Monoclonal Antibodies Opsonize Burkholderia spp. and Reduce Intracellular Actin Tail Formation in a Macrophage Infection Assay.

Melioidosis is a neglected tropical disease caused by the bacterium Burkholderia pseudomallei. The bacterium is intrinsically resistant to various antibiotics, and melioidosis is therefore difficult to treat successfully without a relapse in infection. B. pseudomallei is an intracellular pathogen and therefore, to eradicate the infection, antimicrobials must be able to access bacteria in an intracellular niche. This study assessed the ability of a panel of monoclonal antibodies (MAbs) to opsonize Burkholderia species and determine the effect that each antibody has on bacterial virulence in vitro. Murine macrophage infection assays demonstrated that monoclonal antibodies against the capsule of B. pseudomallei are opsonizing. Furthermore, one of these monoclonal antibodies reduced bacterial actin tail formation in our in vitro assays, indicating that antibodies could reduce the intracellular spread of Burkholderia thailandensis. The data presented in this paper demonstrate that monoclonal antibodies are opsonizing and can decrease bacterial actin tail formation, thus decreasing their intracellular spread. These data have informed selection of an antibody for development of an antibody-antibiotic conjugate (AAC) for melioidosis. IMPORTANCE Melioidosis is difficult to treat successfully due to the causal bacterium being resistant to many classes of antibiotics, therefore limiting available therapeutic options. New and improved therapies are urgently required to treat this disease. Here, we have investigated the potential of monoclonal antibodies to target this intracellular pathogen. We have demonstrated that monoclonal antibodies can target the bacterium, increase uptake into macrophages, and reduce actin tail formation required by the bacterium for spread between cells. Through targeting the bacterium with antibodies, we hope to disarm the pathogen, reducing the spread of infection. Ultimately, we aim to use an opsonizing antibody to deliver antibiotics intracellularly by developing an antibody-antibiotic conjugate therapeutic for melioidosis.

Developing the public health intelligence workforce in the UK.

Recent UK health policies have consistently stressed the importance of basing local action on evidence and local intelligence. A suitably skilled workforce is required to achieve this. In recent years, a new cadre of skilled public health intelligence practitioners has emerged in the UK. This paper describes some of the steps taken by public health observatories and other organizations to grow and train this new workforce, and looks ahead to future developments building on the progress to date.

Radical surgery with omental flap for uncontrolled locally recurrent breast cancer.

BACKGROUND: Uncontrolled chest wall disease due to breast cancer is a highly morbid condition causing pain, ulceration, malodour and the need for frequent dressings. Aggressive surgical approaches are rarely justified because most patients will succumb to metastatic breast cancer within a short period. A highly selected group of patients with minimal or no evidence of metastatic disease and good performance status may benefit from radical chest wall surgery. Omental transposition flaps are ideal for reconstructing extensive surgical defects following chest wall surgery. METHODS: A retrospective review was carried out of 61 female patients treated consecutively between 1980 and 1995. The surgical technique is described herein. RESULTS: All patients were symptomatic preoperatively. Symptoms included ulceration (80%), pain (44%) and malodour (40%). Twenty-nine patients had uncontrolled local recurrence following initial treatment for locally advanced breast cancer and 32 patients developed uncontrolled recurrence after treatment for operable breast cancer by mastectomy or conservation surgery. Median survival following chest wall surgery was 21 months and the median local recurrence-free interval was 20 months. Morbidity was -limited. There were no cases of major flap loss. Twenty-nine patients (48%) had no further local disease. Eighteen patients (30%) developed soft-tissue recurrence at the edge of the omental flap or in surrounding skin and 14 (23%) developed recurrence beneath the flap. CONCLUSION: In a highly selected group of patients with symptomatic uncontrolled chest wall recurrence who are fit and have an expectation of at least moderate-term survival, radical chest wall surgery and omental flap transposition offers excellent palliation and local control in the majority of patients

Life expectancy as a summary of mortality in a population: statistical considerations and suitability for use by health authorities.

OBJECTIVE: To investigate the sampling distribution and usefulness of expectation of life in comparisons of mortality at health district level or below. DESIGN: Derivation of a formula for the variance of the expectation of life, confirmation of the result and generation of the sampling distribution by Monte Carlo simulation; comparison of expectation of life with standardised mortality ratio (SMR) and other summary indices of mortality. SETTING: A health district in Trent Region, England. SUBJECTS: Routinely available mortality statistics at electoral ward level and above. MAIN RESULTS: Given reasonable and simple assumptions the sampling distribution of the expectation of life is approximately normal. Expectation of life shows a high negative correlation with SMR even if the oldest age band for the SMR is open ended. CONCLUSIONS: Where sampling error is an issue, inference concerning differences in mortality rates between populations can be based on expectation of life, which is better for illustrative purposes than SMR. The formula for the variance of the expectation of life is more complex however. If the final age band is open ended, its lower bound should be as high as possible to avoid misleading results caused by hidden differences in age structure.

In-hospital delay in the diagnosis of breast cancer.

BACKGROUND: There is evidence that delay in the diagnosis of breast cancer may prejudice survival. The aim of this study was to determine the incidence, time trends and causes of delay in a dedicated breast clinic. METHODS: The interval between first breast clinic visit and a definitive diagnosis was recorded in all patients with invasive breast cancer between 1988 and 1997. In all patients with a delay of 3 months or more, the case notes were reviewed for evidence of a triple assessment (clinical examination, imaging and needle biopsy). The principal cause of delay was identified. RESULTS: Of 1004 patients with invasive breast cancer, there was a delay in diagnosis of 3 months or more in 42 patients between 1988 and 1997, an incidence of 4.2 per cent. The median delay was 6 months and the median age at diagnosis was 53 (range 27-89) years. Triple assessment was undertaken in 30 patients; ten did not have a needle biopsy performed and three patients had no mammography. The principal cause of delay was: false-negative or inadequate fine-needle aspiration cytology (FNAC) in 19 patients, failure of follow-up in eight, clinical signs did not impress in five, FNAC not carried out in four, false-negative mammogram in three, failure of needle localization in two and one patient did not accept clinical advice. The annual incidence of delay in diagnosis did not change significantly over the 10-year interval. CONCLUSION: Triple assessment is not sufficiently sensitive to detect every breast cancer and a small incidence of diagnostic delay is therefore inevitable with current techniques.

Rectal washout eliminates exfoliated malignant cells.

PURPOSE: Irrigation of the rectal stump before anastomosis after resection for carcinoma is accepted colorectal surgical practice. However, not all surgeons perform this routinely, and it has never been established conclusively that irrigation of the rectal stump eliminates exfoliated malignant cells or even reduces local recurrence. The patients of a surgeon whose standard surgical practice involved rectal irrigation were compared with those of a surgeon who does not routinely practice rectal irrigation. METHOD: Ten patients were given rectal washout with 200 to 500 ml of normal saline introduced via a Foley catheter per rectum. Ten patients were not given rectal washout. In both groups the anastomosis was performed with a circular stapler, and the stapler and donuts were rinsed in 200 ml of normal saline. The saline was sent for cytologic examination and classified as malignant cells seen or no malignant cells seen. The cytopathologist was blinded to the washout status. RESULTS: Of the ten patients who had rectal washout performed, none had malignant cells seen. Of the ten patients who did not have rectal washout performed, eight had malignant cells seen in the cytology (P = 0.007; two-tailed Fisher's exact probability test). CONCLUSION: Rectal washout eliminates exfoliated malignant cells in the rectum in the vicinity of the anastomosis.

Rectal cancer following colectomy and ileorectal anastomosis for familial adenomatous polyposis.

BACKGROUND: Familial adenomatous polyposis (FAP) has historically been treated by colectomy and ileorectal anastomosis (IRA). Preservation of the rectum allows the subsequent development of cancer in the rectum. The risk of rectal cancer following ileorectal anastomosis in the Australian population has not been published to date. METHODS: An audit of the Familial Adenomatous Polyposis Registry of Western Australia was undertaken to assess patients who had undergone colectomy and ileorectal anastomosis. Fifty-five patients ranging in age from 13 to 65 years were studied. RESULTS: Seven patients (13%) developed cancer of the rectum with a median follow-up of 10 years (range: 1-31 years). Median interval to diagnosis of carcinoma of the rectum following colectomy and IRA was 10 years. All patients who developed cancer in the retained rectum had rectal polyps. Colon cancer was present in the initial colectomy specimen in 13 patients (of these, five patients developed rectal cancer). Flat polyps were noted in five patients. Four patients with flat polyps developed cancer of the rectum. CONCLUSIONS: Total colectomy and IRA should be considered as part 1 of a staged procedure in the patient with FAP. With the exception of the patient with no evidence of rectal polyps, completion proctectomy should be undertaken within 10 years of the initial colectomy.

Intratumoral chemotherapy with fluorouracil/epinephrine injectable gel: a nonsurgical treatment of cutaneous squamous cell carcinoma.

BACKGROUND: We attempted to originate a nonsurgical treatment alternative for cutaneous squamous cell carcinoma (SCC), and we evaluated intratumoral modified-release chemotherapy with fluorouracil/epinephrine injectable gel (5-FU/epi gel). OBJECTIVE: To assess the safety and efficacy, we conducted an open-label pilot study of 5-FU/epi gel in 25 patients with biopsy-proven SCC lesions on the face, head, neck, trunk, arms, and hands. METHODS: Each tumor site was injected intradermally with up to 1.0 ml of 5-FU/epi gel. One SCC per patient was treated weekly for up to 6 weeks, then observed for 4 months at which time the tumor site and margins were excised for histologic examination. RESULTS: Overall, 96% (22 of 23) of evaluable treated tumors had histologically confirmed complete tumor clearing. No clinically significant systemic reactions or unexplained treatment-related adverse medical events occurred. The evaluations of the cosmetic appearance of the treated sites, judged subjectively by clinicians and patients, were mostly good to excellent and generally in close agreement. CONCLUSION: Treatment of superficial SCC with 5-FU/epi injectable gel results in a high rate of histologically confirmed complete tumor responses and may provide a nonsurgical treatment alternative in selected patients.

High dose loperamide suppositories: a novel approach for improving clinical function after restorative proctocolectomy.

The effect of loperamide suppositories on patients following restorative proctocolectomy was studied by means of a randomized, double-blind, crossover trial comparing active suppositories (20 mg b.d. x 1 week) with placebo. Ten patients (8 male, 2 female; 7 J pouch, 3 W pouch; 8 ulcerative colitis, 2 familial adenomatous polyposis) were studied 3-60 months (median, 31.5) after ileostomy closure. Ages ranged from 24 to 63 years (medium, 41.5). All patients kept a diary of their bowel habits and eight underwent a standardized test of pouch compliance. Urgency volume (UV) and maximum tolerable volumes (MTV) and the volume at onset of large isolated pouch contractions (LIC) were recorded. Statistical analysis was by the Wilcoxon test for paired data. Mean daily stool frequency during the placebo phase ranged from 3.7 to 7.8. It was reduced during the active phase in only seven patients (P > 0.1) but was reduced in all six patients whose placebo phase stool frequency was five or more. Urgency volume was increased by use of active suppositories in six of the eight patients tested (P > 0.01). There was no consistent effect on MTV. Large isolated pouch contractions were not seen in either test in one patient. In all of the remaining seven patients LIC were recorded after the placebo phase. After the active phase LIC first appeared at higher volumes in three but were not seen at all in four patients. High dose loperamide suppositories suppress pouch contractions and tend to lower stool frequency especially when high initially. They represent a novel therapeutic approach to high stool frequency in pouch patients.

A controlled evaluation of a fitness and nutrition intervention program on cardiovascular health in 10- to 12-year-old children.

BACKGROUND: Programs to improve cardiovascular health in schoolchildren need careful scientific evaluation. METHOD: In a randomized controlled trial of nutrition and fitness programs over a period of about 9 months, 1,147 10- to 12-year-olds from 30 schools were allocated to one of five health programs: fitness, fitness + school nutrition, school-based nutrition, school + home nutrition, home-based nutrition, or a control group. Nutrient intake, fitness, anthropometry, blood pressure, and blood cholesterol were measured before and after intervention. RESULTS: Fitness increased and diastolic blood pressure and triceps skinfolds decreased significantly for girls in the fitness groups. Baseline consumption of sugar, fat, and fiber was outside national guidelines; blood cholesterol exceeded recommendations in one-third of children. In girls, fat intake decreased significantly in the two home nutrition groups and fiber intake increased in the school + home nutrition and fitness groups. Boys in the fitness, fitness + school nutrition, and school + home nutrition group reduced sugar intake. Change in sugar intake correlated negatively with change in fat intake in both boys and girls. CONCLUSIONS: Teacher-implemented health packages are feasible with minimal training but programs should differ between boys and girls. Fitness programs were more successful than nutrition education particularly in girls. Clearer nutrition messages should prevent reciprocal changes in sugar and fat. For girls, the 3 mm Hg reduction of diastolic blood pressure, less obesity, and increased fitness could translate into a substantial reduction in cardiovascular risk in adult life.

The common molecular genetic alterations in Dukes' B and C colorectal carcinomas are not short-term prognostic indicators of survival.

Our study was undertaken to determine the prognostic significance of several common genetic alterations observed in colorectal carcinomas. We have previously analysed loss of heterozygosity of the MCC, APC, p53 and DCC tumour suppressor gene loci as well as p53 gene mutations and protein over-expression in a series of 100 Dukes' stage B and C colorectal tumours obtained at surgery. To extend our observations of alterations that may occur in these tumours, mutations to the c-Ki-ras oncogene and APC tumour suppressor gene were detected by PCR single-strand conformation polymorphism analysis. Short-term follow-up revealed no significant association between overall patient survival and any single, or combination of, genetic alteration(s). Surprisingly, patients whose tumours showed evidence of p53 protein over-expression/accumulation by immunocytochemistry (ICC) had a significantly better prognosis (p = 0.039) than those whose tumours had no p53 ICC reactivity.

Blood pressure and body composition in children: importance of allowing for cuff size.

A cross-sectional study of blood pressure (BP) was conducted in a sample of 1312 Australian school children aged 11-12 yrs. In line with current recommendations for the measurement of BP, a cuff of appropriate size was chosen for each subject based on his or her arm circumference. Analyses revealed statistically significant independent relationships between measured BP and cuff size. Observed relationships in the sample between measured BP and body composition variables were substantially affected when BP measurements were adjusted to remove the effect of cuff size. Cuff-associated biases in estimates of relationships between BP and body composition variables may still exist even though cuffs are carefully chosen to suit arms of different circumferences. It would appear that this potential problem is largely unrecognised. Some previous studies of relationships between BP and body composition may have been affected.

Relationships between blood pressure and measures of dietary energy intake, physical fitness, and physical activity in Australian children aged 11-12 years.

STUDY OBJECTIVE: The aim was to examine associations between blood pressure and dietary energy intake, physical activity, and physical fitness in Australian children. DESIGN: The study was a survey of year 7 children attending schools in metropolitan Perth. SETTING: Survey schools were located in suburbs representative of the range of socioeconomic strata in metropolitan Perth. PARTICIPANTS: Data were obtained on 1311 out of 2045 eligible children (64%). The sample included 681 boys and 630 girls. Mean age was 12.0 (SD 0.4) years. MEASUREMENTS AND MAIN RESULTS: Triplicate blood pressure measurements were obtained using a Dinamap oscillometric recorder. Dietary energy intakes were computed from two week day 24 h records. Physical activity was assessed using questionnaires. Physical fitness was measured using a shuttle run test. Additional measurements included weight, height, and skinfold thickness at four sites. A previous observation of an inverse relationship between diastolic blood pressure and dietary energy intake in boys was confirmed. There was evidence of an inverse relationship in girls but not in boys between blood pressure and physical activity. There was little evidence of relationships between blood pressure and physical fitness. CONCLUSIONS: Compared with weight and body mass index, dietary energy intake and the chosen measures of physical activity and physical fitness are poor predictors of blood pressure in the population studied.

Clinical experience with the second metatarsal artery neurovascular flap.

Three cases of innervated web space flaps from the foot based on the second metatarsal artery and the second common digital nerve are described. This flap provides glabrous skin for digital reconstruction with good sensory recovery and minimal donor site morbidity.

Reflotron cholesterol measurements in 1112 Australian children aged 10-12 years.

OBJECTIVE: To study the distribution of blood cholesterol concentration in a sample of Western Australian schoolchildren. DESIGN: The Reflotron dry-chemistry system (Boehringer Mannheim, Germany) was used to measure cholesterol in finger-prick blood samples obtained from Year 6 children aged 10-12 years attending government schools in metropolitan Perth. Recruitment methods ensured that children from different socioeconomic strata were represented. PARTICIPANTS: For the 1239 children enrolled in Year 6 in 30 government schools, parental consent to participate was obtained for 1144; of these cholesterol concentration was measured in 1112. RESULTS: The measurement method proved practicable and was generally well accepted. Cholesterol levels were somewhat lower than those obtained using a laboratory reference method and a bias, relative to national quality assurance standards, was estimated to be -0.32 mmol/L. Mean cholesterol level in boys (4.28 mmol/L, SD 0.69) was not significantly different from that in girls (4.35 mmol/L, SD 0.72). Allowing for measurement bias, 52% of boys and 57% of girls had cholesterol levels exceeding the National Heart Foundation's designated "desirable" level of 4.5 mmol/L for children. CONCLUSIONS: The distribution of cholesterol levels in Australian children compares unfavourably with distributions in children in countries with low rates of cardiovascular disease and indicates a need for widespread, appropriate diet and lifestyle changes.

Urinary catecholamines, plasma insulin and environmental factors in relation to body fat distribution.

The relationship of body fat distribution to insulin and the catecholamines, hormones that affect lipolysis differentially by fat site, was examined within an environmental context, including factors of medication use, physical activity, dietary intake, educational attainment, and age. Four cross-sectional body fat areas (cm2) were determined by three computed tomography (CT) scans (subcutaneous chest fat at the level of the nipples, subcutaneous and intra-abdominal fat at the level of the umbilicus, and subcutaneous left mid-thigh fat) in 191 second-generation Japanese-American men aged 45-74 years. The site-specific fat measurements were first examined in relation to use of beta-adrenergic antagonists, then to fasting plasma insulin and C-peptide levels and to urinary epinephrine and norepinephrine levels from a 24-h urine collection made during usual daily activities. Greater fat stores in the intra-abdominal area, even after adjustment for body mass index (BMI, weight/height2) and presence of coronary heart disease, were found to be related to use of beta-adrenergic antagonists. In men taking no adrenergic antagonists (n = 157), after adjustment for BMI, truncal fat measurements of the chest (partial r = -0.16, P less than 0.05) and intra-abdominal area (partial r = -0.21, P less than 0.05) were found to be inversely related to epinephrine, and intra-abdominal fat (partial r = 0.25, P less than 0.01) alone was directly related to fasting plasma insulin. With respect to other environmental variables, the significant inverse relationship of intra-abdominal fat (adjusted for BMI) with physical activity (partial r = -0.17, P less than 0.05) and the significant difference in intra-abdominal fat by educational attainment (college 102.3 +/- 5.7 vs no college 115.7 +/- 6.1 cm2, P = 0.03) became non-significant with adjustment, using multiple regression analysis, for insulin in the case of physical activity and epinephrine in the case of educational attainment. Thus, intra-abdominal fat showed a unique set of relationships to metabolic parameters which could be further related to certain environmental variables.

A plasmid cassette system that allows optimization of primers for use in the amplification refractory mutation system.

A plasmid "cassette" system is presented that allows the development of amplification refractory mutation system (ARMS) primers. Primer development may be accomplished even in the absence of appropriate clinical material from patients demonstrating mutated alleles. The use of such cassettes is demonstrated here in the assessment of primers prepared for ARMS analysis of the gene coding for the normal and Z variant alleles of the human serine protease inhibitor alpha-1-antitrypsin.

A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones.

The recent development of yeast artificial chromosome (YAC) vectors has provided a system for cloning fragments that are over ten times larger than those that can be cloned in more established systems. We have developed a method for the rapid isolation of terminal sequences from YAC clones. The YAC clone is digested with a range of restriction enzymes, a common linker is ligated to the DNA fragments and terminal sequences are amplified using a vector specific primer and a linker specific primer. Sequence data derived from these terminal specific products can be used to design primers for a further round of screening to isolate overlapping clones. The method also provides a convenient method of generating Sequence Tagged Sites for the mapping of complex genomes.