Use of urinary gram stain for detection of urinary tract infection in childhood

In this study, urinary culture, urinary Gram stain, and four tests within the urinalysis, leukocyte esterase, nitrite, microscopyfor bacteria, and microscopyforpyuria, were examined in 100 children with symptoms suggesting urinary tract infection. Our purpose was to determine the validity of the urinary Gram stain compared with a combination of pyuria plus Gram stain and overall urinalysis (positiveness of nitrite, leukocyte esterase, microscopy for bacteria, or microscopy for white blood cell). Of 100 children, aged two days to 15 years, 70 (70 percent) had a positive urinary culture: 40 girls (57 percent) and 30 boys (43 percent). Escherichia coli was the most common isolated agent. The sensitivity and specificity of the urinary Gram stain were 80 percent and 83 percent, and that of the combination of pyuria plus Gram stain 42 percent and 90 percent, and that of the overall urinalysis 74 percent and 3.5 percent respectively. Our findings revealed that neither method of urine screen should substitute for a urine culture in the symptomatic patients in childhood.     

尿液中细菌L型检出情况及耐药性分析

目的了解泌尿感染患者尿液标本细菌L型的检出情况,分析尿常规结果、病原菌分布情况及耐药性特点,为临床提供诊疗依据。方法对2014年1月至2015年12月1 532例住院和门诊泌尿感染患者的清洁中段尿标本的尿常规结果和微生物培养结果进行回顾性分析。严格按照《全国临床检验操作规程》要求采集患者尿液标本,2 h内完成尿液普通培养、高渗培养、尿常规检查及尿液离心后沉渣镜检。培养出细菌L型进行菌株鉴定和药敏试验,结果采用SPSS 13.0统计软件进行分析处理。结果共检出细菌L型132例,检出率为8.6%。132例细菌L型阳性病例中,白细胞酯酶阳性19例,阳性率14.4%;尿沉渣镜检白细胞阳性105例,阳性率79.5%。细菌L型检出率排名前三位的分别为大肠埃希菌、粪肠球菌、葡萄球菌,分别占40.9%、22.7%、12.1%。大肠埃希菌对氨苄西林、环丙沙星、左氧氟沙星、氨苄西林/舒巴坦、头孢唑啉、复方新诺明耐药率较高;粪肠球菌对克林霉素、奎奴普丁/达福普汀、红霉素、四环素耐药率较高;葡萄球菌对青霉素G、红霉素、头孢西丁、甲氧西林、环丙沙星耐药率较高。结论尿常规、尿沉渣镜检有助于细菌L型感染的辅助诊断,临床医生应根据尿培养结果合理、足疗程选用抗菌药物。     

Descriptive urological record of chimpanzees (Pan troglodytes) in the wild and limitations associated with using multi-reagent dipstick test strips

Ten urine chemistry parameters were measured on 74 voided urine samples from 34 wild chimpanzees (Pan troglodytes). Multi-reagent urine dipstick tests were performed and results determined using colorimetric scales. Urine pH measured between 8 and 9 units in 91% of the chimpanzees. Test pads detected protein, erythrocytes, leukocyte esterase activity, and nitrites, ketones and bilirubin in 47, 32, 29, and <10% of the chimpanzees, respectively. No apparent association between positive test results for blood in adult females and reproductive status was found. Overall, 17 of the 34 chimpanzees had positive urine test results for protein, hemoglobin, erythrocytes, leukocytes, nitrites, ketones, and/or bilirubin. Dipstick urinalysis alone is an unreliable method for assessing health and physiological status of wild chimpanzees. However, if combined with other diagnostics it could prove to be a valuable health-monitoring tool. Limitations associated with this methodology need to be considered when interpreting urinary dipstick test results.     

Purification and Characterization of A Human-Specific Esterase From Urine

Abstract

A human-specific esterase was isolated from urine and partially characterized by ammonium sulfate precipitation, starch block electrophoresis, and gel filtration. Its molecular weight was estimated to be 136,000 by polyacrylamide gel electrophoresis in 0.17. SDS. A diffusion coefficient of 8.6 X 10^?7 was determined by the L-plate method. Immunologic identity was shown between the urinary esterase and a human tissue esterase.     

Purification and characterization of rat urinary esterase A1

An enzyme, esterase A1, which hydrolyzes tosyl-arginine methyl ester (Tos-Arg-OMe) was separated from esterase A2 and kallikrein of male rat urine and purified by a procedure involving ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography and gel filtration. The resulting preparation was apparently homogeneous, as assessed by polyacrylamide gel electrophoresis. The molecular weight of the preparation was estimated to be 27,000 by SDS-polyacrylamide gel electrophoresis and 30,000 by gel filtration. The enzyme was more specific for arginine methyl esters than for lysine methyl esters. The optimum pH determined with Tos-Arg-OMe as a substrate was 8.0 and the Km was 11.8 mM. The Tos-Arg-OMe esterolytic activity of esterase A1 was inhibited by soybean trypsin inhibitor, but not by aprotinin. In immunodiffusion analysis, the antiserum to esterase A1 formed immunoprecipitin arcs with this enzyme and the urine collected from rat bladder, but not with esterase A2, kallikrein, plasma and the urine collected from ureters. These results indicate that rat urinary esterase A1 differs from esterase A2 and kallikrein. The esterase A1 appears to be produced by accessory sex glands and excreted via the spermiduct into the urine.     

Estimation of ouabain specifically bound to dog renal (Na+ + K+)-ATPase in vivo.

Suspensions of viable renal cortical cells hydrolyzed a synthetic ester substrate (alpha-N-tosyl-L-arginine methyl ester, Tos-Arg-OMe) and generated kinins from a kininogen substrate. This kallikrein-like esterase activity increased linearly with cell number, or time of exposure to substrate. No radiolabelled substrate or product was found within the cells. Most of the activity appeared to be on cell surfaces as supernatant media had less than 20% of the Tos-Arg-OMe esterase activity on the cell suspensions. Cell surface Tos-Arg-OMe esterase activity was inhibited by aprotinin, benzamidine, pentamidine, and a tris-amidine derivative (alpha,alpha',alpha'-tris(3-amidinophenoxy)mesitylene). Preincubation of cells with phospholipase A2 increased renal cell surface esterase activity up to 76% while only slightly increasing supernatant activity. In contrast, preincubation with deoxycholate caused clearing of suspensions and a marked increase in supernatant esterase activity. Renal cell kininogenase (EC 3.4.21.8) activity was inhibited by preincubation with aprotinin, the tris-amidine derivative, or anti-rat urinary kallikrein antibody. Kallikrein elaborated by renal cells formed a single precipitin line with an antibody to rat urinary kallikrein but the two enzymes were not immunologically identical. We conclude that kallikrein's active sites are facing the external environment of renal cortical cells in suspension with access to substrates, inhibitors, and antibody.     

Isolation and partial characterization of rat urinary esterase A2

An enzyme, esterase A2, which hydrolyzes tosyl-arginine methyl ester was isolated from the urine of female, inbred, Dahl-salt-resistant rats using DEAE-Sephadex ion-exchange, aprotinin-agarose affinity and molecular sieve column chromatography. The purest preparation obtained showed four closely migrating bands on polyacrylamide gel electrophoresis. All four bands of the esterase A2 preparation had enzyme activity since all were stainable on zymograms using N-acetyl-L-methionine alpha-naphthyl ester as substrate. Three of these four bands showed decreased electrophoretic mobility following treatment with neuraminidase, indicating that variable sialic acid content accounts for part of the microheterogeneity. The preparation of esterase A2 used was free of rat urinary kallikrein as shown by radioimmunoassay, electrophoretic and isoelectric focusing experiments. The relative kinin-generating ability of rat urinary kallikrein and esterase A2 was highly dependent on the assay used. Using canine plasma as a source of kininogen and the rat uterus to bioassay kinins, esterase A2 was 47% as active as kallikrein; using pure bovine low-molecular-weight kininogen and a radioimmunoassay to measure generated kinins, esterase A2 was only 6% as active as kallikrein. Esterase activity of A2 was activated non-specifically by proteins and detergents. Esterase A2 was 50% inhibited by an 8-fold molar excess of aprotinin and by a 26.5-fold molar excess of soybean trypsin inhibitor, but ovomucoid inhibitor was not inhibitory.     

Interactions of the complex between human urinary trypsin inhibitor and human leukocyte elastase with alpha 1-proteinase inhibitor and alpha 2-macroglobulin

The urinary trypsin inhibitor was recently shown to inhibit human leukocyte elastase. Complexes of human urinary trypsin inhibitor with human leukocyte elastase or human trypsin were produced and subjected to gel filtration. The complexes were found to be sufficiently stable up to 24 h incubation (at least 70% recovery). When human serum was added, elastase and trypsin dissociated from the urinary trypsin inhibitor and associated with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The addition of alpha 1-proteinase inhibitor to a complex of urinary trypsin inhibitor and leukocyte elastase caused a rapid dissociation of the complex (kdiss = 3.2 X 10(-2) s-1).     

Isolation of tissue kallikrein in rat spleen by monoclonal antibody-affinity chromatography

Rat spleen kallikrein was identified and purified by DEAE-cellulose and monoclonal antibody-affinity chromatography. The purified enzyme has Tos-Arg-OMe esterase activity and kinin-releasing activity from a purified low-molecular-weight kininogen substrate. In the direct radioimmunoassay for tissue kallikrein, the splenic enzyme displays parallelism with standard curves of rat urinary kallikrein. The pH profiles of the Tos-Arg-OMe esterase activities of spleen and urinary kallikrein were identical with optima at 9.0 Rat spleen kallikrein was inhibited strongly by aprotinin and affinity-purified kallikrein antibody and weakly by soybean trypsin inhibitor. The IC₅₀ values were similar to those observed against rat urinary kallikrein. Neither the urinary nor the splenic enzyme was inhibited by lima bean trypsin inhibitor or preimmune serum immunoglobulins. Spleen kallikrein was labeled with [¹⁴]diisopropylphosphorofluoridate and visualized by fluorography on a sodium dodecyl sulfate-polyacrylamide gel. The electrophoretic mobility of the splenic enzyme was indistinguishable from that of urinary kallikrein A with an estimated Mr of approx. 38 000. With Western blot analyses using a rabbit anti-kallikrein antibody followed by ¹²⁵I-labeled protein A binding, the spleen and urinary kallikreins were again visualized at identical positions by autoradiography. The data show that there is a rat splenic tissue kallikrein which is indistinguishable from a renal kallikrein with respect to physicochemical properties, immunological character and susceptibility to inhibitors.     

Leukocyte migration inhibitory factor: a serine esterase released by stimulated human lymphocytes. Kinetic analysis and inhibition by cyclic GMP.

Lymphocytes, stimulated with concanavalin A, release small amounts of non-immunoglobulin, highly reactive proteins called lymphokines. One of these, a serine esterase, termed leukocyte migration inhibitory factor according to its function in vitro, is found in supernatants of stimulated human lymphocytes at concentrations less than 1 ng/ml. The esterase was purified in good yield and its esterolytic activity was measured by a sensitive radioenzymic assay. The kinetics of the esterolytic activity were studied and the effect of various nucleotides examined. Competitive inhibition of esterolysis was seen with cyclic GMP at concentrations down to 10(-7) M, and with 2',3'-cyclic CMP at a concentration of 10(-3) M. A role of this esterase, not only as a mediator acting upon polymorphonuclear leukocytes, but also as an intracellular regulator of lymphocyte activation, is discussed.     

Acetylcholine esterase as a probe for erythrocyte-membrane intactness

Erythrocyte acetylcholine esterase can be assayed in intact cells and was tested as a probe for membrane changes. Acetylcholine esterase activity correlated with the erythrocyte relative volume. Antihemolytic acyl sorbitols, fatty acids and phenothiazines inhibit to varying extents the activity of acetylcholine esterase. The inhibition of acetylcholine esterase by linolenoyl sorbitol was further characterized and found to be non-competitive and critically dependent on cell intactness over a wide temperature range. Neither solubilized nor ghost acetylcholine esterase was affected by the acyl sorbitol while under conditions optimal for ghost resealing, the enzyme resumed the sensitivity to the acyl sorbitol. Acetylcholine esterase sensitivity thus appears to be a promising tool to follow the dynamics of membrane integrity.     

Urinary kallikrein and non-kallikrein arginine esterase activities in beagle dogs

The activity of urinary kallikrein and non-kallikrein arginine esterase was measured in the 16hrs urine of 16 beagle dogs. Enzyme activity was assayed using D-valyl-L-leucyl-L-arginine-p-nitroanilide as a substrate at 30 degrees C, pH 8.0, and the unit of measurement was defined as the activity which catalyzes the hydrolysis of 1 mumol of the substrate per minute. Kallikrein activity ranged from 3.87 to 48.92 (28.48 average) mU/ml of urine, or from 1.05 to 12.51 (4.56 average) U/16 hrs; whereas that of non-kallikrein arginine esterase ranged from 0.06 to 24.28 (7.11 average) mU/ml, or from 0.01 to 9.78 (1.43 average) U/16 hrs. The ratio between the activity of these two enzymes was 1: 0.002-1: 2.98 (1: 0.42 average). Male dogs had a tendency to show a higher enzyme activity than females for urinary kallikrein and non-kallikrein arginine esterase.     

Micro-leukocyte adherence inhibition. I. Cellular basis of the mechanism of reactivity.

To study the cellular basis for specific antigen-induced leukocyte adherence inhibition, enriched populations of B cells, T cells, and monocytes were prepared by a two-stage adherence separation procedure from spleen cells of normal C57BL/6J mice and mice bearing progressively growing MCA-38 tumors. The reactor cell undergoing specific antigen-induced adherence inhibition was identified as a monocyte (esterase positive, did not respond to mitogens, and did not bear Thy 1.2 antigen or surface immunoglobulin). Furthermore, an enriched population of MCA-38 sensitized B cells could program normal monocytes to undergo specific antigen-induced adherence inhibition. This programming could be abolished by pretreatment of the MCA-38 sensitized B cells with anti-immunoglobulin and complement (indirect cytotoxicity method). In contrast, enriched populations of MCA-38 sensitized T cells could not program normal nylon wool adherent cells to undergo antigen-specific adherence inhibition; and anti-Thy 1.2 serum and complement had no effect on specific antigen-induced adherence inhibition. Thus, in this murine tumor model, leukocyte adherence inhibition appears to be due to the programming of monocytes by sensitized B cells.     

Purification and preliminary characterization of human leukocyte elastasel.

Affinity chromatography permits the purification of 1–3 mg of human leukocyte elastase from the leukocytes contained in 500 ml of whole blood. Lysosomal granule proteins are extracted from polymorphonuclear leukocytes and subjected to chromatography on a column of elastin-Sepharose. Contaminating proteins are eluted with buffer containing 1 m NaCl and then elastase activity is eluted with buffer containing 8 m urea. The enzyme retains all of its esterase activity against N-t-BOC-l-alanine p-nitrophenyl ester after exposure to 8 m urea and retains 22% of its activity in the presence of 1% sodium dodecyl sulfate. In sodium dodecyl sulfate and 2-mercaptoethanol leukocyte elastase undergoes autolysis giving rise to several low molecular weight fragments. The molecular weight of the native enzyme is found to be 22.000 by both gel filtration and sodium dodecyl sulfate—acrylamide gel electrophoresis. A characteristic set of four isozymes is seen after acrylamide disc gel electrophoresis at pH 4.5. All bands are active against elastin and also contain carbohydrate by the periodic acid-Schiff stain. On the basis of stain intensity, the slower moving isozymes appear to be richest in carbohydrate. Active leukocyte elastase forms a complex with α₁-antitrypsin in a 1:1 molar ratio. The elastase must be enzymatically active for complex formation to occur.     

Silymarin improves metabolism and disposition of aspirin in cirrhotic rats

The profile of urinary salicylate metabolites was determined after an i.p. administration of acetylsalicylic acid (ASA) to CCl4-cirrhotic rats to rats which in addition to CCl4 received an oral dose of silymarin throughout the CCl4 treatment to produce cirrhosis and to control groups. ASA esterase activity was determined in serum and livers. The time course of plasma concentration of salicylates in similar groups was followed after the i.p. injection of ASA. The cirrhotic animals showed a lack of urinary glucuronides and an increase in urinary gentisic and salicylic acids. The activities of plasma and serum ASA esterase were significantly increased in cirrhosis and the plasma half-life of ASA was reduced. The simultaneous administration of silymarin (50 mg/kg of b.w.) along with CCl4, completely prevented all the alterations. The mechanism by which silymarin prevented those alterations is not completely known but our results establish the potential use of silymarin in cirrhotic patients to prevent disorders in drug metabolism and disposition frequently found in patients with liver diseases.     

Specific identification of tissue kallikrein in exocrine tissues and in cell-free translation products with monoclonal antibodies.

A panel of six mouse monoclonal antibodies (IgG1) has been prepared against purified rat urinary kallikrein (EC 3.4.21.35) and characterized. In radioimmunoassay, the antibody titres of ascitic fluid giving 50% binding to 125I-kallikrein range from 1:2 X 10(3) to 1:1 X 10(6). Antibodies from four of the clones show no cross-reactivity with human urinary kallikrein, rat urinary esterase A or tonin. However, antibodies from a fifth clone cross-react with tonin and, from a sixth, with both urinary esterase A and tonin. Three of the kallikrein affinity-purified monoclonal antibodies inhibited, whereas one of the antibodies stimulated, kallikrein activity. Tissue kallikrein from rat submandibular-gland and pancreatic extracts and urine were labelled with [14C]di-isopropyl phosphofluoridate, immunoprecipitated with each of the six monoclonal antibodies and identified to be 38 kDa proteins, similar in size to purified rat urinary kallikrein. Western-blot analysis shows that 125I-labelled kallikrein monoclonal antibodies (V4D11) bind directly to a 38 kDa protein in submandibular-gland and pancreatic extracts and urine. Cell-free translation products of submandibular-gland polyadenylylated[poly(A)+]mRNA were immunoprecipitated with affinity-purified sheep anti-kallikrein antibodies and three monoclonal antibodies (V4D11, V4G6 and V1C3). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these immunoprecipitates revealed that two kallikrein precursors with Mr values of 37 000 and 35 000 are encoded by submandibular-gland mRNA. The third monoclonal antibody, V1C3, which binds to active kallikrein, did not recognize either precursor form. Collectively, the data show that these monoclonal antibodies comprise a set of powerful and specific reagents for studies of tissue kallikreins.     

Single-cell RNA-Seq analysis identified kidney progenitor cells from human urine

Dear Editor, Urine passes through the entire kidney and urinary tract system starting from the glomerulus and ending to the urethra.Cells in the kidney and urinary tract could be exfoliated from the epithelium into the urine, while leukocyte could infiltrate from the local tissue into the urine, which makes the urine a useful subject for clinical evaluation of relevant diseases.Among them, renal tubular cells and podocytes have been identified and 2D or 3D cultured from human urine specimens (Oliveira Arcolino et al., 2015;Schutgens et al.,2019).Particularly, kidney stem cell/progenitor cells were successfully recovered from pediatric patient urine and then cultured for kidney regenerative purpose by the Romagnani group.However, they also showed that such cells cannot be recovered from healthy individuals (Lazzeri et al., 2015).It remains unknown whether similar types of progenitor cells can be found in different individuals, either healthy or diseased.     

Variations circadiennes des activités estérases de l'hémolymphe de Penaeus japonicus

The esterase activities of Penaeus japonicus hemolymph, revealed by a mixture of α- and β-naphtylacetate exhibit tricircadian variations under an LD:12-12 photoperiod. During the 24 h period, the total esterase activities can vary by 0,12 to 0,64 V/μg of protein. Under the experimental conditions adopted, circadian variations of the hemolymph esterase activities are observed whatever the sex and molt stage; the sex factor seems to be predominant for the esterase activities studied. Concurrently, a circadian rhythm of individual variabilities is observed for both sexes. The variabilities are assessed from the ratio of standard deviations on the esterase activity means, for each hour of experimentation. The minimum variabilities correspond to maximum total or specific esterase activities for the females whereas they precede the maximum esterase activities for males.     

A study for the improvement of the cytological urine examination performances in upper tract infection diagnosis

Diagnosis of the location of upper and lower urinary tract infection (UTI) is necessary in defining the therapeutic conduct that has a different period and intensity according to the infection location and in prognosis. Many studies show the lack of clinical criteria peculiarity in revealing the different location of UTI. As a result, the correct location of the level in which UTI develops is the necessity of paraclinical investigations. Urinary sample examination, in which urinary sediment microscopy is essential, is a reliable technique in fast detection and localization of UTI. Finding, in pyuria context, the classic significant bacteriuria (> or = 10(5) CFU/ml) or lower value bacteriuria (< or = 10(4) CFU/ml) confirms the UTI diagnosis. The upper tract infection prognosis increases when leukocyte cylinders, characteristic for pyelonephritis, appear together with intact or degraded leukocytes, single or grouped. We settled an algorithm to examine the urine samples in order to: Concentrate and preserve the structural integrity of leukocytes and cylinders, examining the conventional urinary sediment Precisely identify and differentiate these elements by vital coloration (leukocyte peroxidase coloration and Sternheimer - Malbin coloration) to establish more accurate the UTI level. The vital coloration for leukocyte peroxidase has cytological specificity, confirming the pyuria and the cylinders that contain leukocytes (leukocytary, granular, mixed) and obviously ameliorates the reliability and reproducibility of the urinary sediment cytological exam.     

Studies on rat renal cortical cell kallikrein. I. Separation and measurement.

A technique has been developed to separate and measure kallikrein in a heterogeneous population of rat renal cortical cells in suspension. After rat kidneys were perfused in situ in anaesthetized rats, viable, counted cortical cell suspensions were obtained. Cells were suspended in a sucrose/Tris buffer containing 0.5% deoxycholate, homogenized, centrifuged, dialyzed, and gel filtered on Sephadex G-25. Column chromatography on DEAE-cellulose resulted in a single peak of esterase activity between 0.20 to 0.25 M NaCl/sodium phosphate buffer. Subsequent elution yielded an alkaline esterase which was identical to kallikrein isolated from rat urine, insofar as pH optimum, effects of inhibitors, bioassay activity and immunological properties were concerned. Calculated yields were about 70% of the total esterase activity present in the parent cell homogenates. Recoveries of a purified rat urinary kallikrein added to the cell homogenates, the DEAE-cellulose columns, or the eluates from the columns ranged from 83-108% (mean 96%). Using this technique, it was found that the amount of kallikrein activity present in non-incubated renal cortical cells ranged from 0.6-10(-2) to 4.6 - 10(-2) alpha-N-tosyl-L-arginine methyl ester (Tos-Arg-OMe) esterase units per 10(8) cells. However, cells incubated in a nutrient medium at 37 degrees C for 3-8 h contained no measurable kallikrein activity, whereas the surrounding medium had kallikrein activity which could be significantly increased by aldosterone and decreased by spironolactone.