mmol creatinine, p<0.05). In children with acute PVW there was no association between uEPX and serum IgE levels or markers of clinical severity. Respiratory questionnaires were returned for 25/55 eligible children. There was no difference in uEPX level during acute PVW when stratified by "current wheeze" (n=18) or "no wheeze" (n=7) 2 years later. CONCLUSIONS: Systemic eosinophil activation is associated with PVW but is not associated with serum IgE, clinical severity, or persistence of wheeze into the early school age period.

Eosinophil granule proteins in the assessment of airway inflammation in pediatric bronchial asthma.

Eosinophil granule proteins such as eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and eosinophil protein X (EPX) in serum and urine are indirect measures of eosinophil activity. The measures have been evaluated for prediction, diagnosis and monitoring of anti-inflammatory treatment modalities in children with asthma. Assessments in serum and urine are highly dependent on sampling procedures and must be performed under strictly controlled conditions using standardized sampling and laboratory procedures. The measures are influenced by circadian and seasonal variations. Measurement of the eosinophil granule proteins does not improve the predictive value of a family history of atopy. Due to insufficient sensitivity and specificity, the measures are not useful in the diagnosis of asthma in children, and the clinical use of eosinophil proteins in the individual child for assessment of asthma severity has not been sufficiently validated. Serum and urine eosinophil granule proteins, however, may be useful in extending our knowledge of suppressive effects on eosinophil activity of various doses, devices and administration regimens of inhaled glucocorticoids in children. Such evaluations may be performed in randomized, double-blind trials of well-defined age groups and they should include measures of compliance. One important aspect to look at would be the distinction between suppressive effects on eosinophil activity and clinically important anti-inflammatory effects. Considering the complexity of airway inflammation and the heterogeneity of childhood asthma, however, it may be too simplistic to look for a single measure of the inflammatory processes. In the future, perhaps, a combination of products of inflammatory cells may give more clinically relevant information with respect to prediction, diagnosis, monitoring and outcome of childhood asthma.

Eosinophil degranulation during pregnancy and after delivery by cesarean section.

BACKGROUND: The physiological roles of eosinophils that accumulate in the uterus during pregnancy and of uterus-dwelling mast cells remain unknown. In the present study, we investigated the degranulation of eosinophils and mast cells within the normal course of pregnancy and after delivery by measuring the urinary concentrations of eosinophil-derived neurotoxin (EDN) and N-methylhistamine. METHODS: Spot urine samples from 65 pregnant women and 15 nonpregnant, age-matched women were examined. Urinary EDN and N-methylhistamine concentrations were measured by radioimmunoassay and standardized with urinary creatinine concentration. RESULTS: A significant increase in the urinary EDN concentration was observed until the second trimester in the normal pregnancies. The elevated urinary EDN levels decreased after the onset of labor in the third trimester and normalized within 1 month after normal vaginal delivery. In women who underwent a cesarean section, the urinary EDN concentration was significantly higher for up to 1 month after delivery, compared to that in women who underwent a vaginal delivery. In contrast, the urinary N-methylhistamine concentration did not change until the second trimester and was significantly decreased during the third trimester. No significant correlation between the peripheral blood eosinophil count and the urinary EDN concentration was observed in these subjects. CONCLUSIONS: Eosinophils appear to play a role in the progression of pregnancy and recovery after a cesarean section through the degranulation of eosinophils. In addition, mast cell degranulation does not appear to be related to the contraction of uterine smooth muscle during labor.

Nasal blockage and urinary leukotriene E4 concentration in patients with seasonal allergic rhinitis.

BACKGROUND: Cysteinyl-leukotrienes have been reported to have a primary role in the induction of nasal blockage of allergic rhinitis. However, there has been little experimental evidence that substantiates the relationship between nasal blockage severity and urinary leukotriene E4 (U-LTE4) concentration in patients with seasonal allergic rhinitis (SAR). METHODS: The concentrations of urinary mediators in 20 SAR patients were measured using an enzyme immunoassay to determine the relationship between nasal blockage severity and U-LTE4 concentration in patients with SAR. RESULTS: The basal U-LTE4 concentration was significantly higher in SAR patients with severe nasal blockage than in those with mild nasal blockage and in healthy control subjects. Although U-LTE4 concentrationwas significantly higher in patients with both asthma and SAR than in SAR patients with mild nasal blockage, no significant difference in the U-LTE4 concentration between patients with both asthma and SAR and SAR patients with severe nasal blockage was found. There was a significant correlation between U-LTE4 and urinary 9alpha11beta-prostoglandin F2 (9alpha11betaPGF2) concentrations (rs = 0.51, P = 0.02) in SAR patients. CONCLUSIONS: Although specific sites and cells of cysteinyl-leukotriene biosynthesis could not be determined in this study, severe nasal blockage is associated with the increased excretion level of U-LTE4.

Estradiol in premenstrual asthma: a double-blind, randomized, placebo-controlled, crossover study.

STUDY OBJECTIVES: To characterize asthma symptoms and pulmonary function throughout two menstrual cycles, with and without exogenous estradiol administration, in women with premenstrual asthma, and to determine the effect of estradiol administration on asthma symptoms, pulmonary function, quality of life, and biomarkers of airway inflammation. DESIGN: Double-blind, randomized, placebo-controlled, crossover study. SETTING: Respiratory clinic and clinical research center. SUBJECTS: Twelve women with documented premenstrual asthma (> or = 20% premenstrual worsening of asthma symptoms and/or of peak expiratory flow [PEF] during a 1-month screening phase). INTERVENTION: Each woman received either estradiol 2 mg or placebo orally between cycle days 23 and 28 (i.e., premenstrually, or before the onset of menses) in the first cycle and then crossed over to the other arm in the second cycle. Throughout both cycles, the women recorded daily morning and evening PEF readings and asthma symptoms. MEASUREMENTS AND MAIN RESULTS: Spirometry testing and measurement of serum estradiol and biomarkers of airway inflammation were performed on days 8 (follicular phase), 22 (luteal phase), and 28 (premenstrually) of both the estradiol and placebo cycles. During the two premenstrual visits, the Asthma Quality of Life Questionnaire was administered. No notable differences were observed between the estradiol and placebo cycles in daily PEF recordings or composite asthma symptoms scores. The area under the curve (AUC) for the composite asthma symptoms versus time profile was numerically, but not statistically, lower (denoting less severe symptoms) during the estradiol cycle than during the placebo cycle. Likewise, no significant difference in AUC values for morning PEF or evening PEF was found between the estradiol cycle and the placebo cycle. Despite differences (p<0.05) in day-28 estradiol concentrations for estradiol and placebo cycles, no significant differences were found in forced expiratory volume in 1 second, serum endothelin-1, serum and urine eosinophil protein X, urine leukotriene E4, or quality-of-life scores. CONCLUSION: Exogenously administered estradiol did not have a significant effect in women with premenstrual asthma whose asthma was classified predominantly as mild and under excellent control. As in the case of premenstrual syndrome, the placebo effect may be prominent in premenstrual asthma. Further trials, involving women with more severe asthma under poorer control, are warranted to discern underlying mechanisms for the worsening of asthma in relation to menstruation.

Circadian variations in serum eosinophil cationic protein, and serum and urine eosinophil protein X.

Biochemical evaluation of inflammation may be a useful adjunct to measures of pulmonary function and symptoms in children with asthma. However, little data have been provided to validate the markers in children. The aim of the present study was to assess circadian variations in serum eosinophil cationic protein (ECP), and serum and urine eosinophil protein X (EPX) in children. Five girls and two boys aged 10-14 years were studied. The first sample of urine consisted of urine collected from 24.00 hours the night before until 08.00 hours on the morning of the day of investigation. Thereafter urine was collected at 4-h intervals until 24.00 hours and in another 8-h interval from 24.00 to 08.00 hours. Blood samples for assessment of serum ECP and serum EPX were collected every 2 h during the 24 h. Statistically significant circadian variations in serum ECP (F=3.2, p=0.002), serum EPX (F=3.1, p=0.002) and in urine EPX/creatinine (F=5.4, p=0.003) were detected. The concentrations were higher during the night compared to daytime. Peak levels of serum ECP (mean [+/- SEM]) were found at 06.00 hours (16.3 [5.3] micro g/l), trough levels at 08.00 hours (3.9 [0.7] micro g/l) (p=0.01). Peak levels of serum EPX were seen at 06.00 (43.7 [9.5] micro g/l) with trough levels at 12.00 hours (22.0 [3.5] micro g/l) (p=0.01). Peak levels of urine EPX/creatinine occurred in urine collected from 24.00 to 08.00 hours (90.0 [27.7] micro g/mmol), trough levels in the 16.00-20.00 hours sample (29.7 [8.9] micro g/mmol) (p=0.02). Serum ECP, serum EPX and urine EPX exhibit a circadian variation in children with nocturnal and early morning peak levels. To avoid confounding influence from circadian variations in ECP and EPX in clinical studies blood or urine should be sampled at consistent times.

Asthma attack severity and urinary concentration of eosinophil X protein in children.

OBJECTIVE: Determination of the urinary concentration of eosinophil protein X (U-EPX) may objectively predict the severity and activity of asthma in children. METHODS: Concentrations of U-EPX in 80 non- atopic asthmatic children were compared with those in 25 healthy control children. The patients were studied during attacks and two weeks later. The severity of asthma attacks was determined according to a pre-existing score. U-EPX was measured by the specific radioimmunoassay technique (Pharmacia, Uppsala, Sweden). This measurement was correlated with the clinical and radiological investigations as well as with other variables such as blood oxygen saturation, peak expiratory rate and eosinophil count. RESULTS: U-EPX concentrations were significantly higher in all asthmatic children during attacks (139.6 11.7 microg/mmol of creatinine) than those in the control group (35.3 6.2 microg/mmol of creatinine) (p < 0.001). Two weeks after resolution of the exacerbation, U-EPX significantly decreased (66.5 9.3 microg/mmol of creatinine) (p < 0.001). U-EPX concentrations were highest in patients with severe attacks (191.5 11.3 microg/mmol of creatinine) (p < 0.001). No statistically significant differences were found between mild (88.2 7.2 microg/mmol of creatinine) and moderate attacks (119.6 8.5 microg/mmol of creatinine). At the two-week follow-up, U-EPX concentrations in patients with mild or moderate attacks was similar to those in controls but were persistently elevated in the subgroup with severe attacks (103.8 9.4 microg/mmol of creatinine) (p < 0.001). No significant correlation was found between U-EPX concentrations and blood oxygen saturation, peak expiratory rate or eosinophil count. CONCLUSION: A statistically significant correlation was found between U-EPX concentrations and the severity of attacks in asthmatic children. This substance could be useful in quantifying bronchial inflammation. This result could further be used as a marker of severity of disease exacerbation and would not only facilitate early diagnosis and staging of inflammatory and allergic disorders but would also allow therapy and interventions to be monitored.

Which factors predict success after discontinuation of inhaled budesonide therapy in children with asthma?

Urinary eosinophil protein X (UEPX) concentration, lung function, and nonspecific bronchial hyperreactivity were determined in 40 asthmatic children (asymptomatic for 6.4 +/- 3.0 months) (mean age 9.8 +/- 2.9 years) receiving inhaled budesonide, in order to establish whether measurement of these parameters is useful in determining discontinuation of inhaled corticosteroid therapy. After the discontinuation of therapy, patients were asked to come to the Outpatient Clinic if symptoms recurred and did not respond to beta2 mimetic usage in 24 hr. Otherwise they were to be seen 2-3 months later for a follow-up visit. UEPX concentration was determined and spirometry was performed on this visit. While UEPX concentrations had increased (p < 0.0001), FEV1, FEF 25-75 and PEF had decreased significantly 2.3 +/- 0.53 months after the cessation of inhaled budesonide therapy in all children (p = 0.004, p = 0.02, p = 0.02, respectively). Due to clinical deterioration, inhaled corticosteroid therapy had to be restarted in 19 (48%) of the children (Group I), while the remaining 21 (52%) (Group II) continued to be asymptomatic during the 2.3 +/- 0.5 months follow-up period. Although the initial UEPX concentrations, spirometer variables, and methacholine PC20 values of these two groups were not statistically different, the duration of clinical remission before discontinuation of budesonide prophylaxis was significantly longer in group II (p = 0.0037). We concluded that, in determining discontinuation of inhaled corticosteroid prophylaxis, duration of clinical remission seems to be a more useful criterion than measurement of UEPX levels, lung function test, and assessment of bronchial hyperreactivity.

Human non-pregnancy ribonuclease with anti-Kaposi's sarcoma activity.

Our demonstration of a 19kDa anti-Kaposi's sarcoma (KS) ribonuclease (RNase) in urine from a non-pregnant female may provide at least part of the explanation for the low incidence of KS in human females. N-terminal sequence analysis and isoelectric focusing of the purified RNase, coupled with the very low levels of anti-KS activity noted for recombinant forms of human eosinophil derived neurotoxin and human eosinophil cationic protein, suggest that the 19kDa enzyme is an eosinophilic protein whose potent anti-KS activity is dependent upon post-translational modifications that occur only in human cells.

Circadian variation of exhaled nitric oxide and urinary eosinophil protein X in asthmatic and healthy children.

Asthmatic symptoms and the frequency of admissions to hospital because of acute asthma tend to increase in the early morning hours, and it is therefore possible that airway inflammation increases during the night. To elucidate the hypothetical circadian variation of airway inflammation, we measured concentrations of exhaled nitric oxide (FeNo), urinary eosinophil protein X excretion (EPX), and forced expiratory volume in the first second (FEV1) in 20 asthmatic and 6 nonatopic nonasthmatic children every 3 h during a 21-h period. Compared with control subjects, asthmatic subjects had higher FeNo (median, 22.7 versus 10.3 ppb, p = 0.016) and lower FEV1 % predicted (median, 91.0 versus 101.9%, p = 0.045), but did not differ significantly in EPX (median, 153.8 versus 148.7 microg/mmol creatinine, p = 0.83) at 7 AM. However, differences in gender and age do not allow direct comparisons between asthmatic and control children. FeNo and EPX demonstrated a cosinelike circadian rhythm (log FeNo, p = 0.0001; log EPX, p = 0.0001) with lowest levels at 7 PM and highest at 7 AM. This was also the case for FEV1 % (p = 0.01). No difference in the amplitude of circadian rhythm was observed between asthmatic and healthy control children for log FeNo (p = 0.35), log EPX (p = 0.57), and FEV1 % (p = 0.17). A stratified analysis showed a significant circadian rhythm in the control group for log FeNo (p = 0.014) and log EPX (p = 0.0001). Our results therefore suggest a circadian rhythm of inflammatory markers, which peaks in the early morning. Rhythmicity of EPX excretion and FeNo in healthy children suggests a physiologic mechanism; however, pathologic effects during the night might occur under conditions of asthma-specific inflammation.

Increased urinary leukotriene E4 and eosinophil protein X excretion in patients with interstitial cystitis.

PURPOSE: The role of cysteinyl containing leukotriene C4, D4 and E4, and eosinophil protein X in interstitial cystitis is unknown. Leukotriene E4, the end product of cysteinyl containing leukotrienes, and eosinophil protein X are markers of the activation of mast cells and eosinophils, respectively. Cysteinyl containing leukotrienes are potent and specific chemoattractants for eosinophils. We compared the urinary excretion of leukotriene E4 and eosinophil protein X in patients with interstitial cystitis and in healthy controls. MATERIALS AND METHODS: Morning spot urine samples from nine patients with interstitial cystitis who fulfilled National Institute of Diabetes and Digestive and Kidney Diseases criteria were collected on the day of cystoscopy with biopsies. Aliquots of urine specimens were immediately centrifuged and the supernatants were stored at -80C until use. Urine samples from 9 healthy women served as controls. Urinary leukotriene E4 and eosinophil protein X were measured by enzyme immunoassay and radioimmunoassay, respectively. All determinations were performed in duplicate and normalized to urine creatinine. RESULTS: Leukotriene E4 and eosinophil protein X were significantly increased in the morning urine of patients with interstitial cystitis compared with controls. The mean urinary excretion of leukotriene E4 plus or minus standard deviation was 148.8 +/- 62.5 and 62.2 +/- 17.5 ng./mmol. creatinine in patients and controls (p = 0.003), while the mean urinary excretion of eosinophil protein X was 109.7 +/- 70.4 and 43.7 +/- 22.0 microg./mmol. creatinine, respectively (p = 0.01). All urine cultures were negative. The mean mast cell count in detrusor biopsies in the interstitial cystitis group was 41 cells per mm.2 (range 5 to 84). Eosinophilic granulocytes were occasionally observed in the submucosa but not in the detrusor. CONCLUSIONS: Our study shows that patients with interstitial cystitis and detrusor mastocytosis have increased urinary leukotriene E4 and eosinophil protein X. It is possible that cysteinyl containing leukotrienes and eosinophil protein X are involved in the pathogenesis of interstitial cystitis. Urinary leukotriene E4 and eosinophil protein X may be useful markers for assessing the grade of activation of mast cells and eosinophils in patients with interstitial cystitis and/or for confirming the diagnosis. However, it remains to be investigated whether the increase in urinary leukotriene E4 and eosinophil protein X correlates with interstitial cystitis symptoms.