Analysis of risk factors for tuberculous infection following exposure at a homeless shelter.

SETTING: A homeless shelter for men aged ⩾ 50 years in Seattle, Washington, USA. OBJECTIVES: We examined risk factors for tuberculous infection following exposure to an active pulmonary tuberculosis (TB) case residing in a homeless shelter setting. METHODS: A contact investigation identified shelter clients exposed to the index case; these contacts were then assessed for tuberculous infection. Risk factors, including proximity and duration of exposure to the index case, were evaluated for association with infection. A retrospective cohort study was conducted and a multivariate logistic regression model determined the magnitude of the association between tuberculous infection and significant risk factors. RESULTS: Of the 64 contacts evaluated, 25 (39%) had latent tuberculous infection and one had active TB. The multivariate logistic regression model found that duration of exposure and birthplace were significantly associated with odds of infection. CONCLUSIONS: Birthplace and duration of exposure were significant risk factors for tuberculous infection, underscoring the importance of this information when prioritizing contact investigations after TB exposure in congregate settings. We recommend that public health agencies work with homeless shelters to ensure that clients' attendance records contain the necessary information to facilitate contact tracing during public health TB investigations.

Pregnancy outcome after ultrasound diagnosis of fetal intra-abdominal umbilical vein varix.

OBJECTIVES: Fetal intra-abdominal umbilical vein (FIUV) varix is a focal dilatation of the intra-abdominal portion of the umbilical vein, which has been reported to be associated with intrauterine death and other anomalies. Our aim was to examine our experience with this diagnosis at a single tertiary-care center and to correlate it with clinical outcome. METHODS: This was a retrospective case series study. Our ultrasound database was searched for all cases with a diagnosis of FIUV varix identified at our facility between 1997 and 2007. We reviewed all ultrasound examinations, maternal antenatal records, delivery records and newborns' medical records. RESULTS: We identified 52 cases of FIUV among a population of approximately 68,000. Three cases of trisomy 21 were identified, all of which were accompanied by other anomalies. There was intrauterine death of one fetus with trisomy 21 at 35 weeks of gestation. We did not find an association between FIUV varix and other obstetric complications. CONCLUSIONS: The outcome of pregnancies with FIUV varix is generally favorable. The finding of a FIUV varix should prompt the search for other anomalies, especially markers of aneuploidy.

Comparison of two oxytocin regimens to prevent uterine atony at cesarean delivery: a randomized controlled trial.

OBJECTIVE: To determine if high-dose oxytocin reduces the need for additional uterotonic agents at cesarean. METHODS: A randomized, double-masked trial of two oxytocin regimens was performed to prevent postpartum uterine atony in laboring women. The pharmacy prepared sequentially numbered oxytocin solutions containing either 10 U/500 mL or 80 U/500 mL of lactated Ringer's solution infused over 30 minutes after cord clamping. The need for additional uterotonic agents was determined by the surgical team. Hypotension was diagnosed and treated with crystalloid or a pressor agent. To detect a 50% decrease in the need for additional uterotonic agents and considering a beta error of 0.2, 220 patients would be required in each group (alpha = 0.05, two-tailed chi(2) test). RESULTS: The low-dose group (n = 163) received 333 mU/min, and the high-dose group (n = 158) received 2667 mU/min of oxytocin. The groups were similar with respect to risk factors for atony. Women in the low-dose group received additional uterotonic medication significantly more often than those in the high-dose group (39% compared with 19%, P <.001, relative risk 2.1, 95% confidence interval 1.4, 3.0). Moreover, more women in the low-dose group received methylergonovine, 15-methyl prostaglandin F(2alpha) or both (9% compared with 2%, relative risk 4.8, 95% confidence interval 1.4, 16) after additional oxytocin (median 20 U) had been added to the study solution. The incidence of hypotension was similar in both groups. CONCLUSION: Compared with an infusion rate of 333 mU/min, oxytocin infused at 2667 mU/min for the first 30 minutes postpartum reduces the need for additional uterotonic agents at cesarean delivery.

The involvement of genome researchers in high school science education.

The rapid accumulation of genetic information generated by the Human Genome Project and related research has heightened public awareness of genetics issues. Education in genome science is needed at all levels in our society by specific audiences and the general public so that individuals can make well-informed decisions related to public policy and issues such as genetic testing. Many scientists have found that an effective vehicle for reaching a broad sector of society is through high school biology courses. From an educational perspective, genome science offers many ways to meet emerging science learning goals, which are influencing science teaching nationally. To effectively meet the goals of the science and education communities, genome education needs to include several major components-accurate and current information about genomics, hands-on experience with DNA techniques, education in ethical decision-making, and career counseling and preparation. To be most successful, we have found that genome education programs require the collaborative efforts of science teachers, genome researchers, ethicists, genetic counselors, and business partners. This report is intended as a guide for genome researchers with an interest in participating in pre-college education, providing rationale for their involvement and recommendations for ways they can contribute, and highlighting a few exemplary programs. World Wide Web addresses for all of the programs discussed in this report are given in Table 1. We are developing a database of outreach programs offering genetics education () and request that readers submit an entry describing their programs. We invite researchers to contact us for more information about activities in their local area.

Prenatally diagnosed hypoplastic left heart syndrome--outcomes after postnatal surgery.

OBJECTIVE: To identify prenatally diagnosed cases of hypoplastic left heart syndrome (HLHS) and then to determine postnatal outcomes after surgical interventions. METHODS: An ultrasound and pediatric cardiology database was used to identify all fetuses diagnosed prenatally from 1991-1996 with HLHS. Fetal karyotypes were performed on cultured amniocytes. After diagnosis, parents were given several management options: pregnancy termination before 22 weeks, postnatal hospice care, or surgery using the Norwood procedure or cardiac transplantation. Ultrasound and echocardiography findings were later compared to karyotype results and postnatal outcome data. RESULTS: Fifteen fetuses with HLHS were identified. Two (16%) chromosome abnormalities and three (20%) structural defects were detected. Three mothers (20%) opted for pregnancy termination, two (13%) chose postnatal hospice care, and one aneuploid fetus had an intrauterine death. Nine parents (60%) chose surgery for their infants; however, one infant was not an appropriate surgical candidate due to a coexisting diaphragmatic hernia. Eight infants underwent surgery and two survived (25%). Of the four infants scheduled to undergo the Norwood procedure, one died preoperatively, two died intraoperatively, and one infant survived and is doing well at age 8 months. Of the four infants scheduled for cardiac transplantation, two died awaiting transplant and one died postoperatively. One infant survived cardiac transplantation but has microcephaly and developmental delay at age two. CONCLUSIONS: In prenatally diagnosed HLHS at our institution, the survival rate following surgery for infants felt to be the best candidates was only 25%.

Pneumonia as a complication of pregnancy.

OBJECTIVE: To identify risk factors for the development of antepartum pneumonia and to describe maternal and perinatal outcome in pregnant women with pneumonia. METHODS: The study group consisted of 59 women with antepartum pneumonia. Pneumonia was defined by the presence of lower respiratory tract symptoms, radiographic findings, no other source of infection, and at least two of the following: oral temperature > or =38 degrees C, white blood cell count > or =15,000/ml, auscultatory findings, and/or positive sputum cultures. For comparison, a control group (n = 118) of pregnant women was formed by selecting the first mother who delivered immediately before and after an index study subject. RESULTS: Mothers in the study group were significantly more likely than women in the control group to have either a history of asthma (P = 0.022) or an admission hematocrit < or =30% (P < 0.001). Women with pneumonia were also more likely to receive a tocolytic agent (P < 0.001) and/or beta-methasone to enhance fetal lung maturity (P < 0.001). In addition, study subjects delivered at an earlier mean gestational age (P = 0.002) and had infants who weighed significantly less (P = 0.003) than mothers in the control group. Multivariate analysis indicated that women with asthma or anemia had more than a five-fold increase in the risk of developing pneumonia during pregnancy (P = 0.013), and mothers with pneumonia were significantly more likely to deliver before 34 weeks gestation (P = 0.04). CONCLUSIONS: Pneumonia during pregnancy was associated with maternal anemia and asthma. In addition, preterm labor with tocolysis and/or beta-methasone was more common in women with pneumonia, and these women were more likely to deliver preterm and have low birthweight infants compared to women without pneumonia.

Organophosphate and carbamate insecticides in agricultural waters and cholinesterase (ChE) inhibition in common carp (Cyprinus carpio).

Cholinesterase (ChE) activity was used as a biomarker for assessing exposure of common carp (Cyprinus carpio) to organophosphate and carbamate insecticides from irrigated agricultural waters. Carp were collected from a lake (Royal Lake) that receives most of its water from irrigation return flows and from a reference lake (Billy Clapp Lake) outside of the irrigation system. Results indicated that the mean whole-brain ChE activity of carp from Royal Lake (3.47 micromol/min/g tissue) was 34.2% less than that of carp from Billy Clapp Lake (5.27 micromol/min/g tissue) (p = 0.003). The depressed ChE activity in brain tissue of Royal Lake carp was in response to ChE-inhibiting insecticides detected in water samples in the weeks prior to tissue sampling; the most frequently detected insecticides included chlorpyrifos, azinphos-methyl, carbaryl, and ethoprop. Neither sex nor size appears to be a covariable in the analysis; ChE activity was not correlated with fish length or weight in either lake and there was no significant difference in ChE activity between the two sexes within each lake. Although organophosphate and carbamate insecticides can break down rapidly in the environment, this study suggests that in agricultural regions where insecticides are applied for extended periods of the year, nontarget aquatic biota may be exposed to high levels of ChE-inhibiting insecticides for a period of several months.

Intraoperative hypothermia and post-cesarean wound infection.

OBJECTIVE: To determine whether intraoperative hypothermia during cesarean delivery is a risk factor for wound infection. METHODS: Eighteen cases with wound infection and 18 controls matched for age, weight, presence of gestational hypertension, and surgery length were selected from a cohort of 900 women who underwent cesarean delivery and who were assessed for wound infection according to strict criteria. Because immediate postoperative temperatures reflect intraoperative temperature nadir accurately and were available universally, we compared the mean immediate postoperative temperatures between cases and controls. RESULTS: In addition to the intentionally matched factors, the groups were well-matched for race, parity, presence of labor, presence of meconium, and duration of membrane rupture. The mean initial postoperative temperatures were similar between the two groups (36.3+/-0.9C versus 36.6+/-1.0C, respectively; P=.8). This study had a power of 90% to detect an intergroup difference of 1C. CONCLUSION: In this case-control study of cesarean delivery, intraoperative hypothermia was not a risk factor for wound infection.

The antiphospholipid antibody syndrome: an overview.

The antiphospholipid antibody syndrome is characterized by the presence of maternal anticardiolipin antibodies and/or the lupus anticoagulant in association with recurrent pregnancy loss, thrombotic events, and/or thrombocytopenia. This disorder occurs rarely, but pregnant patients with antiphospholipid antibodies are at risk for adverse maternal and perinatal outcomes. This article reviews the antiphospholipid antibody syndrome, including its pathophysiology, clinical sequelae, diagnostic criteria, medical treatment, and nursing care.

Interaction of the UvrABC endonuclease with DNA containing a psoralen monoadduct or cross-link. Differential effects of superhelical density and comparison of preincision complexes.

The effect of negative supercoiling on UvrABC incision of covalently closed duplex DNA circles containing either a furan-side monoadduct or a cross-link of 4'-hydroxymethyl-4,5',8-trimethylpsoralen at a unique site was examined. The rate of UvrABC incision of these DNA substrates was measured as a function of superhelical density, sigma, for values of sigma between 0 and -0.050. The monoadducted DNA substrate was incised at close to the maximum rate at all superhelical densities, with only a slight stimulation of activity between sigma = 0 and -0.035. In contrast, efficient UvrABC incision of the cross-linked DNA substrate required the DNA to be underwound, and activity showed a linear dependence on superhelical density up to sigma = -0.035. DNase I protection studies show that in the presence of both UvrA and UvrB a protein complex binds to the site of a psoralen monoadduct or cross-link in linear DNA. This UvrA-UvrB-dependent complex binds with similar affinity to both the monoadducted and the cross-linked DNA helices. However, differences in the DNase I footprint on these two DNA substrates indicate that the interaction of this protein complex is different at these two lesions. The addition of UvrC to linear DNA molecules that are saturated at the site of the lesion with the UvrA-UvrB-dependent complex resulted in efficient nicking of the monoadducted DNA, but not the cross-linked DNA. Thus, the properties of a DNA lesion site that lead to UvrAB recognition and binding are not necessarily sufficient to allow incision when all three Uvr subunits are present. We propose that after recognition and binding of a lesion site by the UvrAB complex and prior to incision, the damaged DNA helix undergoes a conformational change such as unwinding or melting that is induced by the lesion-bound Uvr complex.

The T4 DNA polymerase accessory proteins form an ATP-dependent complex on a primer-template junction.

The DNA polymerase holoenzyme of bacteriophage T4 contains, besides the DNA polymerase itself (the gene 43 protein), a complex of the protein products of T4 genes 44 and 62 (a DNA-dependent ATPase) and of gene 45. Together, the 44/62 and 45 proteins form an ATP-dependent "sliding clamp" that holds a moving DNA polymerase molecule at the 3' terminus of a growing DNA chain. We have used a unique DNA fragment that forms a short hairpin helix with a single-stranded 5' tail (a "primer-template junction") to map the binding sites for these polymerase accessory proteins by DNA footprinting techniques. In the absence of the DNA polymerase, the accessory proteins protect from DNase I cleavage 19-20 nucleotides just behind the 3' end of the primer strand and 27-28 nucleotides on the complementary portion of the template strand. Detection of this DNA-protein complex requires the 44/62 and 45 proteins plus the nonhydrolyzable ATP analogue adenosine 5'-O-(thiotriphosphate). The complex is not detected in the presence of ATP. We suggest that ATP hydrolysis by the 44/62 protein normally activates the accessory proteins at a primer-template junction, permitting the DNA polymerase to bind and thus form the complete holoenzyme. However, when the polymerase is missing, as in these experiments, ATP hydrolysis is instead followed by a release (or loosening) of the accessory protein complex.

DNA footprinting studies of the complex formed by the T4 DNA polymerase holoenzyme at a primer-template junction.

We have used DNA footprinting techniques to analyze the interactions of five DNA replication proteins at a primer-template junction: the bacteriophage T4 DNA polymerase (the gene 43 protein), its three accessory proteins (the gene 44/62 and 45 proteins), and the gene 32 protein, which is the T4 helix-destabilizing (or single-stranded DNA-binding) protein. The 177-nucleotide-long DNA substrate consisted of a perfect 52-base pair hairpin helix with a protruding single-stranded 5' tail. As expected, the DNA polymerase binds near the 3' end of this molecule (at the primer-template junction) and protects the adjacent double-stranded region from cleavage. When the gene 32 protein binds to the single-stranded tail, it reduces the concentration of the DNA polymerase required to observe the polymerase footprint by 10-30-fold. Periodic ATP hydrolysis by the 44/62 protein is required to maintain the activity of the DNA polymerase holoenzyme (a complex of the 43, 44/62, and 45 proteins). Footprinting experiments demonstrate the formation of a weak complex between the DNA polymerase and the gene 45 protein, but there is no effect of the 44/62 protein or ATP on this enlarged footprint. We propose a model for holoenzyme function in which the complex of the three accessory proteins uses ATP hydrolysis to keep a moving polymerase tightly bound to the growing 3' end, providing a "clock" to measure polymerase stalling.

Requirement for the replication protein SSB in human DNA excision repair.

Replication and repair are essential processes that maintain the continuity of the genetic material. Dissection of simian virus 40 (SV40) DNA replication has resulted in the identification of many eukaryotic replication proteins, but the biochemistry of the multienzyme process of DNA excision repair is less well defined. One protein that is absolutely required for semiconservative replication of SV40 DNA in vitro is human single-stranded DNA-binding protein (SSB, also called RF-A and RP-A). SSB consists of three polypeptides of relative molecular mass 70,000, 34,000 and 13,000, and acts with T antigen and topoisomerases to unwind DNA, allowing the access of other replication proteins. Human SSB can also stimulate the activity of polymerases alpha and delta, suggesting a further role in elongation during DNA replication. We have now found a role for human SSB in DNA excision repair using a cell-free system that can carry out nucleotide excision repair in vitro. Monoclonal antibodies against human SSB caused extensive inhibition of DNA repair in plasmid molecules damaged by ultraviolet light or acetylaminofluorene. Addition of purified SSB reversed this inhibition and further stimulated repair synthesis by increasing the number of repair events. These results show that a mammalian DNA replication protein is also essential for repair.

Identification of an immunostimulating protein from Mycobacterium leprae.

Despite the recent identification of a number of Mycobacterium leprae proteins, the major immunogenic determinants of this organism remain obscure. We isolated from M. leprae a potent immunostimulatory preparation, designated the MLP fraction, which contains a major protein of 35 kilodaltons (kDa). This protein was precipitated by monoclonal antibody ML03-A1, which recognizes a 35-kDa protein of M. leprae, and by sera obtained from patients with lepromatous leprosy. Neither sera from healthy controls nor sera from patients with pulmonary tuberculosis recognized the 35-kDa protein, and only one of four serum samples from patients with borderline tuberculoid leprosy reacted with this protein. The MLP fraction stimulated T-cell proliferation in patients with leprosy whose T cells proliferate in response to whole M. leprae cells. Apparently, the T-cell epitope associated with MLP is also expressed on M. tuberculosis and M. bovis BCG, since patients with pulmonary tuberculosis and BCG-vaccinated individuals demonstrated significant responses to the MLP fraction. The 35-kDa M. leprae protein, purified to homogeneity in the laboratory of P. J. Brennan, stimulated T-cell proliferative responses in all MLP-responsive subjects. These findings suggest that the 35-kDa protein present in MLP is an immunostimulatory component of M. leprae. In addition to serving as a useful probe for study of the T-cell anergy associated with lepromatous disease, this protein may ultimately be useful as a component of a vaccine designed to provide protection against infection with M. leprae.

Effective vaccination of mice against leprosy bacilli with subunits of Mycobacterium leprae.

Model vaccines against leprosy bacilli have consisted of nonvirulent, live, attenuated Mycobacterium bovis BCG and irradiated, heat-killed, or autoclaved intact M. leprae. We report that immunization with various cell wall fractions of M. leprae, progressively depleted of lipids, carbohydrates, and soluble proteins, as well as a partially purified protein(s) derived from a pellet fraction of sonicated M. leprae, conferred significant protection against subsequent infection with live leprosy bacilli. Moreover, lymphocytes from regional lymph nodes and spleens of mice immunized with these M. leprae-derived subunits responded by proliferation when stimulated with M. leprae in vitro. Our results provide the first evidence that vaccination with M. leprae-derived fractions protects mice against leprosy bacilli.

Localization of DNA repair synthesis by human cell extracts to a short region at the site of a lesion.

Double-stranded bacteriophage M13 DNA molecules were constructed containing a single specifically placed 2-(acetylamino)fluorene adduct or a single 4'-hydroxymethyl-4,5',8-trimethylpsoralen monoadduct. These circular DNA molecules were used to analyze in vitro DNA repair synthesis by cell extracts from normal human lymphoid cell lines. Both types of lesions stimulate DNA repair synthesis at the site of the adduct. DNA repair synthesis induced by the 2-(acetyl-amino)fluorene adduct took place in the damaged strand and was confined to a region within a 31-base pair restriction fragment around the adduct.

UvrABC incision of N-methylmitomycin A-DNA monoadducts and cross-links.

The Escherichia coli UvrABC endonuclease is a multisubunit enzyme that initiates the repair of a wide variety of DNA lesions in vivo by making dual incisions on a damaged strand at the eighth or ninth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the modified base. It has been hypothesized that UvrABC is able to recognize a broad spectrum of lesions because it does not recognize the lesion per se but rather gross helical distortions that the lesion induces in the DNA. Several lesions have recently been studied which are thermal stabilizing and are not believed to distort the DNA grossly, including the CC-1065-N-3-adenine and anthramycin-N-2-guanine adducts. We have studied the activity of UvrABC in vitro on another thermal stabilizing and nondistortive adduct, N-methylmitomycin A (NMA), a bifunctional DNA-alkylating agent that reacts with guanine on the side facing the minor groove, yielding either monoadducts or interstrand cross-links. NMA adducts increase the thermal stability of DNA, and theoretical calculations indicate that NMA adducts do not grossly distort the DNA helix. Our results show that UvrABC makes incisions at the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to an NMA monoadduct, consistent with the incision pattern observed for the majority of other lesions that are also recognized by UvrABC. DNA containing a site-specific NMA cross-link was also recognized and incised by UvrABC. The rate of incision of NMA cross-linked DNA was about 200-fold higher in supercoiled molecules than in relaxed molecules, whereas the rate of incision of DNA containing NMA monoadducts was stimulated approximately 2-fold by supercoiling. The signal for UvrABC recognition and incision of damaged DNA is discussed in relation to the ability of UvrABC to incise NMA adducts as well as other nondistortive lesions.

Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates.

Sequence-specific pausing occurs during DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase holoenzyme in the presence of the T4 helix destabilizing protein (gene 32 protein). Two of the six strongest pause sites on a double-stranded bacteriophage fd DNA template are in regions where hairpin helices are predicted to form when the DNA is single stranded. However, the other pause sites are in regions that are not obviously involved in secondary structure. The positions of the DNA chain ends produced at one pause site of each type were determined to within +/- 2 nucleotides. At this resolution, a clustering of sites is observed, suggesting that the polymerase holoenzyme may become destabilized when moving along selected regions of the DNA and then pause at one or more of several closely spaced positions. The addition of the T4 gene 41 protein (a DNA helicase that forms part of the T4 primosome) to the above replication system greatly increases the rate of fork movement and eliminates detectable pausing. In contrast, the addition of the T4 dda protein (a second DNA helicase that increases the rate of fork movement to a similar extent) has no affect on replication fork pausing. This difference could either be due to specific protein-protein interactions formed between the polymerase holoenzyme and the 41 protein or to the highly processive movement of the 41 protein along the displaced DNA strand.