Biomedical subjects

R A Welch

Publications and source records attributed to R A Welch.

At least 55 records · Page 3Linked to original sources

PubMed1991

Cytotoxicity of the HpmA hemolysin and urease of Proteus mirabilis and Proteus vulgaris against cultured human renal proximal tubular epithelial cells.

Proteus mirabilis, a common agent of nosocomially acquired and catheter-associated bacteriuria, can cause acute pyelonephritis. In ascending infections, bacteria colonize the bladder and ascend the ureters to the proximal tubules of the kidney. We postulate that Proteus species uses the HpmA hemolysin and urease to elicit tissue damage that allows entry of these bacteria into the kidney. To study this interaction, strains of Proteus mirabilis and P. vulgaris and their isogenic hemolysin-negative (hpmA) or isogenic urease-negative (ureC) constructs were overlaid onto cultures of human renal proximal tubular epithelial cells (HRPTEC) isolated from kidneys obtained by immediate autopsy. Cytotoxicity was measured by release of soluble lactate dehydrogenase (LDH). Two strains of P. mirabilis inoculated at 10(6) CFU caused a release of 80% of total LDH after 6 h, whereas pyelonephritogenic hemolytic Escherichia coli CFT073 released only 25% at 6 h (P less than 0.012). Ten P. mirabilis isolates and five P. vulgaris isolates were all hemolytic and cytotoxic and produced urease which was induced by urea. The HpmA hemolysin is apparently responsible for the majority of cytotoxicity in vitro since the hemolysin-negative (hpmA) mutants of P. mirabilis and P. vulgaris were significantly less cytotoxic than wild-type strains. P. mirabilis WPM111 (hemolysin negative) was used to test the effect of urease-catalyzed urea hydrolysis on HRPTEC viability. In the presence of 50 mM urea, WPM111 caused the release of 42% of LDH versus 1% at 6 h in the absence of substrate (P = 0.003). We conclude that the HpmA hemolysin of Proteus species acts as a potent cytotoxin against HRPTEC. In addition, urease apparently contributes to this process when substrate urea is available.

Bacterial Proteins↗

PubMed1991

Detection of prenatal drug exposure in the pregnant women and her newborn infant.

Although urine testing remains the standard method for identifying and monitoring drug addicts, recent data suggest that it seriously underdiagnoses the prevalence of fetal exposure to various drugs. Results obtained with other body fluids and tissues (meconium and hair) are reviewed and the laboratory methodologies used to detect drugs are discussed in details.

Female↗

PubMed1991

Cocaine abuse is associated with abruptio placentae and decreased birth weight, but not shorter labor.

Many of our patients report having ingested cocaine hoping to decrease the duration of labor. We reviewed the computerized records of 592 women who abused cocaine. Compared with 4687 controls, women who ingested cocaine were older and had higher parity. Birth weight, birth weight percentile, and gestational age at delivery were significantly decreased among their neonates, and the incidence of abruptio placentae was nearly doubled among these women. Although these factors tend to shorten labor, the total duration of labor was not significantly different between the two groups. These data add to the accumulating evidence that cocaine abuse is associated with increased obstetric morbidity, but do not support the belief that cocaine shortens labor.

Abruptio Placentae↗

PubMed1990

Characterization of monoclonal antibodies against the Escherichia coli hemolysin.

Twelve monoclonal antibodies (MAbs) produced against the Escherichia coli hemolysin (HlyA) encoded by the hemolysin recombinant plasmid pWAM04 were studied. HlyA derivatives from recombinant strains with different plasmids encoding HlyA amino-terminal and carboxy-terminal truncates, HlyA in-frame deletions, and HlyA frameshift mutations were used in immunoblots to localize the antigenic determinants for the anti-HlyA MAbs. The mapping of the MAb epitopes was also facilitated by immunoblotting analysis of HlyA polypeptide fragments derived by cyanogen bromide cleavage. The HlyA epitopes for 11 of the MAbs were mapped to relatively small linear regions of the cytolysin ranging from 28 to 160 amino acids. Five of the MAbs (C10, G8, E2, B7, and D12) neutralized HlyA hemolytic activity to varying degrees. The epitopes for these neutralizing MAbs were found to reside within the following HlyA regions: C10 and G8, amino acids 2 to 160; E2, amino acids 161 to 194; B7, amino acids 518 to 598; and D12, amino acids 626 to 726. Hemolytically active HlyA was dependent on the action of the hlyC gene product. The D12 MAb recognized only HlyA produced by strains with an intact hlyC function. MAb A10 recognized an epitope within the HlyA region from amino acids 728 to 829 where a glycine-rich repeat domain exists; however, this MAb did not neutralize HlyA hemolytic activity. A HlyA domain map showing the anti-HlyA epitope location was constructed.

Animals↗

PubMed1990

Nonreciprocal complementation of the hlyC and lktC genes of the Escherichia coli hemolysin and Pasteurella haemolytica leukotoxin determinants.

The genetic organization of the Pasteurella haemolytica leukotoxin operon (lktCABD) is similar to that of the Escherichia coli hemolysin (hlyCABD). Their gene products share a sequence similarity of 66, 62, 90.5, and 75.6%, respectively. We investigated the role of the C proteins (LktC and HlyC) by performing reciprocal transcomplementation analyses in an E. coli recombinant background. In the absence of the C genes, neither LktA nor HlyA had their respective cytotoxic activities. When hlyC was provided in trans to lktA, the toxin that was produced had the same activity and target cell specificity as the wild-type leukotoxin; it was leukotoxic for bovine lymphoid cells but not human lymphoblast cells when it was evaluated by a 51Cr-release assay. We also detected a weak hemolytic activity for the active form of LktA against sheep erythrocytes. In contrast, an E. coli strain containing lktC with hlyA produced a form of HlyA which was neither hemolytic nor cytotoxic. A monoclonal antibody (D12) against HlyA which recognized an epitope specific to the active form of HlyA did not cross-react in immunoblots with LktA that was activated by either LktC or HlyC. We conclude that the mechanism for activation of leukotoxin and hemolysin by their respective C proteins (LktC and HlyC) is mechanistically similar but that the exact structural requirements involved in the process are different.

Bacterial Proteins↗

PubMed1990

The HpmA hemolysin is more common than HlyA among Proteus isolates.

Two different hemolysins, HpmA and HlyA, have been reported in Proteus spp. To study the distribution of these hemolysins among Proteus strains, isolates from various infections and normal feces were screened for hemolysin production. All 63 Proteus mirabilis strains and 23 of the 24 Proteus vulgaris strains produced a calcium-independent hemolytic activity detectable in cell-free supernatants. The calcium-independent activity was due to HpmA; this activity correlated with the presence of hpmA sequences and the production of an extracellular 166-kilodalton (kDa) protein that reacted with anti-HpmA antiserum. HpmA- mutants, constructed by deletion of the central portion of the hpmA gene, did not produce the 166-kDa protein and were no longer hemolytic when compared with their respective parent strains. Among the 87 P. mirabilis and P. vulgaris isolates examined, calcium-dependent hemolytic activity was produced by only two P. vulgaris strains. These strains produced a 110-kDa protein which comigrated with the Escherichia coli hemolysin (HlyA) antibodies in immunoblots. These studies show that Proteus spp. produce two distinct extracellular hemolysins, with nearly all strains producing the calcium-independent hemolysin, HpmA, but only an occasional P. vulgaris isolate producing HlyA.

Bacterial Proteins↗

PubMed1990

Cytotoxic activity of the Proteus hemolysin HpmA.

We previously showed that hpmA is the hemolysin determinant most commonly found among Proteus isolates. To assess the potential contribution of HpmA to virulence, we first characterized the toxic activities of this hemolysin. Hemolytic activity was present in total cell cultures and cell-free supernatants of Proteus clinical isolates as well as Escherichia coli containing cloned hpm genes. HpmA also possesses cytotoxic activity which was detected by a chromium release assay against a variety of target cell lines (Daudi, Raji, T24, U937, and Vero). Analysis of the dose response of bacterial cells against both T24 cells and erythrocytes showed that E. coli containing cloned hpm genes was 30-fold more cytotoxic than Proteus mirabilis BA6163. Also, 10(5)-fold more bacterial cells were needed to lyse T24 cells than to lyse erythrocytes. HpmA- mutants of two Proteus strains in which the central portion of hpmA was deleted were constructed. These HpmA- mutants, which have lost the hemolytic and cytotoxic activities exhibited by their respective parent strains, demonstrate that HpmA is needed for both of these activities. In an ascending model of murine urinary tract infection, the hpmA mutant strain WPM111 behaved no differently from its parent strain, BA6163, with respect to either the level of kidney colonization or histopathological changes in the kidney. However, WPM111 had a sixfold higher 50% lethal dose than BA6163 when injected intravenously into C3H mice.

Animals↗

PubMed1990

Calcium is required for binding of Escherichia coli hemolysin (HlyA) to erythrocyte membranes.

The calcium requirement for hemolytic activity of Escherichia coli hemolysin was investigated by using hemolytic assays and immunoblotting of toxin-treated erythrocytes. The hemolytic activity of cell culture supernatants obtained during growth of E. coli in Luria-Bertani (LB) broth or calcium-free LB broth was calcium dependent. The hemolytic activity of culture supernatants obtained during growth in LB broth supplemented with calcium was calcium independent. Osmotic protection experiments using Dextran 4 to prevent cell lysis indicated that calcium was required for the binding of hemolysin to erythrocytes at both 4 and 37 degrees C. The binding efficiency at 4 degrees C was 50% of that occurring at 37 degrees C. The calcium-dependent binding was confirmed by immunoblotting saline-washed, toxin-treated erythrocytes with a monoclonal antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of membrane proteins. Bound hemolysin increased the calcium permeability of the cell membranes as evidenced by calcium-induced membrane protein alterations. The alterations in membrane proteins did not directly cause lysis of the cells. The results were consistent with a mechanism of lysis involving the formation of cation-selective pores in the membranes of target cells.

Animals↗

PubMed1990

Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes.

The primary structure of Escherichia coli hemolysin (HlyA) contains a 9-amino-acid sequence which is tandemly repeated 13 times near the C terminus and which is essential for hemolytic activity. Hemolysin also requires an unknown modification by an accessory protein, HlyC, for hemolytic activity. The role of calcium in the interaction of HlyA with erythrocytes was investigated by using recombinant strains which produced inactive hemolysins unmodified by HlyC or deleted of the repeat sequences. 45Ca2+ autoradiography of the recombinant hemolysins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose showed that full-length, active hemolysin bound calcium. The domain involved in binding calcium was identified as the tandemly repeated sequences, since the deletion derivative missing 11 of the 13 repeats did not bind calcium. Inactive hemolysin, unmodified by HlyC, contained the repeated sequences and bound calcium as efficiently as the active, full-length toxin. The binding of the inactive toxins to erythrocytes was investigated by immunoblotting saline-washed, toxin-treated cells with monoclonal antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of membrane proteins. The binding of full-length, active hemolysin to erythrocytes was calcium dependent. Inactive hemolysin deleted of the repeat units did not bind to cells. The inactive hemolysin, unmodified by HlyC, bound calcium but did not bind to erythrocytes. These results highlight the importance of calcium in the binding of hemolysin to erythrocytes and suggest that the binding of hemolysin to cells requires an interaction between the calcium-binding repeat domain and the modification produced by the HlyC protein.

Animals↗

PubMed1990

Nucleotide sequencing of the Proteus mirabilis calcium-independent hemolysin genes (hpmA and hpmB) reveals sequence similarity with the Serratia marcescens hemolysin genes (shlA and shlB).

We cloned a 13.5-kilobase EcoRI fragment containing the calcium-independent hemolysin determinant (pWPM110) from a clinical isolate of Proteus mirabilis (477-12). The DNA sequence of a 7,191-base-pair region of pWPM110 was determined. Two polypeptides are encoded in this region, HpmB and HpmA (in that transcriptional order), with predicted molecular masses of 63,204 and 165,868 daltons, respectively. A putative Fur-binding site was identified upstream of hpmB overlapping the -35 region of the proposed hpm promoter. In vitro transcription-translation of pWPM110 DNA and other subclones confirmed the assignment of molecular masses for the predicted polypeptides. These polypeptides are predicted to have NH2-terminal leader peptides of 17 and 29 amino acids, respectively. NH2-terminal amino acid sequence analysis of purified extracellular hemolysin (HpmA) confirmed the cleavage of the 29-amino-acid leader peptide in the secreted form of HpmA. Hemolysis assays and immunoblot analysis of Escherichia coli containing subclones expressing hpmA, hpmB, or both indicated that HpmB is necessary for the extracellular secretion and activation of HpmA. Significant nucleotide identity (52.1%) was seen between hpm and the shl hemolysin gene sequences of Serratia marcescens despite differences in the G+C contents of these genes (hpm, 38%; shl, 65%). The predicted amino acid sequences of HpmB and HpmA are also similar to those of ShlB and ShlA, the respective sequence identities being 55.4 and 46.7%. Predicted cysteine residues and major hydrophobic and amphipathic domains have been strongly conserved in both proteins. Thus, we have identified a new hemolysin gene family among gram-negative opportunistic pathogens.

Amino Acid Sequence↗

PubMed1990

Uterine activity compared with symptomatology in the detection of preterm labor.

The relative contribution of uterine activity obtained by home monitoring with a guard ring tocodynamometer compared with seven specific signs and symptoms reported during patient/nurse contact as an aid in detecting preterm labor has not been studied. In this prospective, multicenter study, patients at risk for developing early labor who were randomized to receive home uterine activity monitoring and perinatal nursing support were assessed. The initiator of provider contact (uterine activity detected on routine transmission, patient-perceived signs and symptoms of preterm labor during perinatal nurse contact, or both) resulting in a diagnosis of preterm labor was recorded. Contraction data were then analyzed for an association with preterm labor. There was a strong association of increased uterine activity (four or more contractions per hour) on a repeat monitoring strip with preterm labor (P less than .001). Among patients diagnosed with preterm labor, 31% had increased uterine activity detected on a routine transmission without patient-reported signs and symptoms, compared with 24% who were diagnosed as the result of patient-reported symptoms without increased uterine activity. Daily objective uterine activity data alone have greater incremental value over and above other signs and symptoms as an aid to the physician in diagnosing preterm labor.

Female↗

PubMed1990

The contribution of symptomatology and/or uterine activity to the incidence of unscheduled visits.

Home uterine activity monitoring and perinatal nursing support have been shown to be associated with a decrease in preterm births with no increase in the number of unscheduled patient visits. This prospective, randomized multicenter study compared the frequency of unscheduled visits in patients receiving home uterine activity monitoring and perinatal nursing support with that of patients receiving education regarding the detection and reporting of preterm labor symptomatology. The contribution of patient-reported signs and symptoms versus objective uterine activity data to unscheduled visits is assessed. The overall frequency of unscheduled visits was similar in both groups. In the home uterine activity monitoring and perinatal nursing support group, the contributions of uterine activity versus signs and symptoms to the diagnosis of preterm labor were equal, with 36% of patients diagnosed with preterm labor sent to the physician for increased uterine activity and 36% for signs and symptoms. The sensitivity for the group receiving monitoring and nursing support in detecting preterm labor was 93%. The majority of false-positive visits were associated with patient symptoms. These data show that this combination service does not lead to a clinically significant increase in unscheduled visits. Further, the visits resulting from the combination service provide a sensitive predictive method to aid physicians in detecting early labor.

Clinical Trials as Topic↗

PubMed1990

Relationship of uterine contractility to preterm labor.

An increased uterine contraction rate is associated with preterm labor. The contraction rate in individual patients, however, has not been evaluated critically as to its predictive value in forecasting early labor. In this randomized multicenter study, 105 patients at high risk for preterm delivery monitored their contraction rate at home on a daily basis. An analysis was conducted to determine the association of at least four contractions per hour on a routine strip followed by at least four contractions per hour on a repeat tracing with subsequent preterm labor. Using this threshold, 70% of the patients were correctly classified. This contraction rate resulted in a sensitivity of 57%, a specificity of 80%, a positive predictive value of 72%, and a negative predictive value of 68%. A threshold rate of at least four contractions per hour on a remonitor strip identifies a patient at increased risk for preterm labor (P = .003).

Cardiotocography↗

PubMed1990

Home uterine activity monitoring is associated with a reduction in preterm birth.

The value of home uterine activity monitoring plus perinatal nursing support in the prevention of preterm birth among high-risk patients has been demonstrated by several studies. Other reports with less stringent guidelines and less intensive nursing contact have questioned the contribution of home uterine activity monitoring and perinatal nursing support to a preterm birth prevention program. In this large, prospective, randomized multicenter study, patients were assigned to receive either daily monitoring and nursing contact or a preterm birth prevention program including intensive education and more frequent prenatal visits. Among the monitored patients, there was a significant increase in early detection of preterm labor (2 cm or less), successful tocolysis, and prolongation of pregnancy to term. Thirty-one percent of these diagnoses of preterm labor resulted from evaluation of increased uterine activity without associated patient-perceived symptoms. We conclude that a program of daily uterine activity monitoring and perinatal nursing support results in earlier diagnosis of preterm labor and subsequently more effective tocolysis.

Cardiotocography↗

PubMed1990

Preventing preterm birth in twin gestation: home uterine activity monitoring and perinatal nursing support.

In this investigation, 45 women with twin gestations in four centers were randomly assigned to either daily home uterine activity monitoring and perinatal nursing support (19) or an education group (26). Patients in the home uterine activity monitoring and perinatal nursing support group monitored uterine activity twice daily, transmitted the data each day, and had 24 hour-a-day access to nursing support. Education patients were counseled regarding the common signs and symptoms of preterm labor. Both groups had scheduled prenatal visits at least every 2 weeks. Sixteen (62%) of the education group and 14 (74%) of the home uterine activity monitoring and perinatal nursing support group developed preterm labor, values that were not significantly different. Of the monitored group who experienced preterm labor, all 14 were dilated 3 cm or less at diagnosis of the first preterm labor, compared with ten of 16 in the education group (P = .01; one-tailed Fisher test). The mean cervical dilatation at the first preterm labor episode in the group receiving daily monitoring and contact (1.6 cm) was significantly less (P = .01) than that in the education group (2.9 cm). Thus, fewer preterm births were recorded in the home uterine activity monitoring and perinatal nursing support group and significantly fewer patients delivered because of failed tocolysis (P = .03).

Adult↗

PubMed1990

Management of preterm labor patients at home: does daily uterine activity monitoring and nursing support make a difference?

Daily home uterine activity monitoring and perinatal nursing support were compared with standard care in managing patients with recurrent preterm labor during the current pregnancy in an effort to reduce preterm birth. Sixty-seven successfully treated preterm labor patients were randomly assigned in this multicenter study to daily home uterine activity monitoring and perinatal nursing support or to a standard-care group. Recurrent preterm labor occurred in 15 (45%) of monitored patients and 19 (56%) of standard-care patients. The risk of preterm birth (before 37 weeks) for patients with recurrent preterm labor was significantly reduced (P = .025) in the monitored group (seven, or 47%) compared with the standard-care group (16, or 84%). The relative risk of delivering because of failed tocolysis was 2.8 for the standard-care group versus the monitored group. There were no patients in the monitored group who delivered at the first recurrence of preterm labor for failed tocolysis. The data suggest that home uterine activity monitoring and perinatal nursing support are helpful in achieving term birth through earlier detection and treatment of recurrent preterm labor.

Adult↗

PubMed1990

Diabetic retinopathy in pregnancy: a review.

Diabetic retinopathy is the leading cause of blindness between the ages of 24-64 years. The first half of this period corresponds to peak fertility and the childbearing years. The effects of pregnancy on diabetic retinopathy are unclear, but recent studies suggest that pregnancy may be less harmful to the retina of the diabetic subject than was thought previously. Nevertheless, there is reason to believe that at least some women experience a worsening of their retinopathy as a result of pregnancy. Thus, careful ophthalmic evaluation and follow-up are essential for the pregnant woman with diabetes. This should include a minimum of one complete eye examination every trimester and within 3 months postpartum. Color fundus photography and laser treatment are safe, whereas fluorescein angiography, although commonly used to evaluate retinal vascular disease, can usually be avoided during pregnancy.

Diabetic Retinopathy↗

PubMed1989

Development of a diagnostic test for Johne's disease using a DNA hybridization probe.

A DNA probe, M13 mpHAW71, that detects Mycobacterium paratuberculosis in the fecal material of infected animals was developed for use in the diagnosis of Johne's disease. The probe detected as few as 10(5) M. paratuberculosis when hybridized under stringent conditions to total genomic DNA purified from bovine fecal material. When the probe was used diagnostically, it did not differentiate members of the Mycobacterium avium-M. intracellulare-M. paratuberculosis complex. Compared with culturing, the DNA probe identified 34.4% more mycobacterium-containing fecal samples, and testing took only 72 h to complete.

Animals↗