Subtyping of a large collection of historical Listeria monocytogenes strains from Ontario, Canada, by an improved multilocus variable-number tandem-repeat analysis (MLVA).

Listeria monocytogenes is responsible for severe and often fatal food-borne infections in humans. A collection of 2,421 L. monocytogenes isolates originating from Ontario's food chain between 1993 and 2010, along with Ontario clinical isolates collected from 2004 to 2010, was characterized using an improved multilocus variable-number tandem-repeat analysis (MLVA). The MLVA method was established based on eight primer pairs targeting seven variable-number tandem-repeat (VNTR) loci in two 4-plex fluorescent PCRs. Diversity indices and amplification rates of the individual VNTR loci ranged from 0.38 to 0.92 and from 0.64 to 0.99, respectively. MLVA types and pulsed-field gel electrophoresis (PFGE) patterns were compared using Comparative Partitions analysis involving 336 clinical and 99 food and environmental isolates. The analysis yielded Simpson's diversity index values of 0.998 and 0.992 for MLVA and PFGE, respectively, and adjusted Wallace coefficients of 0.318 when MLVA was used as a primary subtyping method and 0.088 when PFGE was a primary typing method. Statistical data analysis using BioNumerics allowed for identification of at least 8 predominant and persistent L. monocytogenes MLVA types in Ontario's food chain. The MLVA method correctly clustered epidemiologically related outbreak strains and separated unrelated strains in a subset analysis. An MLVA database was established for the 2,421 L. monocytogenes isolates, which allows for comparison of data among historical and new isolates of different sources. The subtyping method coupled with the MLVA database will help in effective monitoring/prevention approaches to identify environmental contamination by pathogenic strains of L. monocytogenes and investigation of outbreaks.

Reliability and validity of the Thai translation (Thai PPS Adult Suandok) of the Palliative Performance Scale (PPSv2).

PURPOSE: the Thai PPS Adult Suandok tool was translated from the Palliative Performance Scale (PPSv2) and had been used in Chiang Mai, Thailand for several years. AIM: to test the reliability and validity of the Thai translation of PPSv2. DESIGN: a set of 22 palliative cases were used to determine a PPS score on Time-1, and repeated two weeks later as Time-2. A survey questionnaire was also completed for qualitative analysis. PARTICIPANTS: a total of 70 nurses and physicians from Maharaj Nakorn Hospital in Chiang Mai participated. RESULTS: The Time-1 intraclass correlation coefficient (ICC) for absolute agreement is 0.911 (95% CI 0.86-0.96) and for consistency is 0.92 (95% CI 0.87-0.96). The Time-2 ICC for agreement is 0.905 (95% CI 0.85-0.95) and for consistency is 0.912 (95% CI 0.86-0.96). These findings indicate good agreement among participants and also were somewhat higher in the Time-2 re-test phase. Cohen's kappa score is 0.55, demonstrating a moderate agreement. Thematic analysis from the surveys showed that 91% felt PPS to be a valuable clinical tool overall, with it being 'very useful' or 'useful' in several areas, including care planning (78% and 20%), disease monitoring (69% and 27%) and prognostication (61% and 31%), respectively. Some respondents noted difficulty in determining appropriate scores in paraplegic patients or those with feeding tubes, while others found the instructions long or difficult. CONCLUSION: the Thai PPS Adult Suandok translated tool has good inter- and intra-rater reliability and can be used regularly for clinical care.

An interdisciplinary computer-based information tool for palliative severe pain management.

OBJECTIVES: As patient care becomes more collaborative in nature, there is a need for information technology that supports interdisciplinary practices of care. This study developed and performed usability testing of a standalone computer-based information tool to support the interdisciplinary practice of palliative severe pain management (SPM). DESIGN: A grounded theory-participatory design (GT-PD) approach was used with three distinct palliative data sources to obtain and understand user requirements for SPM practice and how a computer-based information tool could be designed to support those requirements. RESULTS: The GT-PD concepts and categories provided a rich perspective of palliative SPM and the process and information support required for different SPM tasks. A conceptual framework consisting of an ontology and a set of three problem-solving methods was developed to reconcile the requirements of different interdisciplinary team members. The conceptual framework was then implemented as a prototype computer-based information tool that has different modes of use to support both day-to-day case management and education of palliative SPM. Usability testing of the computer tool was performed, and the tool tested favorably in a laboratory setting. CONCLUSION: An interdisciplinary computer-based information tool can be developed to support the different work practices and information needs of interdisciplinary team members, but explicit requirements must be sought from all prospective users of such a tool. Qualitative methods such as the hybrid GT-PD approach used in this research are particularly helpful for articulating computer tool design requirements.

Chronic Intranasal Oxytocin has Dose-dependent Effects on Central Oxytocin and Vasopressin Systems in Prairie Voles (Microtus ochrogaster).

Oxytocin (Oxt) is a neuropeptide with many functions, including modulation of social behavior(s) and anxiety. Due to its notable pro-social effects, it has been proposed as a treatment in the management of neuropsychiatric disorders, such as autism spectrum disorder (ASD), schizophrenia, and social anxiety; however, effects of long-term daily treatment are still being explored. Previously, we have shown that in male prairie voles (Microtus ochrogaster) exposure to Oxt during the peri-adolescent period impaired adult pair bonding in a dose-dependent fashion. In females, the medium dose used (0.8 IU/kg) appeared to facilitate pair bonding, and the low and medium doses were associated with fewer lines crossed in the open field. In this study, we examined central receptor binding and immunoreactive (IR) protein for Oxt and vasopressin (Avp), a closely related peptide. Voles were treated with saline vehicle, or one of three doses of Oxt (0.08, 0.8, 8.0 IU/kg) for three weeks from postnatal days 21 to 42, and euthanized as adults. We used autoradiography to examine Oxt and Avp receptor binding and immunohistochemistry to examine Oxt and Avp - IR cells in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Females that received the medium dose of Oxt had higher Oxt receptor binding in the nucleus accumbens shell (NAS), while males that received the medium dose had lower Avp-IR cells in the PVN. In summary, we found sex-specific effects of long-term exposure to intranasal Oxt on the Oxt and Avp systems at the weight-adjusted dose currently being used in clinical trials in humans.

Development and reliability testing of the Victoria Bowel Performance Scale (BPS).

With changes in bowel function being a common and often distressful issue for palliative care patients, the ability to easily monitor and record changes in bowel status would be helpful in addressing quality of care. Most bowel tools record either constipation or diarrhea, but not both. A new tool, the Victoria Bowel Performance Scale (BPS), was designed as an ordinal nine-point scale from -4 (severe constipation) to +4 (severe diarrhea) and includes three parameters: visual stool characteristics, bowel pattern, and ability to control defecation. This study tested the reliability of BPS using case scenarios in a test-retest format. Sixty-seven raters in Time Period 1 and 54 raters in Time Period 2 ranked the 18 cases. The intraclass correlation coefficients for absolute agreement were 0.822 and 0.853 for Time Periods 1 and 2, respectively. Results showed that the raters were consistent in their scoring over time, with an average Cohen's kappa of 0.70 over all of the raters. The average Pearson correlation coefficient between Time Periods 1 and 2 scores was 0.92. Further prospective testing in day-to-day clinical care is needed to further confirm the reliability and clinical utility of the BPS. A BPS management guideline has been developed to assist with decision making for each BPS score, which also requires validation.

Use of Palliative Performance Scale in end-of-life prognostication.

BACKGROUND: Current literature suggests clinicians are not accurate in prognostication when estimating survival times of palliative care patients. There are reported studies in which the Palliative Performance Scale (PPS) is used as a prognostic tool to predict survival of these patients. Yet, their findings are different in terms of the presence of distinct PPS survival profiles and significant covariates. OBJECTIVE: This study investigates the use of PPS as a prognostication tool for estimating survival times of patients with life-limiting illness in a palliative care unit. These findings are compared to those from earlier studies in terms of PPS survival profiles and covariates. METHODS: This is a retrospective cohort study in which the admission PPS scores of 733 palliative care patients admitted between March 3, 2000 and August 9, 2002 were examined for survival patterns. Other predictors for survival included were age, gender, and diagnosis. RESULTS: Study findings revealed that admission PPS score was a strong predictor of survival in patients already identified as palliative, along with gender and age, but diagnosis was not significantly related to survival. We also found that scores of PPS 10% through PPS 50% led to distinct survival curves, and male patients had consistently lower survival rates than females regardless of PPS score. CONCLUSION: Our findings differ somewhat from earlier studies that suggested the presence of three distinct PPS survival profiles or bands, with diagnosis and noncancer as significant covariates. Such differences are likely attributed to the size and characteristics of the patient populations involved and further analysis with larger patient samples may help clarify PPS use in prognosis.

Beta-adrenoceptor activation stimulates, and phosphodiesterase inhibition potentiates, placental prorenin synthesis and release.

This study evaluated activation of beta-adrenoceptors and the cAMP pathway on prorenin secretion from human placental explants. For comparative purposes, hCG secretion was also measured. Treatment with selective beta-adrenergic agonists (beta 1-dobutamine and beta 2-terbutaline) produced dose-dependent increases in prorenin secretion, with dobutamine yielding a greater response (10- vs. 6-fold). In contrast, hCG secretion was stimulated only by terbutaline (5-fold). Prorenin and hCG secretory responses were inhibited by corresponding selective receptor antagonists (beta 1-metoprolol and beta 2-ICI 118,551). Selective phosphodiesterase inhibitors were used to evaluate the role of cAMP in mediating these responses. Marked potentiation of beta-adrenoceptor-dependent prorenin secretion was observed with the type III inhibitor, cilostamide (63-76%), and the type IV inhibitor, Ro-201724 (32-43%). Type I (8-methoxymethyl-3-isobutylmethylxanthine) and type V inhibitors (dipyridamole and M&B 22,948) showed no potentiation. These studies demonstrate that activation of both beta 1- and beta 2-receptors stimulates placental prorenin release. The potentiation of beta-adrenergically activated prorenin release by selective inhibitors of phosphodiesterase indicates a coupling of beta-adrenoceptor and adenylate cyclase. The contrast in secretion of prorenin and hCG by selective beta-adrenergic agonists suggests differences in cellular localization. The results indicate that clinically used adrenergic agonists can affect the placental renin-angiotensin system. The role of endogenous activators of beta-adrenoceptors in this system remains to be determined.

Human chorionic gonadotropin stimulates placental prorenin secretion: evidence for autocrine/paracrine regulation.

Functional regulation of the placental renin-angiotensin system remains incompletely defined. Evidence indicates that synthesis and secretion of prorenin in the placenta and gestational sac decrease with advancing gestational age. Possible explanations for this developmental effect include the regulatory influences by locally released hormones, such as CG. To address this question, the effect of CG on prorenin secretion was evaluated in a human placental explant model. In this study, prorenin concentrations were measured in the media and tissue of cultured explants from term placentas. In addition, the role of cAMP in mediating hormone-regulated prorenin secretion was evaluated. Media and tissue concentrations of prorenin increased (2- and 3-fold, respectively) in a concentration-dependent fashion after 48 h of incubation with CG (0.03-300 IU/ml). Selective inhibition of phosphodiesterases by Ro 20-1724 (type IV) and cilostamide (type III) resulted in a marked potentiation of CG-induced prorenin secretion. Media concentrations of cAMP were also elevated with CG treatment and correlated with prorenin values. Prorenin secretion induced by CG was markedly attenuated by the cAMP-dependent protein kinase inhibitor, H-89. These results indicate that placental prorenin secretion may be locally regulated by CG and mediated by cAMP signal transduction mechanisms.

The allogeneic response to cultured human skin equivalent in the hu-PBL-SCID mouse model of skin rejection.

BACKGROUND: Engineered tissues have been proposed for the treatment of a variety of conditions including the partial or complete replacement of human organs. To determine the basis for the rejection of these tissues, we analyzed the immune response to allogeneic human skin equivalent (HSE, also called Apligraf) in the humanized SCID mouse (hu-PBL-SCID). METHODS: Two models of hu-PBL-SCID were used for these studies. In one model, human skin or HSE was transplanted onto humanized mice so that graft survival could be analyzed. In the other model, skin grafts were allowed to heal on naive mice before humanization. This model was used to analyze the immunologic response to the vascularized skin allograft. Humanization was performed by adoptive transfer of human PBL into SCID mice by i.p. injection. RESULTS: Both human foreskin and HSE successfully engrafted onto naive SCID mice and remained stable for more than 6 months. In contrast, human foreskin was rejected by 21 days posttransplant in hu-PBL-SCID, whereas HSE consistently engrafted for more than 28 days. Treatment of HSE grafts with interferon-y for 5 days to induce maximal MHC class II molecule expression before grafting failed to induce rejection. HSE also engrafted onto hu-PBL-SCID mice that were exposed to alloantigen by prior injection with interferon-gamma-treated keratinocytes identical to those used to generate the HSE. In addition, we determined that humanization of SCID mice following engraftment and vascularization of human foreskin resulted in marked CD3+ T cell infiltrates and a lymphocyte-induced vasculitis. In contrast, the response in vascularized HSE was associated with minimal CD3+ T cell infiltration in the absence of vasculitis or morphological features of rejection. CONCLUSION: These results support the use of HSE and other allogeneic engineered tissues in humans provided that such tissues are limited in their antigen presenting capabilities. In addition, our findings suggest a critical function for the donor endothelial cell in rejection.

Evaluation of airway complications in high-risk preterm infants: application of flexible fiberoptic airway endoscopy.

OBJECTIVES: We sought to determine factors that would predict the development of subglottic stenosis (SGS) and tracheomalacia (TM) in preterm infants. The utility of a semiquantitative measurement of airway dimensions was assessed in relation to signs of airway complications. We also sought to determine from a high-risk population of infants those likely to have abnormal findings identified by bronchoscopic examination. METHODS: Prospective airway endoscopy was performed for preterm infants who were intubated for 7 days or more or who demonstrated chronic oxygen needs beyond 28 days after birth and 36 weeks postconceptional age. Subjects were 117 preterm (less than 36 weeks' gestation) infants from two level III intensive care nurseries. Endoscopy was used to classify the type and degree of airway injury. Subglottic stenosis was defined subjectively and compared with an objective measurement using subglottic spatial relations described as a trans-subglottic/vocal cord ratio (TSG/VC). Clinical signs and symptoms and other risk factors were evaluated as significant predictors of SGS and TM, identified by bronchoscopy. RESULTS: Moderate or severe airway abnormalities were identified in 32 patients (27.3%); 13 with SGS, 17 with TM, and 2 with both. All but one infant with TSG/VC less than 0.83 had signs and symptoms of airway dysfunction. Variables more commonly found in patients with SGS included greater number of intubations, use of inappropriately large endotracheal tubes, and longer duration of intubation. Higher averaged mean airway pressure during the first week after birth and lower gestational age were clinical features associated with TM. CONCLUSIONS: Flexible bronchoscopic evaluation of a high-risk population demonstrated a higher incidence of moderate or severe SGS or TM than previously suspected. Subglottic stenosis and TM appear to have different etiologies based on different factors associated with their development. The TSG/VC ratio correlated well with obstructive symptoms and may represent a means to quantitate clinically subglottic narrowing. Infants with chronic lung disease who have persistently elevated partial pressure of carbon dioxide, apnea, or phonation abnormalities are most likely to have airway abnormalities identifiable by bronchoscopy.

Stimulation of placental prorenin secretion by selective inhibition of cyclic nucleotide phosphodiesterases.

Prorenin secretion by human villous placenta is known to be stimulated by activation of adenylate cyclase and enhanced cyclic AMP (cAMP) generation. Placental tissue contains predominantly type III (cGMP-inhibited) and type IV (cAMP-specific) phosphodiesterases (PDEs), which inactivate cAMP. To evaluate the role of PDE subtypes in the regulation of prorenin secretion by human placenta, explants were cultured in the presence of isobutylmethylxanthine (IBMX), a non-selective PDE inhibitor, and selective inhibitors for various PDE subtypes. Inhibition of PDE subtypes with cilostamide (type III), Ro 20-1724 (type IV) and zardaverine (types III and IV) increased prorenin release. Inhibition of type I (Ca(2+)/calmodulin-dependent) PDE by 8-MeoM-IBMX and of type V (cGMP-specific) PDE by zaprinast or dipyridamole did not affect prorenin secretion. The stimulation of prorenin secretion by PDE inhibitors was attenuated by cAMP-dependent protein kinase inhibition. The selective PDE inhibitors caused a parallel increase in media cAMP and prorenin and also increased tissue prorenin levels. These studies demonstrate that cAMP degradation by type III and IV PDE isoenzymes is a major regulatory mechanism for placental prorenin secretion. It is suggested that enhancers of adenylate cyclase activity are constitutively present in placenta and influence prorenin synthesis and release.

Prorenin secretion from villous placenta: regulation by cyclic AMP and angiotensin.

Renin synthesis and secretion from human chorion and decidua have previously been shown to be stimulated by agents which increase cellular cyclic adenosine monophosphate (cAMP). We have now used organ culture of villous placenta, incubated for periods up to 72 h, to investigate the cellular regulation of renin in this tissue. The placental tissues release renin (92-96% in the form of prorenin) and human chorionic gonadotrophin (hCG), but not prolactin. We found that cholera toxin and forskolin markedly stimulate the synthesis and release of renin in a time-dependent manner. This stimulation was potentiated by phosphodiesterase inhibitors and inhibited by an angiotensin II agonist, sar-1-angiotensin II. The inhibitory action of the angiotensin agonist on renin release was blocked by sar-1-leu-8-angiotensin II, a selective angiotensin receptor antagonist. The potential for stimulation of renin expression by cyclic AMP-regulated elements is supported by the dramatic (two-orders of magnitude) increase in renin release observed with cholera and forskolin at 72 h. There are several possible candidates for primary signals for adenylyl cyclase-coupled renin secretion from the placenta, including relaxin and epinephrine. The extremely low concentration of renin in term villous placenta may be related to activation of negative regulatory elements on the renin gene. We propose that angiotensin II is one negative regulator of this system.

Tracheal agenesis with diaphragmatic hernia.

A case is presented of a newborn infant with Type III tracheal agenesis and a right diaphragmatic hernia which represents the first report of this occurrence. Associated multi-organ anomalies and possible embryologic relationships are discussed.

In vivo correlation of Doppler waveform analysis with arterial input impedance parameters.

Previous studies have confirmed that Doppler waveform analysis (DWA) offers a valid reflection of changes in peripheral vascular resistance. However, the ability of the pulsatility index (PI), a parameter of DWA, to reflect the dynamic components of the circulation, as assessed by arterial input parameters, remains uncertain. In addition, the state of the central circulation is considered an important factor influencing the accuracy of this technique. This study evaluated the ability of the aortic PI to reflect alterations of input impedance in a chronically instrumented lamb model that was subjected to pharmacologic alteration of the circulation. Pressure, volumetric flow and continuous-wave Doppler frequency shift measurements were recorded from the infrarenal abdominal aorta. The parameters of input impedance, peripheral vascular resistance (Zpr), characteristic impedance (Zo) and reflection coefficient (Rc), were determined and then correlated with changes in the aortic PI. Initially, perturbations of the circulatory state were created with a vasodilator, hydralazine (HY) and a vasoconstrictor, phenylephrine (PE). During a second set of experiments, the effect of the reflex heart rate (HR) responses on the PI was evaluated. This was accomplished by inhibiting reflex HR responses to these vasoactive agents with either trimethophan (TM) or atropine methyl bromide (AMB). In response to HY and HY with TM, significant decreases in the PI and impedance parameters occurred. Administration of PE and PE with AMB resulted in significant increases in PI and each of the impedance parameters. HY and PE induced changes in PI correlated significantly with changes in volumetric flow (r = 0.82, 0.80; p < 0.001), mean arterial blood pressure (r = 0.64, 0.70; p < 0.001) and Zpr (r = 0.77, 0.80; p < 0.001), but not with Zo (r = 0.34, 0.36) and Rc (r = 0.26, 0.31). However, when reflex HR responses were inhibited during the administration of the vasoactive agents, HY with TM and PE with AMB, induced changes in PI correlated significantly with Zo (r = 0.93, 0.89; p < 0.001) and Rc (r = 0.84, 0.83; p < 0.001), and the correlation with mean arterial pressure (r = 0.78, 0.87; p < 0.05) and Zpr (r = 0.92, 0.91; p < 0.05) was significantly greater. These findings indicate that the PI accurately assesses pharmacologically induced changes in the downstream arterial input impedance. The accuracy of this assessment is enhanced further when central circulatory factors such as changes in HR are considered.

Effects of acute hypoxemia on umbilical arterial Doppler indices in a fetal ovine model.

Changes in umbilical arterial Doppler waveform in response to acute maternal hypoxemia were assessed in a chronic ovine model. Maternal heart rate, blood gases, fetal heart rate and umbilical arterial Doppler indices measured at baseline conditions and during periods of maternal hypoxemia were compared. The indices measured were systolic-diastolic ratio (S/D), pulsatility index (PI) and resistance index (RI). Fetal heart rate decreased (less than 80 beats/min) and umbilical arterial Doppler indices increased (P less than 0.001) during the periods of hypoxemia. Furthermore, the Doppler indices were highly but negatively correlated with alterations in the fetal heart rate (FHR) (P less than 0.001). Analysis of the Doppler waveform phase intervals revealed nearly constant systolic intervals while diastolic intervals varied inversely with the heart rate alterations. Moreover, the umbilical arterial Doppler indices, when corrected for FHR changes, were relatively unchanged from baseline measurements. This study indicates that maternal hypoxemia induced changes in the umbilical arterial Doppler indices may be primarily attributable to the changes in FHR.

An interesting case presentation: pulmonary malformations associated with oculoauriculovertebral dysplasia (Goldenhar anomalad).

Oculoauriculovertebral dysplasia (OAV) is a heterogeneous field defect involving the first and second branchial arches and is characterized by microtia, mandibular hypoplasia, vertebral anomalies, and epibulbar dermoids. We report a case of OAV with pulmonary manifestations and review the literature regarding this association. Anomalies identified were previously undescribed tracheal stenosis, along with tracheoesophageal cleft and unilateral pulmonary agenesis. Recognition of the pulmonary malformations associated with OAV may lead clinicians to consider a diagnostic measure such as flexible fiberoptic endoscopy in the evaluation of infants with craniofacial malformations and respiratory distress.

Doppler velocimetry in obstetrics.

Doppler ultrasound velocimetry offers a noninvasive method for assessing velocities in the various components of the fetal circulation including the cardiac, umbilical, cerebral, and aortic flows. As the volumetric flow measurement in these circumstances is prone to significant errors, the major approach has been to analyze the maximum frequency shift envelope of the Doppler waveform. Numerous studies have demonstrated not only the feasibility, but also the diagnostic efficacy of this approach. The latter is particularly true in relation to the Doppler evaluation of the umbilical circulation in various complications of pregnancy. Although these findings do not establish the role of Doppler technology as a standard of practice, they clearly demonstrate its immense potential as a fetal surveillance technique.

Antepartum fetomaternal hemorrhage. Report of a case with the use of cordocentesis in diagnosis and management.

Clinical recognition of antepartum fetomaternal hemorrhage (FMH) is most often achieved by the observation of characteristic fetal heart rate (FHR) patterns and positive Kleihauer-Betke acid elution staining. Both methods are noted to lack sensitivity and specificity. A case of suspected antepartum FMH occurred with intermittent sinusoidal FHR tracings. Fetal blood sampling by cordocentesis in situations with equivocal antenatal testing, such as in this case, allows not only confirmation of fetal anemia but assessment of fetal acid-base status. In pregnancies of less than 32 weeks' gestation complicated by severe antepartum FMH, intravascular transfusion may be offered via this technique. Cordocentesis is beneficial in the management of pregnancies with uncertain FHR patterns when antepartum FMH is suspected.

Nonimmune hydrops fetalis caused by a massive fetomaternal hemorrhage associated with elevated maternal serum alpha-fetoprotein levels. A case report.

Chronic fetomaternal hemorrhage led to the development of fetal anemia and nonimmune hydrops fetalis in a term neonate. Antenatal maternal serum alpha-fetoprotein levels were abnormally elevated, with normal amniotic fluid levels. Kleihauer-Betke staining was performed as part of the evaluation. Serial ultrasound examinations can be useful for unexplained elevations in maternal serum alpha-fetoprotein because of the associated increased fetal loss in those pregnancies. If fetomaternal hemorrhage is identified, serial ultrasound examinations are indicated for the detection of fetal hydrops.