Descriptors and perception of dyspnea in African-American asthmatics. This study explores self-reported perception of asthma symptoms in African-Americans. Qualitative methodology was used to analyze the responses from African-Americans within focus groups from Nashville, Tennessee. Common symptoms were chest tightness, "breathing problems," and wheeze. Less commonly reported symptoms included cough, chest pain, dizziness, sweating, and "short of breath." A single participant reported nocturnal wheezing. This study provides insight into the descriptors and perception of asthma symptoms in African-Americans. Understanding the descriptors of symptoms and disease severity in African-American patients may lead to more accurate diagnosis, treatment, and reduced mortality within this high-risk population.


Influenza vaccination in adults with asthma: safety of an inactivated trivalent influenza vaccine. Despite recommendations in most countries for giving inactivated influenza vaccine to people with asthma, only a minority currently receive it. One reason for low vaccine coverage has been concern that vaccination may induce exacerbations of asthma. In this randomized trial, 291 patients between 18 and 65 years of age received either an inactivated influenza vaccine followed 14 days later by a saline placebo or placebo followed by the vaccine. Each patient received 1 dose of vaccine and 1 dose of placebo. The percentage of patients reporting at least one asthma exacerbation within 14 days after injection was similar following vaccine or placebo (28.3% and 25.5%, respectively). The combined exacerbation rate during the first 14-day interval was higher (31.5%) than that during the second 14-day interval (22.4%, P = 0.0135), indicating that the occurrence of exacerbations was not likely to be related to the sequence of injections. The percentages of individuals with solicited systemic symptoms were 56.6% and 44.8% after vaccine or placebo injection, respectively. We conclude that influenza vaccination did not increase the incidence of asthma exacerbations compared to placebo in this study and the vaccine was well tolerated. The results thus support annual influenza vaccination in patients with asthma.


Hoarseness in asthmatic patients: the side effect of inhaled steroid or not? Hoarseness is one of the possible side effects of inhaled steroid in asthmatic patients as well as a common presenting feature of laryngeal tuberculosis. We report an asthmatic patient on inhaled corticosteroid treatment who presented with a 3-month history of hoarseness. Laryngeal tuberculosis without pulmonary involvement was diagnosed by laryngeal biopsy and microbiologic studies. Laryngeal tuberculosis should be considered in asthmatic patients who present with hoarseness, at least in regions in which tuberculosis is prevalent.


Disparities in asthma hospitalizations among children seen in the emergency department. We examined the race/ethnicity variation in the risk of hospitalization among children seen in the emergency department (ED) for asthma. ED and hospitalization records for children 1 to 19 years of age in New Jersey for 2004 and 2005 were linked. The dataset identified 47,548 ED and hospitalizations among 37,216 children. ED and hospitalization rates indicated persistent disparities in pediatric asthma. ED admission rates were similar across race/ethnic groups, suggesting similar management of pediatric asthma patients once they are seen in the ED. Integrating existing ED and hospitalization records will enhance asthma surveillance and the targeting of interventions to reduce race/ethnicity disparities.


The potential biases in studying the relationship between asthma and microbial infection. To assess bias in parent reports of asthma status of children and detection bias of medical records-based asthma ascertainment and to examine effects of such bias on the association between asthma status and infections. A prospective cohort study was conducted to compare the correlations between the frequency of acute illnesses and that of medical evaluations between children with or without asthma according to parental report and medical record review by following a group of children who were enrolled in the Mayo Clinic Sick Child Care Program in Rochester, Minnesota. Parents completed a self-administered questionnaire to determine asthma status of their child. Also, comprehensive medical record reviews were conducted to determine asthma status of each subject by applying predetermined criteria for asthma. A convenience sample of 115 parents and their children participated in this study. The mean age of the parents who participated in the study was 32.8 years (standard deviation: 5.4 years); 93% were female (mothers); and 90% were white. Of the 115 children who participated in this study, 84% were reported to be white and 49% were female. The mean age of the children was 2 years (standard deviation: 1.0 year). Parents whose children had asthma by report appeared to be less likely to seek medical evaluations (Spearman's rho: 0.42,p = 0.11) when their children had acute illnesses, compared to those of non-asthmatic children (rho: 0.64,p < 0.001). Concerns that asthmatic patients (rho: 0.62,p < 0.001) are more likely to see health care providers and undergo medical evaluations and laboratory tests when they have acute illnesses than non-asthmatic patients (rho: 0.64,p < 0.001) are not supported by our study. Parental report bias needs to be considered carefully when studying the relationship between asthma and microbial infection.


Air quality and pediatric asthma-related emergencies. Previous studies suggest a relationship between air pollutants, aeroallergens, and asthma exacerbations. To simultaneously examine the role of seasonality, air quality, aeroallergens, and climate on asthma-related pediatric emergency department (ED) visits. A retrospective 4-year study of asthma-related ED visits was conducted. September had the highest number of visits (p < 0.01). There were lower temperatures and precipitation (p < 0.01) and higher tree and weed pollen levels (p = 0.05) on days with more visits (p = 0.05), while grass pollen, mold, ozone, NO2, and PM2.5 levels showed no significant differences. Asthma-related visits were associated with aeroallergens and climatic factors and not air-quality factors.


Influence of house dust mite impermeable covers on health-related quality of life of adult patients with asthma: results of a randomized clinical trial. The aim of this study was to evaluate the effect of house dust mite impermeable covers on asthma-specific health-related quality of life in adult asthmatic patients that were trained in guided self-management. In a 2-year randomized placebo-controlled clinical trial, information on the quality of life was collected. The improvement of Mini Asthma Quality of Life Questionnaire (AQLQ) score in the allergens-avoidance group (0.26) was comparable to the improvement in the placebo group (0.30) and not significant. HDM-impermeable covers for pillows, duvets, and mattresses did not result in improved health-related quality of life.


Risk factors for pediatric asthma in the South Bronx. We identified main asthma risk factors for children living in the South Bronx, where asthma rates are eight times higher than the national average. This case-control study enrolled 261 children at Lincoln Medical and Mental Health Center from 2002 to 2003. We questioned the mothers on medical history and home environment. The most important risk factors for asthma in the South Bronx pediatric population are Hispanic ethnicity, family history of asthma, and exposure to tobacco smoke. South Bronx children limited to breast-feeding during the first 3 months of age are less likely to develop asthma.


The relationship between age of asthma onset and cardiovascular disease in Canadians. To quantify the association between cardiovascular disease (CVD) and asthma in Canadian adults and to determine whether age of asthma onset is a moderator of this association. We used a sample of 74 342 participants with a mean age of 56.4 +/- 12.5 from cycle 1.1 of the Canadian Community Health Survey. Asthma age of onset was categorized into early-onset (0-20 years) and adult-onset (21-54 years). Three major outcomes were used to estimate the relationship between asthma and CVD, namely: high blood pressure, heart disease, and stroke. Multiple logistic regression models revealed that asthmatics were 43% (OR = 1.43, CI = 1.19-1.72) more likely to have heart disease, and 36% (OR = 1.36, CI = 1.21-1.53) more likely to have high blood pressure than non-asthmatics. There were no consistent results for age of onset with high blood pressure, heart disease, or stroke. Using a population-based dataset we confirmed that asthmatics are at increased odds of cardiovascular disease compared to non-asthmatics; furthermore, age of asthma onset did not appear to moderate this relationship. Future research should focus on determining whether asthma severity or allergic/non-allergic phenotypes have a differential effect on the asthma-CVD relationship.


Nepsilon-(carboxymethyl)lysine, a major advanced glycation end product in exhaled breath condensate as a biomarker of small airway involvement in asthma. N(epsilon) -(carboxymethyl)lysine (CML) expression is selectively present in the lower respiratory tract. We compared CML levels in exhaled breath condensate (EBC) between 19 asthmatics and 10 normal control subjects and its levels before and after tiotropium therapy in 11 non-smoking asthmatics and 10 smoking asthmatics. CML levels were significantly lower in asthmatics than in normal control subjects. Moreover, low CML level was associated with small airway dysfunction. After tiotropium therapy, CML level in non-smoking asthmatics was unchanged, while that in smoking asthmatics was significantly increased. Therefore, CML level in EBC is a non-invasive biomarker for evaluating small airway involvements in asthma.


Validation of the Spanish version of the Asthma Control Test (ACT). Validation of the Spanish version of the Asthma Control Test (ACT). A total of 607 asthmatic patients were assessed. The psychometric properties of ACT were evaluated. The ACT capacity to predict the physician's assessment of asthma control was assessed using the area under the receiving operating characteristics (ROC) curve (AUC), sensitivity, specificity, and positive-negative predictive values. ACT's Cronbach alpha was 0.84. The intraclass correlation coefficient was 0.85. The AUC was 0.86, with a sensitivity of 71% and a specificity of 85% for a score of < or =19. The Spanish version of ACT is shown to be a reliable and valid tool for evaluating and discriminating asthma control.


An association between asthma and BMI in adolescents: results from the California Healthy Kids Survey. We examined the relationship between asthma prevalence and BMI in a cross-sectional survey of 471,969 adolescents. The size of the survey allowed us to investigate this relationship with much greater resolution than previously possible. Both lifetime and current asthma prevalence increased monotonically with increasing BMI, starting with individuals as low as the 45th to 55th percentiles of BMI. The pattern was similar between males and females and among six racial/ethnic groups. The results suggest that weight reduction even among persons not classified as overweight or obese may be an important component of asthma management.


The relationship of physical activity and percentage of body fat to the risk of asthma in 8- to 10-year-old children. To evaluate physical activity, obesity and asthma, we analyzed information from children attending a racially diverse middle-class suburban school district. Physical activity in metabolic equivalents (METS) and percent body fat were related to diagnosed asthma. On average the, 636 children were 8.9 years of age, 64.0% black, and 11.8% with reported asthma. Children with asthma were more active: 6,438 versus 5,432 METs/year, p = 0.03. Logistic regression considering METs, percent fat, gender and race showed METs were a significant risk factor for asthma, odds ratio (OR) = 1.24 (95% CI 1.01-1.52, p = 0.045). Higher levels of physical activity were related to more diagnosed asthma.


Does spatial accessibility of pharmacy services predict compliance with long-term control medications? Disadvantaged urban children with asthma are at high risk for undermanagement of their disease and poor compliance with inhaled corticosteroids (ICS). To determine whether spatial accessibility (SA) of retail pharmacy services is associated with ICS compliance. Caregivers of 137 urban high-morbidity asthmatic children attended a comprehensive intervention to improve asthma care and outcomes. Subsequent ICS compliance was determined at 1, 3, and 6 months. We modeled 18 measures of SA to 339 pharmacies using logistic regression and adjusting for numerous covariates. No relationships between SA and compliance were found. In this group of disadvantaged urban children with severe asthma, compliance with ICS was not significantly associated with SA of retail pharmacy services.


Case report: allergic bronchopulmonary aspergillosis and allergic fungal sinusitis successfully treated with voriconazole. Allergic bronchopulmonary aspergillosis and allergic fungal sinusitis are closely related disorders that rarely present in the same individual. The mainstay of treatment for allergic bronchopulmonary aspergillosis is systemic corticosteroids. Itraconazole is used as adjunctive therapy in refractory cases. Allergic fungal sinusitis requires initial sinus surgery followed by systemic steroids. Antifungal therapy has not proven to be beneficial in allergic fungal sinusitis. We report a case of concomitant allergic bronchopulmonary aspergillosis and allergic fungal sinusitis that was refractory to standard therapy but had dramatic clinical response following treatment with voriconazole.


Richard Selzer: premiere American surgeon-writer. Richard Selzer (b.1928) is one of the most prominent American surgeon-authors alive today. Only Sherwin Nuland (b.1930), from Yale University Medical School as Selzer was, carries the same outstanding virtues of good surgery and excellent writing. Recently, Atul Gawande (b. 1965) has taken a road similar to these two distinguished predecessors. Selzer has produced 14 books of superb scholarship and immense value for the active and committed surgeon. He describes, with unusual grace and wit, different aspects of great interest to the surgeon's life. He includes details of instruments, particularly the surgical knife, approaching this essential and many times lifesaving instrument with unparalleled courage and incredible precision. He also advances with savvy understanding and well-defined knowledge the great character of various organs of the human body. Richard Selzer offers a special perspective on being a surgeon and a writer. He has been on both sides of these professions. He practiced general surgery for many years and has been a writer for three decades at least. Dr. Selzer demonstrates writing skills and surgical knowledge that can be extremely helpful to the student and surgeon alike.


William and Charles Mayo: their influence on American medicine. In a little-known Midwest town named Rochester, Minnesota, a talented physician grew in fame and respect: Dr. William Worrall Mayo, who was influential in the evolution of medicine. He was a steadfast learner and raised two sons, William and Charles, to follow in his footsteps and further medical knowledge. They were leaders in surgery and in the creation of advanced and sophisticated medical facilities. Their talents, the issues surrounding medical practice, and unexpected opportunity all came into play for the Mayos. Two hospitals, St. Mary's Hospital and later the Methodist Hospital, witnessed and influenced the advancement of medicine through the Mayo Clinic heritage and dynasty in Minnesota and the rest of the world. In this article, we focus on the role of the Mayo brothers and their influence over the increasing acceptance of hospital care in America and abroad.


Toll-like receptor pathway gene expression is associated with human immunodeficiency virus-associated neurodegeneration. The innate immune system is a significant component of the brain's defense against infection, especially as the blood-brain barrier restricts access of the members of the adaptive immune system, such as T and B cells. The innate immune system includes Toll-like receptors (TLRs) that recognize pathogen-associated molecular patterns. Within the central nervous system, they are expressed on glial cells and their expression can be modulated by pathological states. Although their function is to recognize foreign pathogens and stimulate a protective immune response through the production of cytokines and interferons, there is emerging evidence that activation of these receptors can result in neurodegeneration. In the current study, the authors assessed the expression of TLR-related genes, using a customized Superarray gene chip, and correlated the expression findings with indices of neurodegeneration. We found that, using a stringent threshold for statistical significance to overcome the potential problem of multiple statistical testing, there were significant correlations between the expression of nine TLR-related genes and reduction in dendritic and synaptic staining. Two of these genes, TLR4 and SIGIRR, were validated by quantitative real-time polymerase chain reaction. Additionally, the authors demonstrated in vitro at the protein level that human primary astrocytes exposed to the toxic human immunodeficiency virus (HIV) envelope protein gp120 had a significant increase in TLR4 protein expression. In conclusion, these findings indicate that TLR-related gene expression may contribute to the development of HIV-related neurodegeneration.


Pneumocyte apoptosis induction during cardiopulmonary bypass: effective prevention by radical scavenging using N-acetylcysteine. Cardiopulmonary bypass (CPB) and cardioplegic arrest are associated with pulmonary dysfunction. We sought to investigate whether pulmonary ischemia/reperfusion during standard CPB and cardioplegic arrest is associated with reactive oxygen species (ROS)-mediated pulmonary tissue injury and pneumocyte apoptosis induction, and whether ROS scavenging using N-acetylcysteine (NAC) attenuates these alterations. Twelve pigs (41 +/- 8 kg) were randomized to receive either NAC (100 mg/kg prior to CPB; n = 7) or placebo (n = 5) and subjected to CPB and 60 min of cold (4 degrees C) crystalloid cardioplegic arrest. We collected lung biopsies prior to CPB, at 60 min CPB, as well as at 30, 60, and 120 min post CPB. Lung specimens were immunocytochemically stained against nitrotyrosine, 8-isoprostaglandin-F(2)alpha, and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) as indicators for ROS-mediated tissue injury and active caspase-3, an apoptosis signal pathway key enzyme. Oxidative stress markers were judged using a scale from 1 to 4 (low to intensive staining), and caspase-3-positive pneumocytes were counted per view field. In placebo, the number of caspase-3-positive pneumocytes significantly increased over time to reach a maximum at 120 min post CPB (p = .03 vs baseline). NAC significantly prevented caspase-3 activation in pneumocytes (p = .001 vs Placebo). Pneumocyte nitrotyrosine and 8-OH-dG staining significantly increased over time (p = .003) in the placebo group, but decreased in the NAC group (p = .004). In both groups staining for 8-isoprostaglandin-F(2)alpha showed no significant changes. This yields the conclusion that standard CPB and cardioplegic arrest initiate ROS-mediated tissue injury and apoptosis in pneumocytes that can be reduced by NAC. Thus, ROS scavenging using NAC may represent a novel approach to minimize lung injury associated with CPB.


A single nucleotide change in the mumps virus F gene affects virus fusogenicity in vitro and virulence in vivo. Mumps virus is highly neurotropic, with evidence of infection of the central nervous system in more than half of clinical cases. In the prevaccine era, mumps was a major cause of viral meningitis in most developed countries. Despite efforts to attenuate the virus, some mumps vaccines have retained virulence properties and have caused aseptic meningitis in vaccinees, resulting in public resistance to vaccination in some countries. Ensuring the safety of mumps vaccines is an important public health objective, as the need for robust immunization programs has been made clear by the recent resurgence of mumps outbreaks worldwide, including the United States, which in 2006 experienced its largest mumps outbreak in 20 years. To better understand the molecular basis of mumps virus attenuation, the authors developed two infectious full-length cDNA clones for a highly neurovirulent strain of mumps virus. The clones differed at only one site, possessing either an A or G at nucleotide position 271 in the F gene, to represent the heterogeneity identified in the original virulent clinical isolate. In comparison to the clinical isolate, virus rescued from the A-variant cDNA clone grew to higher cumulative titers in vitro but exhibited similar cytopathic effects in vitro and virulence in vivo. In contrast, virus rescued from the G-variant cDNA clone, in comparison to the clinical isolate and the A-variant, was more fusogenic in vitro but replicated to lower cumulative titers and was less neurovirulent in vivo. These data suggest that nucleotide position 271 in the F gene plays a significant role in virus pathogenesis. This infectious clone system will serve as a key tool for further examination of the molecular basis for mumps virus neurovirulence and neuroattenuation.


Cocaine-mediated enhancement of virus replication in macrophages: implications for human immunodeficiency virus-associated dementia. Injection drug use has been recognized as a major risk factor for acquired immunodeficiency syndrome (AIDS) from the outset of the epidemic. Cocaine, one of the most widely abused drugs in the United States, can both impair the functions of macrophages and CD4(+) lymphocytes and also activate human immunodeficiency virus (HIV)-1 expression in these cells. Because the brain is the target organ for both cocaine and HIV, the objective of the present study was to explore the effects of cocaine on virus replication in macrophages, the target cells for the virus in the central nervous system (CNS). Cocaine markedly enhanced virus production in simian human immunodeficiency virus (SHIV)-infected monocyte-derived macrophages (MDMs) and in U1 cells, a chronically infected promonocytic cell line as monitored by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry. Cocaine treatment also resulted in the activation of nuclear factor (NF)-kappa B and transcriptional activation of the HIV-LTR (long terminal repeat) gag-GFP (green fluorescent protein). Analyses of chemokines in cocaine-treated macrophages by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Luminex assays suggested increased expression of interleukin (IL)-10, a cytokine that is known to promote HIV replication in MDMs. In addition to enhancing IL-10 expression, cocaine also caused an up-regulation of the macrophage activation marker, human leukocyte antigen (HLA)-DR, in MDMs. The synergistic effect of cocaine on virus replication and its enhancement of host activation markers suggest that cocaine functions at multiple pathways to accelerate HIV-associated dementia (HAD).


Wound healing modulators in a tracheoplasty canine model. Postsurgical tracheal stenosis results from fibrosis formation due to ischemia. There are healing modulators, hyaluronic acid (HA) and collagen polyvinylpyrrolidone (CPVP), which reduce collagen fibers formation. Thus we can hypothesize that the topical application of one of these modulators can diminish postsurgical tracheal scarring and stenosis. The aim of this work was to evaluate the macroscopic, microscopic, and biochemical changes of tracheal healing after the application of HA or CPVP in a canine tracheoplasty model. The study design was prospective experimental investigation in a canine model. Eighteen mongrel dogs underwent three cervical tracheal rings resection and end-to-end anastomosis. They were randomized into three groups according to treatment: group I (control group) (n = 6), topical application of saline solution on tracheal anastomosis; group II (n = 6), topical application of 15 microg HA on tracheal anastomosis; and group III (n = 6), topical application of 2.5 mg CPVP on tracheal anastomosis. They were evaluated clinical, radiological and tracheoscopically during 4 weeks. They were euthanized at the end of the study time. Macroscopic, microscopic, and biochemical changes of tracheal anastomosis healing were analyzed. Collagen formation was quantified by the Woessner method. All the animals survived the surgical procedure and study period. Macroscopic, radiologic, and endoscopic studies showed that animals in group I developed tracheal stenosis, inflammation, and firm fibrous tissue formation, and histological studies also showed severe inflammatory reaction and fibrosis formation. Groups II (HA) and III (CPVP) showed well-organized thin collagen fibers with minimal inflammatory response. Biochemical evaluation revealed a higher collagen concentration in group I animals (analysis of variance [ANOVA] p < .05 and Tukey p < .01). Thus, hyaluronic acid or collagen polyvinylpyrrolidone administered after tracheal anastomosis diminished the degree of stenosis and inflammatory reaction. Both modulators improved tracheal healing.


Engineering the Japanese encephalitis virus RNA genome for the expression of foreign genes of various sizes: implications for packaging capacity and RNA replication efficiency. Using the RNA replication machinery of Japanese encephalitis virus (JEV), the authors have established and characterized three strategies for the expression of foreign genes. Initially, approximately 11 kb genomic RNA was engineered to express heterologous genes of various sizes by preferentially inserting a new cistron at the beginning of the 3' nontranslated variable region. RNA transfection yielded recombinant viruses that initiated foreign gene expression after infecting permissive cells. JEV was capable of packaging recombinant genomes as large as approximately 15 kb. However, larger genome size was inversely correlated with RNA replication efficiency and cytopathogenicity, with no significant change in infectivity. Second, a variety of self-replicating propagation-deficient viral replicons were constructed by introducing one to three in-frame deletions into the ectodomains of all the structural proteins of JEV. These replicons displayed a spectrum of RNA replication efficiency upon transfection, suggesting that remnant transmembrane domains play a suppressive role in this process. Third, the authors generated a panel of stable packaging cell lines (PCLs) providing all three JEV structural proteins in trans. These PCLs efficiently packaged viral replicon RNAs into single-round infectious viral replicon particles. These JEV-based virus/vector systems may provide useful tools for a variety of biological applications, including foreign gene expression, antiviral compound screening, and genetic immunization.


Interferon beta treatment: bioavailability and antiviral activity in multiple sclerosis patients. Viral infections have been appointed as the main component of environmental susceptibility to multiple sclerosis (MS). Interferon beta is an immunomudulatory treatment that is able to modify the natural course of the disease; nonetheless, its mechanism of action in not well established yet. The objectives of the present study were (1) to evaluate the bioavailability of interferon beta through the measurement of the expression of myxovirus resistance protein (MxA), metalloproteinase 9 (MMP-9), and its inhibitor (TIMP-1); (2) to analyze its antiviral efficiency through the measurement of human herpesvirus-6 (HHV-6) prevalence; and (3) to correlate both parameters (bioavailability and antiviral efficiency) with the relapse rate in multiple sclerosis (MS) patients treated with interferon beta. Pairs of blood and serum samples were collected from 54 MS patients during five visits in 1 year: one before the start of the treatment and four during interferon beta treatment. Expression of MxA, MMP-9, and TIMP-1 was analyzed by quatitative real-time polymerase chain reaction (qRT-PCR) and HHV-6 genomes were detected by qPCR. The results showed a correlation between MxA and relapse rate (P = .014). MMP-9/TIMP-1 ratio was increased among the patients with relapses, and decreased among the relapse-free patients, although differences were not statistically significant. Furthermore, our results suggest a possible role for HHV-6 in MS, because 42.8% of patients with viral reactivations experienced at least one relapse versus 22.5% of patients without viral reactivations. Lastly, regarding the antiviral effectiveness of the interferon beta, the HHV-6 prevalence decreased from 58% to 36% in PBMCs and from 18.5% to 12.2% in sera; furthermore, a good correlation with the bioavailability of interferon beta was found, because patients with a decrease in HHV-6 prevalence had higher levels of MxA (P = .046, at the third month).


Asialoerythropoietin is a strong modulator of angiogenesis by bone-marrow cells. The angiogenetic effect of asialoerythropoietin (asialoEPO) was investigated in a murine (BALB/c) ischemic hind limb model created by ligating the right femoral artery, and severe ischemia was created by ligating two points on the femoral artery. The mice were allocated to six groups: 1, Control (n = 15), no treatment; 2, BMC (n = 15), Injected with 1 x 10(6) bone marrow cells (BMC); 3, EPO (n = 15), EPO(500 U/kg for 2 weeks); 4, BMC + EPO (n = 15), BMC + erythropoietin (EPO); 5, asialoEPO (n = 15), asialoEPO (500 U/kg for 2 weeks); 6, BMC + asialoEPO (n = 15); BMC + asialoEPO. Blood flow in the ischemic and normal limbs was measured using a Doppler flowmeter on days 7 and 14. Vessel density was evaluated by immunohistochemical staining for Von Willebrand Factor (vWF). In the severe ischemia model, limb survival was investigated. Blood flow was significantly higher in the BMC + asialoEPO group than in the Control, BMC, EPO, BMC + EPO, or asialoEPO group on day 7 (p = .007) and on day 14 (p = .002). Vascular density was also significantly higher in the BMC + asialoEPO (0.067 +/- 0.022) group than in the Control (0.026 +/- 0.007), BMC (0.027 +/- 0.012) EPO (0.029 +/- 0.002), BMC + EPO (0.048 +/- 0.015), and asialoEPO (0.031 +/- 0.001) groups (p = .006). Finally, limb survival at day 14 in the severe ischemia model was significantly better in the BMC + asialoEPO group (83.3%) than in the Control (40.0%), BMC (52.9%), EPO (44.4%), BMC + EPO (64.7%), or asialoEPO (36.4%) group (p = .02). This provides the conclusions that asialoEPO promotes angiogenesis by BMC and that its action is significantly more potent than that of EPO.


Sealing of gastrointestinal anastomoses with a fibrin glue-coated collagen patch: a safety study. Sealing of anastomoses has previously been tested with several methods, including sealing with liquid fibrin glue. Sealing with a collagen patch coated with fibrin glue components has never been systematically examined. The aim of the present study was to determine the safety of sealing gastrointestinal anastomoses with a collagen patch coated with fibrin glue. The study is a prospective, experimental animal study comparing sealed and unsealed gastrointestinal anastomoses. Laparotomy was performed in 11 pigs under general anesthesia. In each pig two anastomoses were performed on the small intestine. One of the anastomoses was sealed with a collagen patch coated with fibrin glue components (TachoSil). The other anastomosis contained no sealing. The pigs were observed for 1 to 6 weeks. The observation period was followed by in vivo examination under general anesthesia and included observation for anastomotic leakage, signs of present or former peritonitis, abscess, adhesions to the anastomoses, and signs of intestinal obstruction. In addition, the anastomotic diameter was measured with barium and radiography. Finally, bursting pressure was measured in each segment. After the pigs were sacrificed, the bowel segments were microscopically examined. There were no differences between the sealed and the unsealed anastomoses with respect to abdominal pathology, in vivo bursting pressure, or degree of stenosis. The collagen fleeces were in situ in all anastomoses. Microscopically, we found no difference in healing or signs of infection.


Identification of host range mutants of myxoma virus with altered oncolytic potential in human glioma cells. The authors have recently demonstrated that wild-type myxoma virus (MV) tagged with gfp (vMyxgfp) can generate a tumor-specific infection that productively infects and clears human tumor-derived xenografts when injected intratumorally into human gliomas transplanted into immunodeficient mice (Lun et al, 2005). To expand the understanding of MV tropism in cancer cells from a specific tissue lineage, the authors have screened a series of human glioma cells (U87, U118, U251, U343, U373) for myxoma virus replication and oncolysis. To assess the viral tropism determinants for these infections, the authors have screened myxoma virus knockout constructs that have been deleted for specific host range genes (M-T2, M-T4, M-T5, M11L, and M063), as well as a control MV gene knockout construct with no known host range function (vMyx135KO) but is highly attenuated in rabbits. The authors report wide variation in the ability of various vMyx-hrKOs to replicate and spread in the human glioma cells as measured by early and late viral gene expression. This differential ability to support vMyx-hrKO productive viral replication is consistent with levels of endogenous activated Akt in the various gliomas. The authors have identified one vMyx-hrKO virus (vMyx63KO) and one nonhost range knockout construct (vMyx135KO) that appear to replicate in the gliomas even more efficiently than the wild-type virus and that reduce the viability of the infected gliomas. These knockout viruses also inhibit the proliferation of gliomas in a manner similar to the wild-type virus. Together these data, as well as the fact that these knockout viruses are attenuated in their natural hosts, may represent environmentally safer candidate oncolytic viruses for usage in human trials.


Synergistic effects in apoptosis induction by taurolidine and TRAIL in HCT-15 colon carcinoma cells. Induction of apoptosis in tumor cells by TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) is a promising therapeutic principle in oncology, although toxicity and resistance against TRAIL are limiting factors. Taurolidine (TRD), an antineoplastic agent with low toxicity, is a potential candidate for combined therapy with TRAIL. The aim of this study was to evaluate the apoptotic effects of a combined treatment with TRD and TRAIL in a human HCT-15 colon carcinoma cell line. HCT-15 cells were incubated with increasing concentrations of recombinant human TRAIL (50 ng/mL to 500 ng/mL) or TRD (50 micromol/L to 1000 micromol/L). In a second experiment, cells were furthermore exposed to a combination of both substances (TRAIL 50 ng/mL and TRD 100 micromol/L). At various time points (3 h to 36 h), cell viability, apoptosis, and necrosis were quantified by FACS analysis (propidium iodide/annexin V-FITC) and confirmed by TUNEL assay. Incubation with TRD resulted in cell death induction with maximum effects observed at 100 micromol/L and 1000 micromol/L after 36 h. TRAIL application led to dose-dependent cell death induction as early as 6 h. Combined treatment of TRD (100 micromol/L) and TRAIL (50 ng/mL) caused a sustained induction of apoptosis that was superior to single-agent application, exceeding a merely additive effect. Combinatory treatment of human colon carcinoma cells with TRD and TRAIL results in a synergistic effect on apoptosis induction with a significant increase of the apoptotic index. Combination of TRAIL with the nontoxic TRD might represent a novel therapeutic strategy in oncological therapy.


Diet and nutrition in polycystic ovary syndrome (PCOS): pointers for nutritional management. PCOS patients are not always markedly overweight but PCOS is strongly associated with abdominal obesity and insulin resistance. Effective approaches to nutrition and exercise improve endocrine features, reproductive function and cardiometabolic risk profile--even without marked weight loss. Recent studies allow us to make recommendations on macronutrient intake. Fat should be restricted to < or =30% of total calories with a low proportion of saturated fat. High intake of low GI carbohydrate contributes to dyslipidaemia and weight gain and also stimulates hunger and carbohydrate craving. Diet and exercise need to be tailored to the individual's needs and preferences. Calorie intake should be distributed between several meals per day with low intake from snacks and drinks. Use of drugs to either improve insulin sensitivity or to promote weight loss are justified as a short-term measure, and are most likely to be beneficial when used early in combination with diet and exercise.


Human immunodeficiency virus infection inhibits granulocyte-macrophage colony-stimulating factor-induced microglial proliferation. It is well known that infection by the human immunodeficiency virus (HIV) dysregulates cell physiology, but little information is available on the consequences of HIV infection in primary macrophages and microglia. The authors examined the relationship between cell proliferation and HIV infection in primary cultures of microglia and in human central nervous system (CNS). In cultures infected with HIV (ADA and BaL), granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated cell proliferation was reduced in productively infected (p24+) cells as compared to p24- cells. The reduction was observed with both Ki67 and BrdU labeling, suggesting a G1/S block. The reduction was insignificant when microglia were infected with a Vpr- mutant virus. In human CNS, proliferating (Ki67+) cells were rare but were increased in the HIV+ and HIV encephalitis (HIVE) groups compared to the HIV- group. A positive correlation between GM-CSF immunoreactivity and Ki67 counts, implicating GM-CSF as a growth factor in human CNS was found. The relationship between total macrophage (CD68+) proliferation and infected macrophage (p24+) proliferation was assessed in HIVE by double labeling. Whereas 1.2% of total CD68+ cells were Ki67+, only 0.5% of HIV p24+ cells were Ki67+ (P < .05). Furthermore, staining for CD45RB (as opposed to CD68) facilitated the identification of Ki67+ microglia, indicating that CD68 could underestimate proliferating microglia. The authors conclude that although there is increased expression of GM-CSF and increased cell proliferation in the CNS of HIV-seropositive individuals, cell proliferation in the productively infected population is actually suppressed. These data suggest that there might be a viral gain in the suppressed host cell proliferation.


Editorial note.

Editorial note.


Anterograde transsynaptic tracing in the murine somatosensory system using Pseudorabies virus (PrV): a "live-cell"-tracing tool for analysis of identified neurons in vitro. The Pseudorabies virus (PrV) strain Bartha is widely used as a tool for retrograde transneuronal tracing in mammals. Traced neurons can be identified in cell culture allowing the analysis of their physiological features ("live-cell"-tracing). Compared to PrV-Bartha, PrV-Kaplan is known for higher virulence and transsynaptic spread in both retrograde and anterograde direction. Herein we assess the authors assess PrV-Kaplan for transsynaptic anterograde "live-cell"-tracing. Following intranasal application in mice, labelled trigeminal and brainstem neurons could be identified in vitro. Detailed electrophysiological analysis indicated that viral infection did not affect neuronal properties, making PrV-Kaplan eligible for functional analysis of identified neurons within somatosensory systems.


Maternity guidelines: aid or hindrance?

Maternity guidelines: aid or hindrance? This questionnaire survey was performed to explore the attitudes of 295 obstetricians and midwives in a teaching and a district general hospital, towards the maternity directorate guidelines, and to document the possible barriers that may hinder their adherence and suggestions for improvement. The response rate was 61%. There was a tendency in both hospitals to agree that guidelines help learning and improve the quality of care, but make practice defensive, stop innovation, narrow the clinical freedom and do not reduce cost. A significantly higher number in the district hospital valued the guidelines as an aid to use the most up-to-date knowledge. Interestingly, about half of the staff in both hospitals were neutral regarding the role guidelines play in increasing professional satisfaction. The major barrier for lack of adherence was the belief that the individual guidelines were not comprehensive enough to cover daily practice. The trend of the responses was similar in both hospitals with the exception of three areas, where there were significant differences; pressure of time in the teaching hospital, and senior disagreement and lack of resources in the district hospital. Using bullet points for clarity within guidelines, shortening the guidelines, announcing new editions in directorate meetings, and frequent updating were the most common suggestions for improvement. The variations in staff response between the two hospitals highlight the importance of tailoring guidelines to match the working environment and the staff views rather than rigidly dictating practice.


Identification of a novel protein encoded by the latency-related gene of bovine herpesvirus 1. The latency-related (LR) RNA encoded by bovine herpesvirus 1 (BHV-1) is abundantly expressed and alternatively spliced in trigeminal ganglia. A mutant BHV-1 strain that contains three stop codons at the beginning of LR open reading frame (ORF)-2 (LR mutant virus) does not express ORF-2 or an adjacent reading frame that lacks an initiating ATG (RF-C). Calves latently infected with wild-type (wt) BHV-1, but not with the LR mutant virus, reactivate from latency, indicating that proteins encoded by the LR gene regulate the latency-reactivation cycle. The LR gene also contains another large ORF (ORF-1) that is approximately 200 bp downstream of stop codons inserted at the N-terminus of ORF-2. To test whether the LR mutant virus can expresses ORF-1, the authors developed antiserum directed against ORF-1. The ORF-1 antiserum recognizes specific proteins in bovine cells productively infected with wt BHV-1. ORF-1 protein expression is reduced, but not blocked, when bovine cells are infected with the LR mutant virus. Confocal microscopy demonstrated ORF-1 is present in the cytoplasm and nucleus of productively infected cells, whereas RF-C or a fusion protein containing RF-C localizes to the cytoplasm. Trigeminal ganglia from calves latently infected with wt BHV-1 contain neurons specifically stained with the ORF-1 antiserum. These studies suggest ORF-1 expression may be important for the BHV-1 latency-reactivation cycle.


Associations of cigarette smoking with viral immune and cognitive function in human immunodeficiency virus-seropositive women. Cigarette smoking alters the immune system and may improve cognitive deficits in neuropsychiatric disorders. Smoking prevalence is high in human immunodeficiency virus (HIV)-infected patients; however, its effect on HIV-associated cognitive impairment remains unknown in the era of antiretroviral treatment. The authors examined associations of smoking with viral immune profile and cognitive function in a cohort of HIV-seropositive women. This observational cross-sectional study included 56 women (36 HIV-seropositive and 20 HIV-seronegative) surveyed with a tobacco questionnaire: the Fagerström Test for Nicotine Dependency. Viral immune status was obtained 6 to 12 months before questioned. Neurocognitive testing (NP) assessed verbal memory, frontal/executive function, psychomotor speed, and motor speed. A reference group of HIV-seronegative women was used to calculate standardized z-scores. Cognitive impairment was classified using a modified American Academy of Neurology criteria, adding an asymptomatic group based on NP tests. Statistics included parametric and nonparametric tests. HIV-seropositive women were more likely to report a history of smoking (P = 0.028). Among them, current smoking correlated with higher plasma viral load (P = 0.048), and history of smoking correlated with lower CD4 cell count (P = 0.027). The authors observed no associations between cognitive impairment and either current or past history of smoking and no differences in neurocognitive domain scores between HIV-seropositive and -seronegative women or between those with and without a history of smoking. However, restricting analysis to HIV-seropositives showed a significant better performance on the frontal/executive domain in those with history of smoking. In summary, history of smoking correlated with better frontal/executive cognitive domain performance in HIV-seropositive women and with worse viral immune profile.


The practice of caesarean birth in the UK. A survey of 231 clinical directors in obstetrics and gynaecology in the British isles in 2005 asking about attitudes and practice relating to Caesarean Section (CS) had a 68.3% response rate. This paper reports on consultants' practice. Since our previous survey done in 1990 relating to births in 1989 the use of epidural anaesthesia had increased and the vast majority of caesarean sections were done under epidural or spinal aneasthesia and the majority of consultants allowed the partner to be present at the birth compared with 55% in 1989. Practice varied considerably between consultants in regard to the risks of surgery routinely mentioned, the proportion who would allow a trial of scar after two previous CS and those who would perform a CS at 24 weeks for fetal distress. A third accepted the NICE guidleines about CS fully and only 5% did not accept these with 61% accepting partially despite the lack of evidence for some of their recommendations. The proportion of women monitored routinely in labour had dropped from 45% to 11% although half still had an admission strip performed despite the 2001 guidelines. Few had read, the FIGO Ethics committee recommendations about non-medically indicated CS and it is suggested that the RCOG should bring such information to the attention of members through O&G news. A leaflet explaining the possible risks of CS and the long term implications should be produced by the RCOG alone or in conjunction with the Dept of Health so that women are better informed about these.


Level of fetal head above brim: comparison of three transabdominal methods of estimation, and interobserver agreement. This study compared three intra-partum transabdominal methods of estimating the level of the fetal head above the brim, and determined interobserver agreement in fifths estimation of the fetal head. The researcher examined 508 women in term labour and estimated level of head in fifths by the Crichton method, in fifths by the Notelowitz finger-breadth method, and by symphysis-to-sinciput measurement (SSM). The attending clinicians also made their estimates, using the methods of their choice. Two-fifths of head or less was considered engaged. When two-fifths was palpable by the Crichton method, the Notelowitz method gave a mean of 2.40 fifths. The researcher and clinicians agreed on the level of head in 42.9% of examinations. Interobserver agreement was poor (kappa = 0.22). In conclusion, the Crichton method overestimates head descent in comparison with the Notelowitz method. SSM was easy to perform but requires validation. The fifths method of determining level of head appears inexact and poorly reproducible.


An evaluation of the use of reported febrile illness in predicting malaria in pregnancy. The object of this study was to evaluate the sensitivity and specificity of reported febrile illness in predicting malaria in pregnant women at booking in NAUTH, Nnewi. This was a case control prospective survey using a structured questionnaire to collect data from pregnant women attending antenatal clinic of Nnamdi Azikiwe University Teaching Hospital between April and September 2001. Peripheral blood smears were examined in 420 pregnant women during their first antenatal visit. This study showed that 57.4% of parasitaemic pregnant women and 54% of aparasitaemic pregnant women reported fever before their first antenatal visit. The sensitivity and specificity of reported febrile illness in predicting malaria were 57.4% and 46%, respectively. However, the predictive value of a positive test was as high as 80.25%, while it was only 22% for the predictive value of a negative test. The efficiency of reported febrile illness as a screening device was 55%. Reliance on reported febrile illness will not be adequate to identify parasitaemic pregnant women because many of those with heavy placental parasitisation may not report fever. This justifies the place of the intermittent presumptive therapy using sulphadoxine-pyrimethamine in pregnant women living in malaria endemic areas such as sub-Saharan Africa.


Ergometrine given during caesarean section and incidence of delayed postpartum haemorrhage due to uterine atony. Delayed postpartum haemorrhage due to uterine atony after caesarean section was occurring in women in our recovery area despite many of them already having an oxytocin infusion running to prevent such a problem. We therefore decided to compare the incidence of such problems for a 2-month period before and after altering our uterotonic policy: in addition to the routine bolus dose of 5 units of oxytocin after delivery of the baby, we added 500 microg of intramuscular ergometrine during abdominal closure. We noticed a reduced number of massive postpartum haemorrhages due to an atonic uterus in the recovery room but an increased incidence of nausea and vomiting. No prophylactic anti-emetic was given during this pilot study. This small study suggests that 50 women would need to be given ergometrine at caesarean section to prevent one delayed massive haemorrhage from uterine atony and four extra women would suffer with vomiting. We feel this is reasonable and now use a prophylactic anti-emetic as well as delaying the ergometrine until closure of the rectus sheath which reduces the incidence of nausea and vomiting.


Timing of planned repeated caesarean section: an enigma. We aimed to explore the current trends among obstetric consultants in timing planned caesarean sections in patients with previous caesarean deliveries. A questionnaire was sent to 129 consultant obstetricians and gynaecologists. The questionnaire included closed questions about the preferred gestational age to perform an elective caesarean section in patients with previous one, two, three or more caesarean deliveries and in patients with a previous classical caesarean scar. The study was confined to NHS hospitals at the Eastern and London Deaneries, UK. The main outcome measure was the gestational age at which a planned caesarean section was preferred. The percentage of consultants who preferred to do caesarean section at 39 weeks or more gestational age in patients with previous one, two, three or more and a classical caesarean section were 93.6%, 87.3%, 71.3% and 35.9%, respectively. A considerable body of obstetricians plan to perform elective caesarean sections at a gestational age <39 weeks in patients with three or more previous abdominal deliveries and in patients with a previous classical caesarean section. There are no randomised controlled trials to address the best practice in these cases. Looking at the available evidence in the literature, it seems unjustified to perform the caesarean section at an earlier gestational age for patients with previous multiple caesarean sections but there is some evidence to support this policy in patients with a previous classical section.


The influence of social support on the duration of breast-feeding among antenatal patients in Ibadan. In Nigeria, breast-feeding has been shown to be very closely related to infant survival. Prolonged and adequate breast-feeding is critical to most infants' nutritional health and growth. This study aims to determine the influence of family support on the duration of exclusive and total breast-feeding of infants of antenatal patients. This cross-sectional study was conducted between 1 September and 30 December 2005. The tool was a structured questionnaire. The main outcome variables were the duration of breast-feeding; both total and exclusive explanatory variables were mainly related to support obtained during breast-feeding from husband and older female relations. Husbands support significantly increased the total duration of breast-feeding by a mean of 1.69 months (95% CI 0.88, 2.51), however, exclusive breast-feeding was not significantly affected by the husband's support (OR 0.94; 95% CI 0.63, 1.39). Female support had a significant influence on both the total duration of breast-feeding which is increased by a mean of 1.08 months (95% CI 0.14, 2.02), and the adequate conduct of exclusive breast-feeding (OR 1.83; 95% CI 1.17, 2.86). The cultural practice of having additional female support in the postpartum period has been shown by this study to be beneficial. Therefore, this cultural practice should be encouraged and catalogued as a beneficial cultural practice. This practice is also cheap and sustainable.


Pregnancy following infertility: a concern? This study is to assess whether a waiting time to pregnancy (WTP) >1 year elevates the risk of adverse pregnancy outcomes in essentially healthy women with a single normal fetus at a gestational age of >20 weeks. Comparisons were made between those who conceived spontaneously with those who conceived after treatment for infertility. A retrospective analysis of 1,020 pregnancies occurring after a delay (WTP) of >1 year was undertaken over a 6-year period. Of these pregnancies, 24 resulted from advanced reproductive techniques and were excluded, as were four first trimester abortions, leaving 992 women. Of these 992, study group A (treated) consisted of 553 (56%) women, of whom 312 (56%) were primigravidae (A1). The pregnancy outcomes were compared with those of the remaining 439 (46%) women who conceived spontaneously, group B (control, untreated). Of these women, 234 (53%) were primigravidae (B1). Results suggested that primigravidae in the study group showed an increased risk of pre-term births and this risk was statistically significant between the gestations of 34 and 37 weeks. This risk remained elevated after adjustment for covariates. Pregnancies of women in group A1 were appreciably shorter than those of primigravidae who got pregnant without treatment (36.1 +/- 0.8 weeks vs 39.2 +/- 2.1 weeks); had lower birth weights (2684 +/- 481 g vs 3481 +/- 703 g) and were more likely to be admitted to the neonatal intensive care unit. Primigravidae among the treated group showed a statistically significant risk of caesarean births.


Delivery outcome after induction of labour using prostaglandin in women with one previous caesarean section.


Sex and reproduction among HIV-infected people in Maiduguri, Nigeria. For HIV-infected people, prevention of transmission of the virus to their spouses and other sexual partners can only be achieved through abstinence and safer sex practices using condoms. New drugs and technologies are now available that can prevent vertical transmission of the virus. A total of 262 people living with HIV/AIDS (PLWHA) were interviewed to explore their sexual and reproductive desires and practices. About 75.6% of them were sexually active and 62.2% never used condoms. Although only 26.3% had no living child, the majority of these (71.4%) wanted to have children. Their knowledge of mother-to-child transmission of HIV and how to prevent it was good. PLWHA engage in unprotected sexual intercourse with the desire to have more children. It is expected that more paediatric HIV infections will be seen in the future in a poor-resource setting like ours.


Attitudes and awareness of female genital mutilation: a questionnaire-based study in a Kenyan hospital. There are considerable efforts in Kenya to increase awareness of the issues and health risks associated with female genital mutilation (FGM) through educational programmes. The Kenyan government formally outlawed FGM in 2001. This questionnaire-based study aimed to explore attitudes and awareness of FGM in Kenya with particular reference to the law, health complications and educational programmes. A significant decline in the prevalence of FGM was demonstrated and awareness of health complications of FGM shown to be the main factor causing this trend. The need for further efforts to eradicate the practice and the importance of religion and culture in shaping social attitudes was evident. The outlawing of FGM was considered a positive advance but may have the detrimental effect of deterring women from seeking medical assistance for complications relating to FGM.


Risk factors for obstetric fistulae in north-eastern Nigeria. This prospective comparative study of obstetric fistulae (OF) was aimed at identifying risk factors. A total of 80 obstetric fistulae treated at the gynaecological unit of the FMCG, and 80 inpatients without fistulae recruited randomly as controls formed the basis of this study. Through interview and case record review, information on age, parity and marital status was collected. Other features were educational status, occupation and booking status of the pregnancy that might have led to this condition. The duration of labour, place of birth and mode of delivery, including its outcome were also collected. The data were analysed using the Epi Info. The majority of the patients were Hausa/Fulani 87.5%, Muslims 91.2%, with large vesicovaginal fistulae (average size 5.0 cm) mainly resulting from obstructed labour (93.7%). Major risk factors included early age at first marriage (average 14 years), short stature (average height 146.2 cm) and illiteracy (96.3%). Also low social class and lack of gainful employment were factors. Failure to book for antenatal care (93.7%), and rural place of residence (95%) were also factors associated with acquiring the fistulae. Living far away (>3 km) from a health facility also contributed or predisposed to the development of an obstetric fistula. Social violence and stigma associated with the fistulae included divorce, being ostracised as a social outcast, and lack of assistance from relations in terms of finding and funding treatment. This study supports improved access to basic essential obstetric care, family planning services, and timely referral when and where necessary. Universal education will provide a long-term solution by improving the standard of living and quality of life. Especially important are media- and community-based programmes on the ills of teenage marriage and child pregnancy using cultural and religiously-based values to give sound advice. In a male dominated society, reaching out to men with traditionally palatable messages that will change their attitude and practices to taking responsibility in reproductive health could be a winning strategy.


Study of the demographic criteria and management of adolescents referred with abnormal cervical smears. This observational study was conducted on the adolescents referred to a colposcopy clinic in a UK general hospital over a period of 10 years (1996 - 2005). It included 155 newly referred adolescents (age < or = 20 years) with abnormal smear results. The aim of the study was to define the demographic risk factors associated with the development of abnormal cervical smears and identify the incidence of high-grade cervical lesions in this age group. All the risk factors associated with development of HPV infection were found among this group, particularly early age of sexual contact and number of sexual partners. A total of 52.9% of the cases had a smear showing moderate or severe dyskaryosis. The incidence increased to 66.7% among those who had their first sexual contact before the age of 16. Colposcopy and histology confirmed the presence of CIN II and CIN III in 40.6% of cases. The study showed that patients can develop high-grade cervical lesions in their early teens. Delaying the age of onset of cervical screening to 25, as it is currently recommended in UK, will delay the detection and treatment of these cases. A new approach is required to deal with this problem.


Injuries to the levator ani in unexplained, chronic pelvic pain. Patterns of injury to the levator ani in parous women with laparoscopy-negative, chronic pelvic pain (CPP) are described. A consecutive series of 26 parous women with laparoscopy-negative CPP, had magnetic resonance imaging (MRI) in axial, coronal and sagittal planes. Similar imaging studies were performed in 32 parous women having MRI scans for other clinical indications and 20 asymptomatic, nulliparous women. Three patterns of injury/defect to the levator ani (LAd) were observed in 20/26 patients with unexplained pelvic pain: avulsion of the pubococcygeus from its origin at the pubis (LAd I, 3/20), avulsion of the pubococcygeus from its origin at the arcus tendineus levator ani (LAd II, 14/20), and, loss of shape and form of the pubococcygeus (LAd III, 6/20). Three patterns of injury to the levator ani have been described with MR imaging in parous women with unexplained CPP.


Contraceptive practices in women with repeat termination of pregnancies. The incidence of repeat termination of pregnancies (TOP) has been increasing in the past decade in-spite-of widespread availability of free and effective contraceptive methods. A retrospective analysis of case notes of women referred for TOP in the Family Planning clinic at Luton, between January and October 2005 was carried out. A total of 159 women were referred for TOP. Of these, 42 (26.4%) were repeat procedures. In this subgroup, 78.5% had used one or more contraceptive methods after the first termination. However, only 61.9% of them continued to use contraception at the time of presentation for a repeat TOP, the most common method being the male condom. Half of the total women seen for a repeat TOP were under 6-weeks' gestation and 14.2% had been treated for sexually transmitted infections in the past. The incidence of repeat TOPs is increasing in the UK. Many studies have shown that the periabortion contraceptive practices are generally poor. In our study, women undergoing repeat termination of pregnancies were either not using contraception or using a method with a high failure rate. We also found the highest number of repeat TOPs occurring in the 20 - 25 age group. Effective contraceptive counselling is vital to combat this worrying trend. Routine 2-week follow-up appointments after TOP and regular auditing of the periabortion contraceptive practices are needed.


Abortion-related mortality in a tertiary medical centre in Enugu, Nigeria. This study reviewed abortion-related maternal deaths, from January 2000 to December 2005 at the University of Nigeria Teaching Hospital, Enugu, Nigeria, to identify maternal characteristics, abortion types, provider characteristics, complications and causes of death. Abortion was defined as termination of pregnancy before 28 completed weeks of pregnancy. There were 93 pregnancy-related deaths, and 11 were abortion-related. Abortion-related mortality accounted for 11.8% of all maternal deaths. Nine (81.8%) had induced abortions; three (27.3%) were teenagers; five (45.5%) were married; and six (54.5%) were unmarried. Five (45.5%) of the women were nulliparous. Six (54.5%) of the procedures were in private medical clinics. The cause of death was sepsis and haemorrhage in eight (72.7%) and three (27.3%) of the women, respectively. Abortion-related mortality is a major contributor to maternal mortality in our institution, with induced unsafe abortion constituting the bulk of the burden. Improved access to family planning and reproductive health services may reduce abortion-related maternal deaths.


Endometrial biopsy: a pilot study of instrument used; Pipelle vs infant feeding tube. A small pilot study to compare the use of infant feeding tubes (IFT) for endometrial biopsy compared with the standard Pipelle tubes was calculated. We assessed patients' pain scores and the number of successful samples obtained. This study highlights that an IFT may be more comfortable for the patient and be equally as effective at obtaining an adequate biopsy sample but a larger randomised study is needed to explore this further.


A survey of the value of routine intimate examination and related practices in subfertile couples. The practice of undertaking routine intimate examinations in the management of subfertile couples varies among clinicians. An anonymous self-administered mailed questionnaire survey was carried out to determine the current practices followed by clinicians and the rationale supporting their practice. In the absence of large comparative studies, this survey provides expert opinion regarding this practice. The questionnaire was mailed to the Fellows and members of the Royal College of Obstetricians and Gynaecologists in the Northern, Yorkshire, West Midlands and Wales regions in the UK. A total of 802 questionnaires were posted, of which 516 were returned, giving an overall response rate of 64%. The responses and comments varied. Some 62% of respondents would routinely perform a pelvic examination on all women presenting with subfertility; 23% would do so only in selected cases and 63% of the clinicians would perform a routine transvaginal ultrasound. The male partners are examined by only 19% of the clinicians. A total of 83% of the respondents were of the opinion that the survey made them think about the role and justification of intimate examinations in greater detail. The practice of intimate examination of subfertile couples varies among clinicians in the survey group. There is a need for large prospective comparative studies to study the value of this examination and related practices in the management of subfertile couples. In the absence of comparative trials, this survey provides expert opinion. It may be suggested that an intimate examination should not be carried out routinely in all subfertile couples. It could be done when it has potential to add value to the management of the patient. A pragmatic approach should be adopted in every clinical situation.


Colposcopy audit--including a subgroup of young women.


Bilateral exudative retinal detachment causing blindness in severe pre-eclampsia.


Idiopathic chronic fetomaternal haemorrhage presenting with reduced fetal movements.


Use of sulindac in polyhydramnios.

Use of sulindac in polyhydramnios.


Spondylometaphyseal dysplasia-Sedaghatian type associated with intra-partum cardiac arrhythmia and neonatal death.


Pregnancy following Whipple's procedure.


Prophylactic catheters placement and intraoperative internal iliac artery embolisation in a patient with placenta accreta.


Negative pregnancy test: could it be a molar pregnancy?


Is labour safe in infective endocarditis patients with septic lung embolism?


A difficult delivery associated with a nuchal cord found nine times around the fetal neck.


Successful outcome for mother and twin babies following peri-mortem caesarean section.


Vaginal triplet delivery.

Vaginal triplet delivery.


Tubal pregnancy four months after cauterisation of the vaginal vault: one year following a vaginal hysterectomy.


Transvaginal bowel evisceration thirteen weeks after total abdominal hysterectomy.


Fetal bone mistaken for intrauterine contraceptive device.


Neglected porcelain pessary causing postmenopausal bleeding and vesicovaginal fistula.


Knife entry into the uterine cavity; overcoming severe cervical stenosis at hysteroscopy.


Uterine rupture at 8 weeks' gestation following 600 microg of oral misoprostol for management of delayed miscarriage.


IUCD induced abdomino-pelvic actinomycosis presenting as acute large bowel obstruction.


Re: Savage W, Francome C. 2007. British consultants' attitudes to caesareans. Journal of Obstetrics and Gynaecology 27:354 - 359.


Primary non-gestational choriocarcinoma of the lung.


Disruption of the plasma membrane integrity by cholesterol depletion impairs effectiveness of TRH receptor-mediated signal transduction via G(q)/G(11)alpha proteins. We monitored the radioligand-binding characteristics of thyrotropin-releasing hormone (TRH) receptors, functional activity of G(q/11)alpha proteins, and functional status of the whole signaling cascade in HEK293 expressing high levels of TRH receptors and G(11)alpha. Our analyses indicated that disruption of plasma membrane microdomains by cholesterol depletion did not markedly influence the binding parameters of TRH receptors, but it altered efficacy of signal transduction. The functional coupling between TRH receptor and G(q/11)alpha was assessed by agonist-stimulated [(35)S]GTPgammaS binding, and results of these measurements pointed out to significantly lower potency of TRH to mediate G protein activation in the plasma membrane fraction isolated from cholesterol-depleted cells; there was a shift in sensitivity by one order of magnitude to the higher concentrations. A markedly lower sensitivity to stimulation with TRH was also observed in our experiments dealing with determination of hormone-induced Ca(2+) response. These data suggest that the intact structure of plasma membranes is an important optimum signal transduction initiated by TRH receptors and mediated by G(q/11)alpha proteins.


Airport-related air pollution and noise. To provide quantitative evidence of the impact on people of a neighboring metropolitan airport, La Guardia Airport (LGA) in New York City, (1) airborne particulate matter (PM) was measured to determine whether concentration differences could be detected between homes that are upwind and downwind of the airport; (2) 24-hr noise measurements were made in 12 homes near the airport; and (3) the impact of noise was assessed by a Community Wellness and Health Promotion Survey. Particulate matter concentrations were higher during active airport operating hours than during nonoperating hours, and the percent increase varied inversely with distance from the airport. Hourly differences between paired upwind and downwind sites were not remarkable. Residents living near the airport were exposed to noise levels as much as four times greater than those experienced by residents in a quiet, comparison home. Impulse noise events were detected from both aircraft and vehicular traffic. More than 55% of the people living within the flight path were bothered by aircraft noise, and 63% by highway noise; these were significantly higher percentages than for residents in the nonflight area. The change in PM concentrations with distance during operating compared with nonoperating hours; traffic-related impulse noise events; and the elevated annoyance with highway noise, as well as aircraft noise among residents in the flight path area, show airport-related motor vehicle traffic to be a major contributor to the negative impact of airports on people in the surrounding communities.


Tamoxifen-induced [Ca2+]i rises and Ca2+-independent cell death in human oral cancer cells. The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca(2+) concentrations ([Ca(2+)](i)) and cell viability in OC2 human oral cancer cells. [Ca(2+)](i) and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). The tamoxifen-induced Ca(2+) influx was sensitive to blockade of L-type Ca(2+) channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca(2+)-free medium, after pretreatment with 1 muM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), tamoxifen-induced [Ca(2+)](i) rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca(2+)](i) rises. Inhibition of phospholipase C with 2 microM U73122 did not change tamoxifen-induced [Ca(2+)](i) rises. At concentrations between 10 and 50 microM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 microM tamoxifen was not reversed by prechelating cytosolic Ca(2+) with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca(2+)](i) rises, in a nongenomic manner, by causing Ca(2+) release from the endoplasmic reticulum, and Ca(2+) influx from L-type Ca(2+) channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca(2+)](i) rise.


The GIRK1 brain variant GIRK1d and its functional impact on heteromultimeric GIRK channels. Four isoforms of GIRK channels (GIRK1-4) have been described in humans. In addition, several splice variants of more or less unknown function have been identified from several tissues and species. In our study, we investigated the structure and function of a new variant of GIRK1 that has been isolated from rat brain. Because of wide similarities with a previously described variant, we also named it GIRK1d. This variant lacks a region corresponding to exon 2 of full-length GIRK1, leading to a truncated GIRK1 that lacks the main part of the C-terminus. To study GIRK1d we used the Xenopus laevis expression system, the two-electrode voltage clamp method, and confocal laser scan microscopy. We found that our GIRK1d variant preferentially binds GIRK2 or GIRK4 over GIRK1. Furthermore, it largely reduces conductances mediated by GIRK1/2 or GIRK1/4 hetero-multimeric channels when coexpressed and nearly totally abolishes currents when replacing GIRK1 in hetero-multimeric channels.


Dimeric DOTA-alpha-melanocyte-stimulating hormone analogs: synthesis and in vivo characteristics of radiopeptides with high in vitro activity. Dimeric analogs of alpha-melanocyte-stimulating hormone (alpha-MSH) labeled with radiometals are potential candidates for diagnosis and therapy of melanoma by receptor-mediated tumor targeting. Both melanotic and amelanotic melanomas (over-)express the melanocortin-1 receptor (MC1-R), the target for alpha-MSH. In the past, dimerized MSH analogs have been shown to display increased receptor affinity compared to monomeric MSH, offering the possibility of improving the ratio between specific uptake of radiolabeled alpha-MSH by melanoma and nonspecific uptake by the kidneys. We have designed three linear dimeric analogs containing a slightly modified MSH hexapeptide core sequence (Nle-Asp-His-d-Phe-Arg-Trp) in parallel or antiparallel orientation, a short spacer, and the DOTA chelator for incorporation of the radiometal. In vitro, all three peptides were more potent ligands of the mouse B16-F1 melanoma cell melanocortin-1 receptor (MC1-R) than DOTA-NAPamide, which served as standard. The binding activity of DOTA-diHexa(NC-NC)-amide was 1.75-fold higher, that of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was 3.37-fold higher, and that of DOTA-diHexa(CN-NC)-amide was 2.34-fold higher. Using human HBL melanoma cells, the binding activity of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was sixfold higher than that of DOTA-NAPamide. Uptake by cultured B16-F1 cells was rapid and almost quantitative. In vivo, however, the data were less promising: tumor-to-kidney ratios 4 hr postinjection were 0.11 for [(111)In]DOTA-diHexa(NC-NC)-amide, 0.26 for diHexa(NC-NC)-Gly-Lys([(111)In]DOTA)-amide, and 0.36 for [(111)In]DOTA-diHexa(CN-NC)-amide, compared to 1.67 for [(111)In]DOTA-NAPamide. It appears that despite the higher affinity to the MC1-R of the peptide dimers and their excellent internalization in vitro, the uptake by melanoma tumors in vivo was lower, possibly because of reduced tissue penetration. More striking, however, was the marked increase of kidney uptake of the dimers, explaining the unfavorable ratios. In conclusion, although radiolabeled alpha-MSH dimer peptides display excellent receptor affinity and internalization, they are no alternative to the monomeric DOTA-NAPamide for in vivo application.


Unifying electrostatic mechanism for receptor-ligand activity. A prior article in skeletal form proposed an electrostatic mechanism for receptor-ligand activity. The present review provides an elaboration, including supporting evidence. The fundamental aspect entails the presence of molecular electrostatic potential associated with ions and dipoles in the ligand. The ligand can be regarded as an electrical link that joins prevalent electrostatic fields present in the surrounding protein matrix. The exact role of these fields is speculative. One possibility is to function as conduits for electrons and radicals in cell signaling. There is increasing support for important participation of these species in signal transduction. There might also be a favorable influence on energetics involving the electron transfer process. A summary of receptor biology is also provided, including receptors for acetylcholine (nicotinic and muscarinic), GABA, adrenergic, and glutamate.


Overview: Workshop on children's inhalation dosimetry and health effects for risk assessment.


Unifying electrostatic mechanism for phosphates and sulfates in cell signaling. Prior proposals suggested the importance of electrochemistry in signal transduction and receptor-ligand activity. Electrostatic fields associated with ions and dipoles were assigned important roles. Little is known concerning the precise mode of action in cell signaling by widespread phosphorylation. According to the hypothetical framework, molecular electrostatic potential associated with phosphate anion is a key element as a link in the communication grid, possibly inducing favorable energetics in the electron transfer process. Similar involvement appears plausible for the sulfate anion. Supporting evidence for the electrostatic mechanism is presented. Representative literature on phosphorylation in the biological domain is reviewed with emphasis on cell signaling. The treatment includes phosphates from protein, lipids, and other molecules, plus the role of reactive oxygen species. Protein sulfation is also discussed.


Focusing on children's inhalation dosimetry and health effects for risk assessment: an introduction. Substantial effort has been invested in improving children's health risk assessment in recent years. However, the body of scientific evidence in support of children's health assessment is constantly advancing, indicating the need for continual updating of risk assessment methods. Children's inhalation dosimetry and child-specific adverse health effects are of particular concern for risk assessment. When focusing on this topic within children's health, key issues for consideration include (1) epidemiological evidence of adverse effects following children's exposure to air pollution, (2) ontogeny of the lungs and effects on dosimetry, (3) estimation and variability of children's inhalation rates, and (4) current risk assessment methodologies for addressing children. In this article, existing and emerging information relating to these key issues are introduced and discussed in an effort to better understand children's inhalation dosimetry and adverse health effects for risk assessment. While much useful evidence is currently available, additional research and methods are warranted for improved children's health risk assessment.


Modeling approaches for estimating the dosimetry of inhaled toxicants in children. Risk assessment of inhaled toxicants has typically focused upon adults, with modeling used to extrapolate dosimetry and risks from lab animals to humans. However, behavioral factors such as time spent playing outdoors may lead to more exposure to inhaled toxicants in children. Depending on the inhaled agent and the age and size of the child, children may receive a greater internal dose than adults because of greater ventilation rate per body weight or lung surface area, or metabolic differences may result in different tissue burdens. Thus, modeling techniques need to be adapted to children in order to estimate inhaled dose and risk in this potentially susceptible life stage. This paper summarizes a series of inhalation dosimetry presentations from the U.S. EPA's Workshop on Inhalation Risk Assessment in Children held on June 8-9, 2006 in Washington, DC. These presentations demonstrate how existing default models for particles and gases may be adapted for children, and how more advanced modeling of toxicant deposition and interaction in respiratory airways takes into account children's anatomy and physiology. These modeling efforts identify child-adult dosimetry differences in respiratory tract regions that may have implications for children's vulnerability to inhaled toxicants. A decision framework is discussed that considers these different approaches and modeling structures including assessment of parameter values, supporting data, reliability, and selection of dose metrics.


Assessing the health effects and risks associated with children's inhalation exposures--asthma and allergy. Adults and children may have different reactions to inhalation exposures due to differences in target tissue doses following similar exposures, and/or different stages in lung growth and development. In the case of asthma and allergy both the developing immune system and initial encounters with common allergens contribute to this differential susceptibility. Asthma, the most common chronic childhood disease, has significant public health impacts and is characterized by chronic lung inflammation, reversible airflow obstruction, and immune sensitization to allergens. Animal studies described here suggest that air pollutants exacerbate asthma symptoms and may also play a role in disease induction. Changes characteristic of asthma were observed in rhesus monkeys sensitized to house dust mite antigen (HDMA) as infants and exposed repeatedly thereafter to ozone (O3) and HDMA. O3 exposure compromised airway growth and development and exacerbated the allergen response to favor intermittent airway obstruction and wheeze. In Brown Norway rats a variety of air pollutants enhanced sensitization to HDMA such that symptoms elicited in response to subsequent allergen challenge were more severe. Although useful for assessing air pollutants effects on initial sensitization, the rodent immune system is immature at birth relative to humans, making this model less useful for studying differential effects between adults and children. Because computational models available to address children's inhalation exposures are limited, default adjustments and their associated uncertainty will continue to be used in children's inhalation risk assessment. Because asthma is a complex (multiple genes, phenotypes, organ systems) disease, this area is ripe for systems biology approaches.


Potential new approaches for children's inhalation risk assessment. The U.S. Environmental Protection Agency (EPA) practice of risk assessment is moving toward more thoroughly considering children's unique susceptibilities and exposure potential. Childhood is assessed as a sequence of life stages that reflects the fact that as humans develop, windows of susceptibility may appear that lead to enhanced sensitivity to exposure of environmental agents, while changes in behavior and physiology may increase exposure and dose. The U.S. EPA developed guidance in the past few years that addresses some aspects of increased susceptibility and exposure and dose. However, when it comes to considering inhalation exposure, dose, and risk, current U.S. EPA practice does not explicitly address children. The purpose here is to begin studying the adequacy of practice for children's health and to explore possible next steps in developing new methods to more accurately assess life-stage-specific differences. The existing guidelines and policies used to address potentially unique susceptibilities of children for inhaled environmental chemicals were considered, as well as what may be learned from examples of approaches that have been applied by state agencies (such as the California Environmental Protection Agency) or in the literature, to incorporate potentially unique susceptibilities and exposures to children. Finally, there is a discussion of possible approaches for considering inhalation exposure and susceptibility in U.S. EPA risk assessments.


Distributional analyses for children's inhalation risk assessments. The discussion here is divided into three parts. First there is a generic introduction to variability and uncertainty and a discussion of some underappreciated features of these concepts that have nontrivial implications for structuring risk analyses. Next are some suggestions for a way forward to build a better quantitative understanding of relevant variability and uncertainty. The process involves building databases of putative analogous cases to represent the concerns that are now reflected in the various point-estimate "uncertainty factors" or "adjustment factors" in the formulas used to derive current reference doses and reference concentrations (RfDs and RfCs). The use of such databases is illustrated with some limited selections from our own databases where some preliminary comparisons between child and adult population variability in activity and respiratory response parameters are possible. A final section reflects on the current evolution of practice in estimating variability in risk assessments.


Nasal contribution to breathing and fine particle deposition in children versus adults. Both the route of breathing, nasal versus oral, and the effectiveness of the nose to filter inhaled, fine particles may differ between children and adults. This study compared (1) the nasal contribution to breathing at rest and during mild to moderate exercise in children (age 6-10 yr) versus young adults and (2) the nasal deposition efficiency (NDE) of fine particles (1 and 2 microm MMAD, GSD < 1.2) under resting and light exercise breathing conditions in the same children and adults. Nasal contribution to breathing was assessed by respiratory inductance plethysmography and a nasal mask with flow meter during incremental exercise on a bicycle ergometer. Fine particle deposition fractions for nasal and oral breathing were assessed by inhalation of monodisperse carnauba wax particles and laser photometry to determine inhaled/exhaled concentrations. There was a trend for children to have a lesser nasal contribution to breathing at rest and during exercise, but the differences from adults were not statistically significant. Children did, however, have significantly decreased NDE for 2-microm particles under light exercise breathing conditions compared to adults, suggesting less efficient nasal filtering for larger particles and higher flow conditions. These results suggest that the lungs of children may be exposed to higher concentrations of inhaled, ambient particles than adults.


Children's response to air pollutants.

Children's response to air pollutants. It is important to focus on children with respect to air pollution because (1) their lungs are not completely developed, (2) they can have greater exposures than adults, and (3) those exposures can deliver higher doses of different composition that may remain in the lung for greater duration. The undeveloped lung is more vulnerable to assault and less able to fully repair itself when injury disrupts morphogenesis. Children spend more time outside, where concentrations of combustion-generated air pollution are generally higher. Children have higher baseline ventilation rates and are more physically active than adults, thus exposing their lungs to more air pollution. Nasal breathing in adults reduces some pollution concentrations, but children are more typically mouth-breathers--suggesting that the composition of the exposure mixture at the alveolar level may be different. Finally, higher ventilation rates and mouth-breathing may pull air pollutants deeper into children's lungs, thereby making clearance slower and more difficult. Children also have immature immune systems, which plays a significant role in asthma. The observed consequences of early life exposure to adverse levels of air pollutants include diminished lung function and increased susceptibility to acute respiratory illness and asthma. Exposure to diesel exhaust, in particular, is an area of concern for multiple endpoints, and deserves further research.


Mechanistic considerations for formaldehyde-induced bronchoconstriction involving S-nitrosoglutathione reductase. Inhalation of formaldehyde vapor has long been suspected of producing airway pathophysiology such as asthma and hyperresponsivity, presumably via irritant mechanisms. Recent studies on asthma and airway biology implicate changes in nitric oxide (NO) disposition in the adverse effects of formaldehyde, principally because enzymatic reduction of the endogenous bronchodilator S-nitrosoglutathione (GSNO) is dependent upon GSNO reductase (formally designated as alcohol dehydrogenase-3, ADH3), which also serves as the primary enzyme for cellular detoxification of formaldehyde. Considering recent evidence that regulation of bronchodilators like GSNO might play a more important role in asthma than inflammation per se, formaldehyde also needs to be considered as influencing ADH3-mediated GSNO catabolism. This is due to changes in ADH3 cofactors and thiol redox state among several potential mechanisms. Data suggest that deregulation of GSNO turnover provides a plausible, enzymatically based mechanism by which formaldehyde might exacerbate asthma and induce bronchoconstriction.


Inducible overexpression of cingulin in stably transfected MDCK cells does not affect tight junction organization and gene expression. Cingulin is a component of the cytoplasmic domain of vertebrate tight junctions (TJ). Mutation or down-regulation of cingulin in cultured cells results in changes in gene expression. Some of these changes are dependent on RhoA, whose activity is regulated by GEF-H1, which is inactivated by binding to cingulin at junctions. To gain further insights on the function of cingulin through dominant-negative effects, we cloned and sequenced canine cingulin, and developed stable MDCK cell lines where either full-length cingulin, or head or rod+tail domains were inducibly overexpressed. Surprisingly, analysis of these clones by immunoblotting, microarray, immunofluorescence, measurement of transepithelial resistance, and cell density showed that the overexpression of either full-length cingulin or its domains does not significantly affect TJ protein levels, gene expression, RhoA activity, cell density, doubling time, and the organization and function of TJ. These results suggest that compensatory mechanisms prevent dominant-negative effects in this model system, and that modulation of cellular functions by cingulin occurs within physiological protein levels.


Membrane interactions of peptides representing the polybasic regions of three Rho GTPases are sensitive to the distribution of arginine and lysine residues. Rho GTPases are a multifunctional family of proteins that are localized at cellular membranes via an isoprenyl group covalently linked to a C-terminal cysteine. Close to this primary site of membrane anchoring there is often found an additional polybasic region (PBR), which plays a secondary role in membrane binding and targeting of the complex. Here, peptides derived from the PBRs of the Rho family proteins Rac1 (K(183)KRKRK), TCL (K(198)KKKKR) and Cdc42 (P(182)KKSRR) were prepared with hexalysine (K(6)) and hexaarginine (R(6)) to study their interactions with multilamellar vesicles of phosphatidylglycerol (DOPG) and headgroup-deuterated dimyristoylphosphatidylcholine (DMPC-d(4)) using (2)H and (31)P NMR. The membranes retained their lamellar architecture after peptide binding, but the (2)H NMR line shapes for DMPC-d(4) indicated that the bound peptides altered the orientation of the choline headgroups, consistent with a change in membrane surface charge. Rac1 and TCL peptides appeared to affect the headgroup orientation similarly to K(6), although the perturbations were weaker and unlike those induced by the Cdc42 peptide and R(6). Magic-angle spinning (31)P NMR spectra of the membranes showed significant and selective broadening of the peak for DMPC after addition of the peptides, with R(6) and the Cdc42 peptide having the greatest effect. The selective broadening may be a consequence of the lipids separating into short-lived domains enriched in peptide-bound DOPG and peptide-free DMPC. These results illustrate that a complex relationship exists between the sequence of PBRs and their behaviour at membrane surfaces, which may have implications for the cellular functions and localization of Rho GTPases.


DNA pre-condensation with an amino acid-based cationic amphiphile. A viable approach for liposome-based gene delivery. A study related to the development and characterization of a new gene delivery system was performed. The approach consists in both the pre-condensation of plasmid DNA with an arginine-based cationic surfactant, arginine-N-lauroyl amide dihydrochloride (ALA), which was found not to be toxic, and the incorporation of the blood protein transferrin (Tf) into the formulations. Two cationic liposome formulations were used, one composed of a mixture of dioleoyl trimethylammoniopropane and cholesterol (DOTAP:Chol) and the other a pH sensitive formulation constituted of DOTAP, Chol, Dioleoyl phosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS). Particles with different ALA/DNA and cationic lipid/DNA charge ratios were produced and a physicochemical characterization of the systems developed was performed. DNA conformational changes in the presence of ALA were studied by Circular Dichroism (CD) and the ALA binding to DNA was followed by surface tension measurements. Insight into the structure and morphology of the various ALA-complexes (complexes composed of ALA, DNA, Tf and liposomes) was obtained by cryogenic-Transmission Electron Microscopy (cryo-TEM) and the sizes of the ALA-complexes were determined through Photon Correlation Spectroscopy (PCS). We found that the transfection capacity of these systems is directly related with the presence of ALA and the lipidic composition. Complexes based on the pH sensitive liposome formulation present better transfection profiles. The correlation between the inner structure, density and size of the ALA-complexes and their biological activity is discussed. Overall, we demonstrate that the presence of ALA improves the transfection efficiency when conjugated with cationic liposome systems.


Palmitoylation participates in G protein coupled signal transduction by affecting its oligomerization. Much in vivo and in vitro evidence has shown that the alpha subunits of heterotrimeric GTP-binding proteins (G proteins) exist as oligomers in their base state and disaggregate when being activated. In this article, the influence of palmitoylation modification of Galpha(o) on its oligomerization was explored extensively. Galpha(o) protein was expressed and purified from Escherichia coli strain JM109 cotransformed with pQE60(Galpha(o)) and pBB131(N-myristoyltransferase). Non-denaturing gel electrophoresis analysis revealed that Galpha(o) existed to a small extent as monomers but mostly as oligomers including dimers, trimers, tetramers and pentamers which could disaggregate completely into monomers by GTPgammaS stimulation. Palmitoylated Galpha(o), on the other hand, only present as oligomers that were difficult to disaggregate into monomers. The effect of palmitoylation on oligomerization of Galpha(o) was further investigated by several other biochemical and biophysical methods including gel filtration chromatography, analytical ultracentrifugation and atomic force microscopy analysis. The results consistently demonstrated that palmitoylation facilitated oligomerization of the Galpha(o) protein. Autoradiography indicated that [(14)C]-palmitoylated Galpha(o) would in no case disaggregate into monomers after treatment with GTPgammaS. [(35)S]-GTPgammaS binding activity assay showed that palmitoylated Galpha(o) was saturated at only 7.8 nmol/mg compared to 21.8 nmol/mg for non-palmitoylated Galpha(o). Fluorescent quenching studies using BODIPY FL-GTPgammaS as a probe showed that the conformation of GTP-binding domain of Galpha(o) tended to become more compact after palmitoylation. These results implied that palmitoylation may regulate the GDP/GTP exchange of Galpha(o) by influencing the oligomerization state of Galpha(o) and thereby modulate the on-off switch of the G protein in G protein-coupled signal transduction.


Aminopeptidase N (APN)/CD13-dependent CXCR4 downregulation is associated with diminished cell migration, proliferation and invasion. Aminopeptidase N (APN/CD13) is a 150 kDa membrane-bound ubiquitously expressed protease with a broad functional repertoire. It hydrolyzes small peptide mediators, modulates cell motility and adhesion to extracellular matrix and also acts as a viral receptor. In order to dissect the function of enzymatically active and inactive APN/CD13, substitutions of different enzymatic active amino acid residues were generated by site-directed mutagenesis and stably transfected into human embryonic kidney cells. All APN variants analyzed exhibited a complete loss of enzymatic activity, whereas wild type APN transfectants exerted a strong aminopeptidase-specific activity. Furthermore, wild type APN expression was associated with a significant decrease in proliferation, migration and also reduced anchorage-independent growth when compared to enzymatically inactive APN variants and controls. This appeared to be due to a downregulated mRNA and protein expression of the chemokine receptor CXCR4 and an inhibition of the stromal cell-derived factor (SDF)-1alpha/CXCL12-mediated migration. Thus, high APN enzyme activity may antagonize the cellular properties regulated by the CXCR4/SDF-1alpha system in embryonic kidney cells.