Floxed-Cassette Allelic Exchange Mutagenesis Enables Markerless Gene Deletion in Chlamydia trachomatis and Can Reverse Cassette-Induced Polar Effects.

As obligate intracellular bacteria, Chlamydia spp. have evolved numerous, likely intricate, mechanisms to create and maintain a privileged intracellular niche. Recent progress in elucidating and characterizing these processes has been bolstered by the development of techniques enabling basic genetic tractability. Florescence-reported allelic exchange mutagenesis (FRAEM) couples chromosomal gene deletion with the insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Similar to other bacteria, many chlamydial genes exist within polycistronic operons, raising the possibility of polar effects mediated by insertion cassettes. Indeed, FRAEM-mediated deletion of Chlamydia trachomatistmeA negatively impacts the expression of tmeB We have adapted FRAEM technology by employing a gfp-bla cassette flanked by loxP sites. Conditional expression of Cre recombinase in ChlamydiatmeA containing a floxed cassette resulted in deletion of the marker and restoration of tmeB expression.IMPORTANCEC. trachomatis infections represent a significant burden to human health. The ability to genetically manipulate Chlamydia spp. is overcoming historic confounding barriers that have impeded rapid progress in understanding overall chlamydial pathogenesis. The current state of genetic manipulation in Chlamydia spp. requires further development, including mechanisms to generate markerless gene disruption. We leveraged a stepwise Cre-lox approach to excise selection marker genes from a deleted gene locus. We found this process to be efficient, and the removal of extraneous elements resulted in the reversal of a negative polar effect on a downstream gene. This technique facilitates a more direct assessment of gene function and adds to the Chlamydia molecular toolbox by facilitating the deletion of genes within operons.

Fowler's syndrome and pregnancy.

We present a favourable outcome of a pregnant woman with underlying Fowler's syndrome. This is a rare disorder affecting young women with painless urinary retention, and as there is no known cure, the main concern is to ensure adequate bladder emptying. Our patient went through self-catheterisation and suprapubic catheters after which she was finally successfully managed on a sacral nerve stimulator (SNS). Upon getting pregnant the stimulator was switched off due to safety concerns, and from then on, bladder emptying was managed with a suprapubic catheter (which was regularly changed over the gestation). The main concern thereafter was recurrent hospital admissions with urinary tract infections and pelvic pain requiring parenteral antibiotics and analgesia. She underwent an uncomplicated elective caesarean section at 36 weeks and delivered a healthy female infant. The SNS was successfully reactivated in the postnatal phase, and the suprapubic catheter was removed upon achieving adequate urethral output.

The fuzzy-boundary arena--a method for constraining an animal's range in spatial experiments without using walls.

A method is described for confining an animal within an experimenter-defined area without the use of physical boundaries. The area of exploration is constrained by the presence of an aversive noise, triggered whenever the animal steps across a computer-controlled boundary. The radius of the invisible boundary is constantly reset so that the boundary becomes "fuzzy" and the animal cannot use it as a spatial localizing cue. The effectiveness of this technique is demonstrated both with behavioural data confirming reliable confinement, and also recordings of hippocampal place cells made from rats exploring the arena. The place cell data reveal that indeed, the cells did not appear to be controlled by the fuzzy boundary, in contrast with the strong control normally exerted by fixed boundaries. This technique is thus promising for studies of spatial behaviour in which the strong influence of walls needs to be removed in order to allow the study of more subtle processes such as landmark use and path integration.

Hospital referrals with possible ectopic pregnancy: prospective observational study.

Women of reproductive age with abdominal pain represent a diagnostic challenge, especially in primary care where the decision whether to refer to hospital needs to be taken. The diagnosis of an ectopic pregnancy hinges on a combination of clinical findings and a positive urinary pregnancy test (UPT). We investigated whether non-pregnant patients with abdominal pain were being referred inappropriately to hospital to exclude ectopic pregnancy because a UPT had not been performed or had been interpreted incorrectly. In this study, of the 81 patients referred by their general practitioners (GPs) on suspicion of ectopic, 38 were not pregnant on admission and only 46% of the UPT results in hospital agreed with those reported to the GP by the patient prior to referral. Given the high sensitivity and specificity of correctly interpreted UPTs, our findings suggest that pregnancy should be confirmed before hospital referral.

Uterine compression sutures: surgical management of postpartum hemorrhage.

BACKGROUND: It has been estimated that worldwide, over 125,000 women die of postpartum hemorrhage each year. The traditional management of this condition includes the use of oxytocics, such as oxytocin, ergometrine, and prostaglandins, before proceeding to ligation of the internal iliac arteries and even hysterectomy. The B-Lynch technique is a surgical procedure that may be used to arrest postpartum hemorrhage resulting from uterine atony. CASES: This paper describes simple modifications of this technique that make this procedure less complex to perform. Three clinical case scenarios illustrate the context in which the sutures may be used. CONCLUSION: Compression sutures placed into the postpartum uterus may provide a simple first surgical step to control bleeding when routine oxytocic measures have failed. We suggest that the technique we have described is a simple procedure and should be tried before more complex interventions are used.

Microvascular vasodilator response to acetylcholine is increased in women with pre-eclampsia.

OBJECTIVES: To evaluate in vivo microvascular responses to incremental doses of the endothelial-dependent vasodilator, acetylcholine, and the endothelial-independent vasodilator, sodium nitroprusside, in women with pre-eclampsia and gestation-matched normotensive pregnant controls. DESIGN: Prospective clinical study. SETTING: Southern Derbyshire Acute Hospitals Trust, and University of Nottingham Division of Vascular Medicine. POPULATION: Thirteen women with pre-eclampsia and 16 normotensive pregnant controls. METHODS: Cutaneous microvascular vasodilator responses to iontophoretic administration of incremental doses of acetylcholine and sodium nitroprusside (25-100 microC) were evaluated under temperature-controlled conditions using laser Doppler fluximetry. RESULTS: Resting skin temperature and blood flow were similar among 13 women with pre-eclampsia [mean blood pressure 151/93 mmHg (4/3); mean gestation 35.6 weeks (1.0); and mean proteinuria 1.1 g/24h (0.2)] and 16 normotensive pregnant controls [mean blood pressure 111/63 mmHg (2/2); mean gestation 34.3 weeks (0.9)]. Peak vasodilator responses to acetylcholine were increased in women with pre-eclampsia (median flux ratio 15.1 [IQR 12.3-17.6] vs 11.7 [IQR 8.4-12.6], P < 0.05), whereas sodium nitroprusside responses were not different between the two groups: 11.4 [IQR 8.6-13.4] vs 9.5 [IQR 8.0-12.3]. CONCLUSION: In vivo microvascular vasodilator responses to acetylcholine are increased in women with pre-eclampsia, while endothelial-independent vasodilation is unchanged. Although the mechanism of acetylcholine induced vasodilation in small vessels is unclear, this study confirms previous animal data and provides in vivo evidence of altered microvascular endothelial cell function in pre-eclampsia.

The preliminary characterization of a vasoactive circulating factor(s) in preeclampsia.

OBJECTIVE: The endothelium-dependent behavior of myometrial resistance vessels from women with preeclampsia differs dramatically from that of healthy pregnant women. Similar functional changes may be induced in vessels from healthy pregnant women by incubation with plasma from women with preeclampsia. STUDY DESIGN: Myometrial arterioles, obtained from healthy pregnant women at elective cesarean section, were incubated with plasma from women with preeclampsia or plasma from healthy pregnant women. Myographic techniques were used to study the endothelium-dependent relaxation to bradykinin. The effects of subjecting plasma from women with preeclampsia to heat treatment, charcoal stripping, protein extraction and digestion, and column fractionation on the inducible changes in endothelial function were likewise investigated. RESULTS: Incubation with plasma from women with preeclampsia resulted in a significant reduction in the vessel's endothelium-dependent relaxation, a change that was reversible. This effect was reduced by heat treating and charcoal stripping, maintained within a plasma protein concentrate, and completely removed by protease digestion. The vasoactive component(s) of the plasma had a molecular weight greater than 100 kd. CONCLUSIONS: Plasma of women with preeclampsia alters the endothelium-dependent relaxation of myometrial vessels. Our findings suggest that such alterations are induced by a high-molecular-weight protein/glycoprotein, with possible contributions from a hydrophobic, lipophilic factor.

Inducible change in the behavior of resistance arteries from circulating factor in preeclampsia: an effect specific to myometrial vessels from pregnant women.

OBJECTIVE: It has previously been observed that a circulating factor or factors may be responsible for the changes in vessel behavior that are postulated to underlie the pathogenesis of preeclampsia. We wished to ascertain whether such changes in endothelial function are dependent on the vascular bed under investigation. STUDY DESIGN: Myometrial and infracolic omental vessels resistance vessels were obtained at cesarean delivery or hysterectomy. After incubation with either plasma from women with preeclampsia or plasma from normotensive pregnant women, myographic techniques were used to assess the endothelium-dependent relaxations of these vessels. RESULTS: Incubation of myometrial vessels from normotensive pregnant women with plasma from women with preeclampsia resulted in a significant reduction in endothelium-dependent relaxation, an effect that was independent of the parity of the patients from whom the vessels had been taken. Incubation of omental vessels from normotensive pregnant women and myometrial vessels from nonpregnant women with plasma from women with preeclampsia had no effect on the endothelium-dependent relaxation observed. CONCLUSIONS: This study demonstrates that the inducible changes in resistance vessel behavior are dependent on the tissue bed under investigation and on the pregnancy status of the patient.

Participation in research: informed consent, motivation and influence.

OBJECTIVE: To investigate the process and quality of informed consent, motivation and influence in parents who were invited to enroll their baby in a research project. METHODOLOGY: A mixed quantitative/qualitative questionnaire was sent to a cohort invited to participate in a physiological research project on sudden infant death syndrome (SIDS) at the Dunedin Public Hospital, Dunedin, New Zealand. Separate questionnaires were used for parents who participated (94) and those who declined to participate (103). Response rates were 69% and 47%, respectively. RESULTS: All consenting parents felt they understood the purpose and procedure of the study. The majority (90%) thought the information about the study was very good; 6.5% felt more detail was required. Eighty-five per cent found the verbal explanation the most useful source of information. All participated for altruistic reasons such as to aid SIDS research. Although 27% had concerns about safety of the tests, after the tests all responders felt happy with the safety of the tests. Inconvenience was the main reason (53%) for declining to participate. Twenty-eight per cent of declining parents were concerned about the safety of the tests. CONCLUSION: Of those who responded to the questionnaire, the process for obtaining informed consent in the SIDS studies was satisfactory. Parents' motives for participating were mostly altruistic. The role of recall bias and selection bias may make the implications of this study unclear.

Factors affecting heart rate variability and heart rate responses to tilting in infants aged 1 and 3 months.

Heart rate variability (HRV) and heart rate (HR) responses following a 60 degree head-up tilt were measured in 60 infants at 1 and 3 mo of age to investigate the effects on these of age, sleep state, sleep position, and mother's smoking status. HRV was determined from Poincaré plots of 500 sequential RR intervals to measure overall variability derived from the SDRR of this plot, and instantaneous variability derived from the SDdeltaRR. HR responses to the tilt were measured as changes in RR interval length from rest to immediately following the tilt and again once a stable pattern was reached. SDRR and SDdeltaRR increased 20 and 40%, respectively, with age (p < 0.0001), SDRR was higher in active sleep (AS) than quiet sleep (QS, +72%, p < 0.0001) but both measures of variability (SDRR and SDdeltaRR) were lower in the prone position compared with supine (-18%, p < 0.0001). However, several findings were dependent on the basal RR interval, thus the age effect disappeared once RR interval was taken into account, sleep state remained an important factor and the lower variability when prone now became a difference of -3% (p = 0.034). The tilt generally provoked a reflex tachycardia followed by a bradycardia and settling to a stable HR level below, at, or above baseline within 30 s. The more unusual responses were no HR change, sustained tachycardia or sustained bradycardia (15% of total). These were more likely to occur in younger infants (p = 0.008) and in AS (p < 0.0001). No changes were seen in any of the cardiac indices related to maternal smoking status. The findings confirm several reports indicating that prone sleeping damps some physiologic responses. The data emphasize the need to consider basal heart rate, and sleep position as well as sleep state in autonomic function testing during infant sleep.

Botulinum toxin type A therapy for palmar and digital hyperhidrosis.

OBJECTIVE: We evaluated the efficacy of subepidermal injections of botulinum toxin type A on recalcitrant palmar and digital hyperhidrosis. METHODS: Twenty patients with recalcitrant palmar and digital hyperhidrosis were treated with subepidermal injections of botulinum toxin. Nineteen patients completed the 12-month study. Injections were performed in 3 stages. The total dose of toxin for each hand, which included the palm, thenar eminence, and digits, was 165 units. Patients were followed up on a monthly basis. RESULTS: Botulinum toxin significantly reduced sweat production in the treated areas. Anhidrosis lasted 9 months in 3 patients, 8 months in 3 patients, 7 months in 8 patients, 6 months in 3 patients, 5 months in 1 patient, and 4 months in 1 patient. Reduced sweating of the palm and digits continued in all patients for the 12-month evaluation period, with the greatest reduction of sweating in the nondominant hand. Mild weakness of the thumb occurred in 4 patients at a mean duration of 3 weeks, with the greatest duration being 6 weeks. CONCLUSION: Botulinum toxin provides a safe and efficacious alternative in the treatment of recalcitrant palmar and digital hyperhidrosis.

Desmin as a possible immunohistochemical marker for feline hypertrophic cardiomyopathy.

Desmin has been suggested as a possible histopathological marker for hypertrophic cardiomyopathy (HCM) in humans. To test whether a similar pattern of desmin staining applies to HCM in cats, we conducted an immunohistochemical study on myocardial samples from 13 cats (HCM 4, other cardiomyopathies (OCM) 4, and control 5). The pattern of staining for desmin in HCM cats was not the same as that reported in humans, but was weaker than in OCM cats and controls. This suggested that desmin may be a possible histochemical marker for feline HCM, but our data was insufficient to clearly confirm this.

Plasma from women with pre-eclampsia induces an in vitro alteration in the endothelium-dependent behaviour of myometrial resistance arteries.

OBJECTIVE: To compare the in vitro effect of plasma from normal pregnant women and women with pre-eclampsia on the endothelium-dependent behaviour of myometrial resistance arteries from normal pregnant women. DESIGN: An in vitro comparative study. SETTING: Nottingham City Hospital. SAMPLE: Uterine biopsy specimens were obtained from normal pregnant women delivered by elective caesarean section at term. Plasma was collected from nulliparous women with pre-eclampsia (n = 18), and from multiparous normal pregnant women (n = 18), all samples being matched for maternal age and gestation at venepuncture. Pools of plasma from women with pre-eclampsia and normal pregnant women were formed from these samples and were used in all the experiments. METHODS: Myometrial resistance vessels obtained from the uterine biopsies were incubated with normal pregnant plasma, plasma from women with pre-eclampsia, or without plasma. Wire myography was employed to study the effect of plasma on the endothelium-dependent behaviour of these vessels. RESULTS: Incubation of vessels from normal pregnant women with plasma from women with pre-eclampsia resulted in a significant reduction in endothelium-dependent relaxation, compared with vessels incubated either with plasma from normal pregnant women or without plasma. This alteration in endothelial function occurred after an incubation period of one hour and required a threshold concentration for its effect to become established. Removal of the vascular endothelium abolished these changes in vessel behaviour. There were no plasma-induced alterations in the endothelium-independent behaviour of the vascular smooth muscle. CONCLUSIONS: This study supports the hypothesis that plasma from women with pre-eclampsia is capable of altering endothelium-dependent myometrial relaxation in vessels from pregnant women.

VEGF via VEGF receptor-1 (Flt-1) mimics preeclamptic plasma in inhibiting uterine blood vessel relaxation in pregnancy: implications in the pathogenesis of preeclampsia.

Preeclampsia is a multisystem disorder characterized by hypertension and proteinuria. There is accumulating evidence that this is a disease of the endothelium, with an as-yet unidentified circulating factor, or factors, causing the observed alteration in vascular function. We previously reported that the function of myometrial vessels is altered on exposure to plasma from women with preeclampsia. Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that acts via two high-affinity receptors (KDR and Flt-1), and its production is increased in preeclampsia. Here we report that VEGF and its Flt-1 receptor may play a pivotal role in the altered vascular function of preeclampsia. Myometrial resistance vessels were obtained at the time of cesarean section. Using the Mulvany wire myograph, the endothelium-dependent behavior of these vessels was studied. Incubation of vessels from pregnant women with VEGF resulted in a reduction of endothelium-dependent relaxation that mimicked the reduction induced by plasma from women with preeclampsia. The altered function that occurred upon exposure of vessels to VEGF or plasma from women with preeclampsia did not occur when plasma was incubated with antibodies to VEGF before vessel incubation. The presence of an anti-KDR receptor antibody had no effect on VEGF response. However, in the presence of an anti-Flt-1 receptor antibody, VEGF or plasma from women with preeclampsia no longer attenuated the endothelium-dependent relaxation (p < 0.05). The changes observed with VEGF and plasma from women with preeclampsia and their subsequent blockade with anti-VEGF antibody and anti-Flt-1 receptor antibody strongly suggest that VEGF acting through the Flt-1 receptor is pivotal in the pathogenesis of this disease.

Relationship of cell adhesion molecule expression to endothelium-dependent relaxation in normal pregnancy and pregnancies complicated with preeclampsia or fetal growth restriction.

OBJECTIVES: To determine the degree of endothelium-dependent relaxation in myometrial and omental resistance arteries from normal pregnancies and pregnancies complicated with preeclampsia or fetal growth restriction (FGR) (compromised pregnancy group), and to correlate the results with the endothelial surface expression of cell adhesion molecules (CAMs) in the same vessels. METHODS: Parallel wire myography was used to assess the relaxation of omentum or myometrial vessels obtained from nonpregnant women (n = 3), women with normal pregnancies (n = 11), and women with pregnancies complicated by preeclampsia or fetal growth restriction (n = 10). These resistance vessels were constricted with incremental concentrations of vasopressin (10(-10) mol/L to 3.3 x 10(-8) mol/L) prior to the addition of incremental concentrations of bradykinin (10(-10) mol/L to 3.3 x 10(-6) mol/L). Immunohistochemistry was used to assess the endothelial expression of the CAMs E-selectin, ICAM-1, VCAM-1, and PECAM. RESULTS: A significant reduction in endothelium-dependent relaxation of myometrial vessels was found in the compromised pregnancy group when compared with both the normotensive pregnant group and the nonpregnant group. This reduction was not noted with omental vessels. All vessels in the nonpregnant group, normal pregnant group, and compromised pregnancy group expressed PECAM and ICAM-1 on the endothelium. There was no difference in intensity of immunostaining between the groups. None of the vessels in any of the groups expressed VCAM-1 or E-selectin. CONCLUSIONS: We found no evidence that impaired relaxation responses to bradykinin are linked to altered expression of CAMs in preeclampsia and FGR. These results suggest that increased CAM expression occurs in a vascular bed separate from those investigated in the present study. Possible sites for this would be in the microcirculation of organs such as the kidney.

Angiotensin-converting enzyme insertion-deletion polymorphism in normotensive and pre-eclamptic pregnancies.

OBJECTIVE: To investigate the hypothesis that pre-eclampsia is associated with a common insertion-deletion polymorphism in the angiotensin-converting enzyme gene. DESIGN: Seventy-two women with pre-eclampsia and 83 normotensive pregnant women participated in the study. Pre-eclampsia was defined as a blood pressure exceeding 140/90 mm Hg in a previously normotensive woman, associated with proteinuria in excess of 300 mg/l in a 24 h collection. Samples for fetal genotyping were available from 66 pregnancies complicated by pre-eclampsia and 79 normotensive pregnancies. METHODS: Maternal and fetal samples were genotyped at the insertion-deletion (I-D) polymorphism in intron 16 of the angiotensin-converting enzyme gene by the polymerase chain reaction followed by agarose electrophoresis. RESULTS: Neither the I-D genotype distributions nor the allele frequencies differed significantly between pre-eclamptic and normotensive pregnancies in maternal or fetal samples (phi2 <0.3, not significant). The odds ratio for pre-eclampsia in women with the DD genotype, compared with the ID and II genotype, was 1.09 (95% confidence interval 0.55-2.16). The odds ratio associated with the DD genotype in the fetus was 1.14 (0.56-2.32). CONCLUSION: This study has found no evidence that the insertion-deletion polymorphism in the angiotensin-converting enzyme gene is associated with pre-eclampsia.

Preeclampsia: the endothelium, circulating factor(s) and vascular endothelial growth factor.

It has been proposed that endothelial cell activation is the primary event in the multisystem disorder of preeclampsia. Evidence for endothelial involvement in this condition abounds. The best-characterized morphologic abnormality of this syndrome, glomerular endotheliosis, involves endothelial cells. Also associated with preeclampsia is a loss of endothelial cell integrity, with the consequent increase in vascular permeability, and an increase in the circulating levels of the endothelial cell markers, fibronectin, von Willebrand factor, tissue plasminogen activator, and plasminogen activator inhibitor-1. It is now well documented that endothelial activation contributes to the coagulation abnormalities observed in this disease. There is much evidence that the endothelial alterations in preeclampsia result from one or more circulating factors. The incubation of cultured endothelial cells with serum or plasma samples, taken from normal pregnant women and women with preeclampsia, results in marked alterations in cell behavior and metabolic processes. More recently, experiments employing myographic techniques have demonstrated convincingly the effects of a circulating factor(s) on the function of endothelial cells of resistance arteries. Vascular endothelial growth factor (VEGF) possesses many of the characteristics required of a candidate circulating factor. It contains a hydrophobic secretory signal sequence, exerts in vitro effects specific to vascular endothelial cell, and promotes endothelial expression of procoagulant activity. Circulating VEGF concentrations are elevated in women with preeclampsia, and VEGF increases microvascular endothelial cell prostacyclin production in a dose-dependent manner, analogous to the acute effects of plasma from patients with preeclampsia. Similarly, in myographic studies, when myometrial resistance arteries are incubated with VEGF, there are dose-dependent alterations in endothelium-dependent behavior, mirroring those found after incubation with plasma from patients with preeclampsia.

Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta.

SMAD3 is one of the intracellular mediators that transduces signals from transforming growth factor-beta (TGF-beta) and activin receptors. We show that SMAD3 mutant mice generated by gene targeting die between 1 and 8 months due to a primary defect in immune function. Symptomatic mice exhibit thymic involution, enlarged lymph nodes, and formation of bacterial abscesses adjacent to mucosal surfaces. Mutant T cells exhibit an activated phenotype in vivo, and are not inhibited by TGF-beta1 in vitro. Mutant neutrophils are also impaired in their chemotactic response toward TGF-beta. Chronic intestinal inflammation is infrequently associated with colonic adenocarcinoma in mice older than 6 months of age. These data suggest that SMAD3 has an important role in TGF-beta-mediated regulation of T cell activation and mucosal immunity, and that the loss of these functions is responsible for chronic infection and the lethality of Smad3-null mice.