R. Phillip Heine

Cysteine Proteases of Trichomonas vaginalis Degrade Secretory Leukocyte Protease Inhibitor

Sexually transmitted diseases, including trichomoniasis, are risk factors for acquisition of human immunodeficiency virus (HIV) infection. Enhancement mechanisms are unknown. Secretory leukocyte protease inhibitor (SLPI) from saliva appears to prevent transmission of HIV through inhibition of virus entry into monocytic cells in vitro. This study was undertaken to determine if secreted cysteine proteases of Trichomonas vaginalis degrade SLPI and render it nonfunctional. It was determined if SLPI levels were decreased in vaginal fluids from pregnant women infected with T. vaginalis. Isolated proteases were incubated with recombinant human SLPI, and the degradation was followed by Western analysis with SLPI antiserum. SLPI levels were measured by ELISA in vaginal fluids from women infected with T. vaginalis and uninfected controls. Cysteine proteases cleaved SLPI and rendered it nonfunctional. Median levels of SLPI from infected patients were 26% of those of controls (P <.005). The degradation of SLPI in association with trichomonal infection may increase the risk of HIV acquisition.

Serial salivary estriol to detect an increased risk of preterm birth

Objective To evaluate serial measurements of salivary estriol (E3) to detect increased risk of spontaneous preterm labor and preterm birth. Methods A masked, prospective, multicenter trial of 956 women with singleton pregnancies was completed at eight United States medical centers. Saliva was collected weekly, beginning at the 22nd week of gestation until birth, and tested for unconjugated E3 by enzyme-linked immunosorbent assay. Women were separated into high-risk and low-risk groups using the Creasy scoring system. Results A single, positive (at or above 2.1 ng/mL) salivary E3 test predicted an increased risk of spontaneous preterm labor and delivery in the total population (relative risk [RR] 4.0, P < .005), in the low-risk population (RR 4.0, P ≤ .05), and in the high-risk population (RR 3.4, P = .05). Two consecutive positive tests significantly increased the RR in all study groups, with a dramatic improvement in test specificity and positive predictive value but only a modest decrease in sensitivity. In women who presented with symptomatic preterm labor, salivary E3 identified 61% of those who delivered within 2 weeks, using a threshold of 1.4 ng/mL. Conclusion Elevated salivary E3 is associated with increased risk of preterm birth in asymptomatic women and symptomatic women who present for evaluation of preterm labor.

Accuracy of salivary estriol testing compared to traditional risk factor assessment in predicting preterm birth

OBJECTIVE The objective was to compare the predictive accuracy (percentage of correct vs incorrect predictions) of salivary estriol levels (SalEst; Biex, Inc, Dublin, Calif) with that of the modified Creasy score for predicting preterm labor followed by preterm delivery. STUDY DESIGN A triple-blinded prospective trial was conducted at 8 US centers. RESULTS Among 601 evaluable patients, serial salivary estriol testing correctly predicted the appropriate outcome 91% of the time and the Creasy scoring method correctly predicted the appropriate outcome 75% of the time (McNemar test P <. 001). Among subjects with Creasy scores >/=10 (high-risk group, n = 152), use of salivary estriol testing correctly predicted the end point 87% of the time, compared with only 7.2% correctly predicted by modified Creasy scoring (McNemar test P <.001). CONCLUSION Salivary estriol assessment was more accurate in predicting outcome than was modified Creasy scoring.

Amniotic Fluid Defensins: Potential Markers of Subclinical Intrauterine Infection

Human neutrophil peptides 1-3 (defensins) are granule constituents released from activated neutrophils. We hypothesized that amniotic fluid (AF) defensin levels are elevated in preterm labor (PTL) patients with subclinical intrauterine infection (IUI). AF samples were obtained from 203 pregnant patients with varying clinical characteristics. Defensin levels were measured by enzyme-linked immunosorbent assay. Median AF defensin levels were fourfold to 24-fold higher in patients with IUI than in preterm and term controls. Among patients with subclinical IUI, the degree of AF defensin elevation was greater in those with a positive AF culture. AF defensin levels increased exponentially with increasing severity of histologic chorioamnionitis. An AF defensin level of > 2,500 ng/mL identified 88% of patients with a positive AF culture, whereas a level of > 400 ng/mL identified 85% of all infected patients. AF defensin levels accurately identify patients with subclinical IUI, as defined by a positive AF culture or placental histology.

Association between Elevated Neutrophil Defensin Levels and Endometritis

The role of host defenses in the pathogenesis of pelvic inflammatory disease (PID) remains largely uncharacterized. The antimicrobial peptides defensins are important components of innate host defense. To explore the relationship between neutrophil defensins and upper genital tract infection, 377 women who were at risk for PID were enrolled in a study. Women infected with Neisseria gonorrhoeae, Trichomonas vaginalis, or Chlamydia trachomatis had higher median levels of neutrophil defensins (human neutrophil peptides 1-3) in the vagina than did uninfected women. Neutrophil defensins were strongly associated with the presence of endometritis after the analysis was controlled for the presence of sexually transmitted diseases. Vaginal neutrophils were associated with endometritis only in the presence of elevated defensin levels, which highlights the importance of neutrophil activation, rather than the presence of neutrophils alone, in this inflammatory process. Neutrophil defensins appear to participate in the host defense in ascending pelvic infection and the pathogenesis of PID.

Levels of Antimicrobial Molecules Defensin and Lactoferrin Are Elevated in the Cerebrospinal Fluid of Children With Meningitis

Objective: To measure levels of defensins and lactoferrin in the cerebrospinal fluid (CSF) of children with meningitis. Study Design. Prospective descriptive study involving children undergoing lumbar puncture during evaluation for meningitis. Methods. CSF concentrations of defensins and lactoferrin were determined using enzyme-linked immunosorbent assays on 19 children with bacterial meningitis, 31 children with aseptic meningitis, and 32 control children found to have normal CSF during evaluation for meningitis. Pertinent clinical and laboratory data were gathered on all children. Results. CSF concentrations of both defensins and lactoferrin were elevated markedly in children with bacterial and aseptic meningitis, compared with control children. No control subject had detectable levels of defensins in the CSF. Lactoferrin was undetectable in the CSF of 31 of 32 control subjects. Defensin and lactoferrin levels were significantly higher in the CSF of children with bacterial meningitis than in those with aseptic meningitis. Defensin levels in the CSF of children with bacterial meningitis ranged from 128 ng/mL to 99 430 ng/mL with a mean of 30 311 ng/mL (SD ± 28 865) and a median of 23 042 ng/mL. Defensin levels in the CSF of children with aseptic meningitis ranged from 0 ng/mL to 1675 ng/mL with a mean of 227 ng/mL (SD ± 433) and a median of 23 ng/mL. A significant correlation was found between defensin levels in the CSF and the total leukocyte count and the absolute neutrophil count in the CSF of children with bacterial meningitis. Lactoferrin levels in the CSF of children with bacterial meningitis ranged from 184 ng/mL to 31 412 ng/mL with a mean of 13 209 ng/mL (SD ± 9644) and a median of 10 382 ng/mL. Lactoferrin levels in the CSF of children with aseptic meningitis ranged from 0 ng/mL to 2715 ng/mL with a mean of 1042 ng/mL (SD ± 878) and a median of 852 ng/mL. No correlation was found between lactoferrin level in the CSF and the total leukocyte count or the absolute neutrophil count in the CSF of children with bacterial meningitis. In our study population, the sum total of CSF defensins and lactoferrin was found to be highly sensitive and specific in delineating bacterial from aseptic meningitis when compared with standard CSF studies. Conclusions. Significant elevations of defensins and lactoferrin, indicative of endogenous local antimicrobial peptide and polypeptide release, are found in the CSF of children with meningitis. We speculate that elevations in these antimicrobial molecules may reflect the intensity of the host response. Defensins seem to parallel neutrophil activation more closely than lactoferrin. Cumulative levels of CSF defensins and lactoferrin clearly distinguished bacterial meningitis from aseptic meningitis and control patients. Further investigation is warranted to determine the usefulness of measuring defensins and lactoferrin as a diagnostic tool and therapeutic monitor in the evaluation of children with meningitis.

Plasma concentrations of defensins and lactoferrin in children with severe sepsis.

Background. We hypothesized that systemic release of endogenous leukocyte-derived polypeptide antimicrobial defensins (polymorphonuclear leukocyte-specific) and lactoferrin (polymorphonuclear leukocyte and epithelial cell derived) occurs in nonneutropenic children with severe sepsis. Methods. We performed a prospective cross-sectional and longitudinal study in a university children’s hospital pediatric intensive care unit. Ninety-two consecutive children meeting criteria for sepsis and 14 critically ill children without sepsis (controls) were enrolled, and plasma defensins and lactoferrin concentrations were measured on Days 1 and 3 of sepsis. Results. Nonneutropenic sepsis patients (n = 71) had increased defensins and lactoferrin plasma concentrations compared with critically ill control patients [defensins, 450 ng/ml vs. 150 ng/ml; lactoferrin, 332 ng/ml vs. 176 ng/ml (median values);P < 0.05] and neutropenic sepsis patients [n = 21; defensins, 450 ng/ml vs. 50 ng/ml; lactoferrin, 332 ng/ml vs. 20 ng/ml (median values);P < 0.05]. Neutropenic sepsis patients had similar plasma defensin concentrations and a decrease in plasma lactoferrin concentrations compared with control patients (P < 0.05). Defensins and lactoferrin plasma concentrations correlated to total white blood cell and absolute neutrophil count (P < 0.05). There was no association between plasma defensin concentration and organ failure or outcome; however, increased plasma lactoferrin concentrations were observed with the development of organ failure (P < 0.05). Conclusion. These data suggest that increased circulating defensins and lactoferrin release are dependent in part on neutrophil count and might play a role in host defense in children with severe sepsis.

Trichomonas vaginalis Weakens Human Amniochorion in an In Vitro Model of Premature Membrane Rupture

Objective: Trichomonas vaginalis (TV) infection is associated with preterm rupture of membranes (PROM) and preterm birth. We evaluated the effects of TV growth and metabolism on preparations of human amniochorion to understand and characterize how TV may impair fetal-membrane integrity and predispose to PROM and preterm birth. Methods: Term fetal membranes were evaluated using an established in vitro fetal-membrane model. Fresh TV clinical isolates were obtained from pregnant women. The protozoa (5.0×105 to 1.5×106/ml) were incubated with fetal membranes in modified Diamonds medium for 20 h at 37°C in 5% CO2.The effects of fetal-membrane strength (bursting tension, work to rupture, and elasticity) were measured using a calibrated Wheatstone-bridge dynamometer. Tests were also performed to evaluate the effects of 1) inoculum size; 2) metronidazole (50 μg/ml); and 3) cell-free filtrate. Results: The TV-induced membrane effects were 1) isolate variable; 2) inoculum dependent; 3) incompletely protected by metronidazole; and 4) mediated by both live organisms as well as protozoan-free culture filtrates. Six of 9 isolates significantly reduced the calculated work to rupture (P ≤ 0.02); 7 of 9 reduced bursting tension; and 1 of 9 reduced elasticity. One isolate significantly increased the work to rupture and bursting tension (P ≤ 0.002). Conclusions: In vitro incubation of fetal membranes with TV can significantly impair the measures of fetal-membrane strength. This model may be used to delineate the mechanisms of TV-induced membrane damage. This study suggests that there are enzyme-specific effects as well as pH effects.

Maternal Effects of Respiratory Syncytial Virus Infection during Pregnancy.

Clarifying these effects could show potential benefits of RSV vaccination of pregnant women.

Amniotic-fluid Lactoferrin: A Marker for Subclinical Intraamniotic Infection Prior to 32 Weeks Gestation.

Objective: Lactoferrin is a glycoprotein released from the secondary granules of activated neutrophils in the setting of infection. The purpose of this study was to determine if amniotic-fluid (AF) lactoferrin levels are elevated in preterm labor (PTL) patients with subclinical intraamniotic infection (IAI). Methods: AF samples were obtained from 186 pregnant patients with the following characteristics: group 1 - term, no labor; group 2 - preterm, no labor; group 3 - PTL with IAI; group 4 - PTL without IAI. Lactoferrin levels were measured with an enzyme-linked immunosorbent assay (ELISA). Results: AF lactoferrin levels were elevated in normal gestation after 31 weeks (P < 0.0001). Lactoferrin levels were also higher in infected PTL patients compared with noninfected PTL patients at gestations ≤31 weeks (P = 0.005). An AF lactoferrin level of >2.5 μg/ml is highly suggestive of infection in PTL patients at <32 weeks, with an overall sensitivity of 82% and a specificity of 83%, when infection is defined as a positive AF culture or positive placental histology. Conclusions: AF lactoferrin levels increase after 31 weeks in normal gestations, but lactoferrin levels >2.5 μg/ml in PTL patients before this gestational age are highly suggestive of IAI. AF lactoferrin levels may be a useful clinical tool for selecting those PTL patients who might benefit from antimicrobial therapy, closer observation, or early delivery.