Isolation and characterization of a species-specific DNA probe for the detection of Candida krusei.

In an attempt to design species-specific primers for the detection of Candida krusei by polymerase chain reaction, a partial genomic DNA library from Candida krusei was screened for hybridization with radiolabeled genomic probes from a broad variety of fungal and bacterial species and from human. Species-specific candidate DNA inserts were then tested for hybridization with dot blots of DNA from various organisms. One 570-basepair insert from Candida krusei DNA that hybridized under stringent conditions only with DNA from Candida krusei and human was sequenced. It revealed considerable homology with the gene for the mitochondrial inner membrane protease I of Saccharomyces cerevisiae, and the 147 amino acid residues deduced from an open reading frame showed considerable homology with the N-terminal portion of the enzyme from Saccharomyces cerevisiae. From the sequence of the Candida krusei DNA fragment, a pair of 21-base oligonucleotide primers enclosing a 501-basepair sequence was designed for polymerase chain reaction. When these primers were tested with a broad range of genomic DNAs, the expected amplification was obtained only with Candida krusei DNA and not with DNA from any other source, including human. Experiments with DNA from mixed cultures of Candida krusei and other yeasts and bacteria showed that the polymerase chain reaction was specific for Candida krusei and that as few as ten cells could be detected.

Activity of some Mannich bases of conjugated styryl ketones against Candida albicans.

A number of Mannich bases of conjugated styryl ketones displayed activity against Candida albicans strains 3153A and B311. Antifungal potency was influenced by the relative hydrophobicities of the molecules and on occasions by the electronic nature of the aryl substituents. The compounds inhibited one or more of the following enzymes in the glutathione metabolic pathway namely glutathione S-transferases, glutathione reductase, gamma-glutamyl transpeptidase and glutathione peroxidase. Nearly half of the compounds examined on the yeast-to-mycelium transition in C. albicans 3153A prevented this conversion from occurring.

Urinary excretion of Tamm-Horsfall protein in elderly women.

The incidence of urinary tract infection is higher in the geriatric population than in younger adults despite the exclusion of patients with known risk factors. Tamm-Horsfall protein, a renal glycoprotein excreted in urine, may constitute a natural defense mechanism against ascending urinary tract infection by binding mannose-sensitive fimbriated microorganisms. We hypothesized that the quantity of Tamm-Horsfall protein excreted is decreased in the elderly. Native aggregated Tamm-Horsfall protein was measured in urine samples from 24 young women (group 1, mean age 33 years) and 47 female nursing home patients (group 2, mean age 84 years) using enzyme-linked immunosorbent assay techniques. Another 16 elderly women (group 3, mean age 85 years) had active urinary tract infection. The aggregated Tamm-Horsfall protein was then disaggregated by dilution and quantified. Significant differences in mean urinary disaggregated Tamm-Horsfall protein concentrations were found between groups 1 (64.22 mg./l.) and 2 (35.07 mg./l.), and between groups 1 and 3 (34.71 mg./l.), respectively. In contrast, mean aggregated Tamm-Horsfall protein levels were significantly higher in group 2 (1.56 mg./l.) than in group 1 (0.92 mg./l.) or group 3 (0.97 mg./l.). Our studies show that urinary disaggregated Tamm-Horsfall protein concentration is decreased in the elderly, and that aggregated Tamm-Horsfall protein is increased compared to younger adults. The aggregated Tamm-Horsfall protein concentration is decreased in the elderly during episodes of urinary tract infection.

Effect of Tamm-Horsfall protein on chemoluminescence response of polymorphonuclear leukocytes to uropathogenic Escherichia coli.

Mannose residues on the outer membranes of polymorphonuclear leukocytes (PMNL) are capable of binding mannose-sensitive Escherichia coli. Tamm-Horsfall protein, a major urinary glycoprotein, has also been shown to bind mannose-sensitive E. coli via mannose-containing side chains. The effect of Tamm-Horsfall protein on the interaction between PMNL and mannose-sensitive E. coli was studied by measuring luminol-dependent chemoluminescence after bacterial stimulation. In the presence of 0.475, 4.75, 47.5, and 475 mg/l Tamm-Horsfall protein, chemoluminescence responses were reduced in a dose-dependent fashion by 8.7%, 38.1%, 60.3%, and 96.1%, respectively. Addition of 0.003 units/ml alpha-mannosidase reversed the effect of increasing concentrations significantly. Thus, urinary Tamm-Horsfall protein seems to compete with PMNL for mannose-sensitive E. coli in a mannose-sensitive fashion, thereby significantly reducing the role of PMNL as a defense mechanism in the urine of patients with urinary tract infection.

Effect of urine and urine components on the chemiluminescent response of bacteria-stimulated polymorphonuclear leukocytes.

The role of polymorphonuclear leukocytes (PMN) found in urine during infectious episodes is still unknown. Opsonophagocytosis of Escherichia coli by normal blood PMN in the presence of urine was measured using a chemiluminescence (CL) assay. PMN were challenged by a type I fimbriated E. coli strain shown to elicit a CL response through attachment to the mannose-containing receptors on the leukocytes. In the presence of urine the CL response decreased significantly. Urine osmolality due to inorganic salts partially caused this decrease. A higher inhibitory effect was elicited by urea. Under otherwise similar conditions, the presence of an additional CL-inhibiting factor, most probably a protein, was detected in urine; however, its identity has not yet been defined. In vitro and in vivo urine dilution improved PMN function. No difference in effect on CL response was found between urine obtained from 25 children with recurrent urinary tract infections and urine from 15 age-matched controls.

Quantitation of Tamm-Horsfall protein binding to uropathogenic Escherichia coli and lectins.

In quantitative experiments using ELISA, binding of Tamm-Horsfall protein (THP) to uropathogenic Escherichia coli was studied with monoclonal antibody to THP. Adherence to E. coli bearing type 1 fimbriae was proportional to THP concentration and size of the bacterial inoculum. Type 1 fimbriae-bearing E. coli bound 50 times more THP than did non-type 1-fimbriated or P-fimbriated strains. Concanavalin A and wheat germ agglutinin bound THP in a dose-dependent fashion, whereas pokeweed mitogen and Vicia villosa B4 isolectin did not. Addition of mannose and N-acetylglucosamine reduced adherence of THP to concanavalin A and wheat germ agglutinin by 50%-80%. Sugar inhibition studies suggested that the fimbrial receptor site for THP has lectin-like properties and that THP binds to fimbriae via its mannose side chains. This quantitative assay is useful for studying the interaction between THP, uroepithelial cells, and bacteria in vitro.

Urinary excretion of Tamm-Horsfall protein in women with recurrent urinary tract infections.

Since MS-fimbriated bacteria adhere to Tamm-Horsfall protein, it has been suggested that Tamm-Horsfall protein may trap urinary pathogens and prevent them from colonizing the mucosal surfaces of the urinary tract. To test the hypothesis that low urinary Tamm-Horsfall protein excretion rates predispose to urinary tract infection we obtained serial urine samples from 17 women with and 18 without a history of recurrent urinary tract infection. None of the women had known structural abnormalities of the urinary tract. Concentrations of Tamm-Horsfall protein in urine were measured with a sensitive enzyme-linked immunosorbent assay method. On the average, 3 urine samples per person collected within 3 to 6 months were analyzed. The mean Tamm-Horsfall protein excretion of women with recurrent urinary tract infection was 57.0 mg./l. and that of controls was 66.3 mg./l.; this difference was not statistically significant. The mean coefficient of variation was 44.2 and 62.1%, respectively. We conclude that urinary Tamm-Horsfall protein concentration is not significantly decreased in women with recurrent urinary tract infection compared with controls, and that excretion varies widely in repeat samples obtained from the same individual.

Saethre-Chotzen syndrome associated with defective neutrophil chemotaxis.

An 11-month-old male infant with Saethre-Chotzen syndrome and recurrent respiratory infections is described. Persistent extremely high leukocytosis warranted evaluation of neutrophil functions. It was found that the opsonophagocytic activity was normal, but neutrophil chemotaxis was markedly decreased. Further studies pointed to an intracellular neutrophil defect causing this motility dysfunction.

Effect of an intravenous gammaglobulin preparation on the opsonophagocytic activity of preterm serum against coagulase-negative staphylococci.

Recent reports have described cases of septicaemia caused by coagulase-negative staphylococci in preterm neonates, mainly due to the use of artificial intravenous devices. It was of interest to examine if intravenous immunoglobulin therapy, known to be effective in group B streptococcal infections of neonates, had a similar beneficial effect in coagulase-negative staphylococcal infections. Opsonophagocytosis of coagulase-negative staphylococci by normal polymorphonuclear leukocytes in the presence of cord blood serum supplemented with the commercial IgG preparation 'Sandoglobulin' was investigated, using luminol-dependent chemiluminescence. It was found that with two different coagulase-negative staphylococcal strains, Sandoglobulin had a concentration-dependent enhancing effect on the chemiluminescent response. This effect was demonstrated in the presence of native as well as inactivated cord blood serum and in the presence of sera from preterm infants (28-33 weeks). It is concluded that intravenous Sandoglobulin therapy may be effective in the treatment of preterm infants with severe coagulase-negative staphylococcal infections.

A new ELISA method for the rapid quantification of Tamm-Horsfall protein in urine.

The authors present a simplified enzyme-linked immunosorbent assay (ELISA) technique for the quantitative measurement of urinary Tamm-Horsfall protein (THP). Microtiter plates are coated with THP and urine samples at various dilutions without the need for a capture antibody. The bound glycoprotein is then incubated with a monoclonal anti-THP antibody and an alkaline phosphatase conjugated anti-IgG antibody. The assay was validated and gave reproducible results over a wide range of absorbance values. The sensitivity of the assay for THP was 2-5 micrograms/L, the coefficient of variation between assays 7.5%, and the day-to-day variability 11.1% for THP concentrations between 6.25 and 200 micrograms/L. THP excretion was assayed in five volunteers over five days comparing THP concentration in spot urines and in 24-hour urine collections.

Chemiluminescence response of polymorphonuclear leucocytes to serum-opsonized zymosan--a predictive parameter for infections and prognosis in multiple myeloma patients.

Serum opsonization capability and phagocytosis by polymorphonuclear leucocytes (PMNL) were investigated in 37 patients with multiple myeloma (MM), 32 patients with chronic lymphocytic leukaemia (CLL), and 30 healthy controls. Opsonophagocytosis was assessed by measuring the chemiluminescence (CL) response of controls' and patients' PMNL to control- and patient-serum-opsonized zymosan particles. Control PMNL CL responses were significantly reduced both by MM and CLL sera. Normal sera restored the CL responses in MM patients, but only partially corrected the CL responses in CLL patients. Significant correlations were found between the CL responses in MM patients and four clinical parameters: clinical stage, presence of disease-related symptoms, incidence of bacterial infections, and outcome. The findings suggest a defect in MM and CLL sera opsonization ability which, at least in part, could account for the increased susceptibility to infections observed in these patients. Furthermore, the assessment of the CL response in MM patients is recommended as a simple and useful clinical tool for prediction of susceptibility to infection, and prognosis.

Escherichia coli adherence to human transitional cell tumors: a simple research model of bacterial attachment to the lower urinary tract.

A simple model for assaying bacterial adherence to the urinary tract is presented. Cell suspensions of freshly excised transitional cell tumors were used as substrata for attachment of 3H-adenine-labeled Escherichia coli urine isolates. Quantitative radioactive counts were confirmed by microscopic observations in replicate assays of each condition tested. This model provided for species and organ specificity in studies of the bacterial adherence role in urinary tract infections.

Phagocytosis of mucoid and nonmucoid strains of Pseudomonas aeruginosa.

In order to evaluate the role of mucoid coating of clinical isolates of Pseudomonas aeruginosa as a virulence factor, opsonophagocytosis of mucoid and nonmucoid strains of P. aeruginosa were studied by three different methods: uptake of [3H]adenine-labeled bacteria, oxygen consumption, and chemiluminescence production by phagocytosing polymorphonuclear leukocytes. All three methods showed a strong correlation with regard to phagocytosis of the bacterial isolates. As a group, the mucoid strains of P. aeruginosa demonstrated significantly reduced phagocytic uptake by leukocytes when compared to nonmucoid strains, although occasionally mucoid strains did exhibit normal uptake. In vitro growth of mucoid strains under nonstationary conditions was associated with reduced mucoid coating and with a corresponding increase in susceptibility to leukocyte phagocytosis. Although the overall bactericidal activity of human polymorphonuclear leukocytes for mucoid strains was diminished, this appears to be a function of reduced ingestion since the rate of intracellular killing was similar for mucoid and nonmucoid strains. The enhanced susceptibility of mucoid strains to spontaneous bactericidal activity of pooled human serum was confirmed. Mucoid-producing strains produced less protease in vitro; however, the role of this phenomenon in influencing phagocytosis is unknown.

Role of hydrophobicity in adherence of gram negative bacteria to epithelial cells.

Agglutination of yeast, human group A and guinea pig erythrocytes by multiple clinical isolates of E. coli, K. pneumoniae, and P. aeruginosa was investigated and correlated with hydrophobicity measurements of each bacterial strain. Hydrophobicity of the isolates, as measured by hydrophobic interaction chromatography, was similarly correlated with in vitro adherence of the microorganisms to buccal epithelial cells. Agglutination and adherence studies were done in the presence and absence of 0.046 M D (+) mannose. Results showed a wide variability of these parameters among the three general of bacteria. Although E. coli designated mannose sensitive by agglutination showed significantly greater hydrophobicity and attachment to buccal cells, there was no direct correlation between hydrophobic retention and adherence to epithelial cells (p greater than 0.5). As a group, K pneumoniae strains adhered in higher numbers than other gram negative species, but this was unrelated to the hydrophobicity or the designated mannose sensitive/mannose resistant adhesin status of the strain. P. aeruginosa isolates failed to agglutinate yeast and erythrocytes and also adhered poorly to buccal cells. A relationship between bacterial hydrophobicity and in vitro adherence was not found.

Effects of subinhibitory concentrations of ketoconazole on in vitro adherence of Candida albicans to vaginal epithelial cells.

The in vitro adherence of multiple 14C-glucose labeled isolates of Candida albicans to exfoliated vaginal epithelial cells in the presence of subinhibitory concentrations of ketoconazole was studied with a new method utilizing differential centrifugation in a gelatin-PBS solution as well as by the standard method of direct microscopy measurement. Pre-incubation of stationary-phase candida in ketoconazole at concentrations of 0.002 to 0.1 microgram/ml for 4 h at 37 degrees C had no effect on adherence. The addition of ketoconazole to logarithmic phase Candida albicans failed to reduce the total number of cell-associated adherent yeast but exposure to subinhibitory concentrations of ketoconazole was associated with a dystrophic morphology of the blastospores, extensive clumping and reduced germination resulting in fewer individual candida blastospores directly attached to the cell membranes. Germination inhibition and a marked reduction in adherent candida was observed when 0.01 microgram/ml ketoconazole was added to Candida albicans incubated in germination promoting medium. The diminished adherence of Candida albicans to vaginal epithelial cells after exposure to subinhibitory concentrations of ketoconazole may have clinical relevance in preventing recurrent candida vaginitis.

In vivo adherence of Pseudomonas aeruginosa to rat bladder epithelium.

To date, the study of bacterial adherence to uroepithelial cells has utilized considerable variation in methodology, resulting in highly divergent conclusions. In an attempt to standardize methodology, a modified method is described to more accurately measure the in vivo bacterial adherence to rat bladder uroepithelium utilizing Pseudomonas species labeled with 2-[3H]adenine. Two isolates of P. aeruginosa were selected for more intensive study; one showed consistently good adherence; the second strain always adhered poorly, thus providing a negative control. Maximum adherence was detected after 60 min of incubation. Neither of the two Pseudomonas isolates when examined by transmission electron microscopy showed evidence of pili formation. The morphological features of Pseudomonas adherence to bladder mucosa as studied by scanning electron microscopy are described.

Lysozyme concentration in tears of patients with sicca syndrome.

Fifty-three patients with sicca syndrome and 34 healthy controls had their tear lysozyme concentration examined. Lysozyme level was significantly decreased (P less than .0005) in all patients with low Schirmer test values as compared with healthy controls. Even in rheumatoid arthritis patients with normal Schirmer test results a low concentration of tear lysozyme was found. Tear lysozyme can be used to detect subclinical involvement of lacrimal glands in collagen diseases.