Urinary C-peptide analysis in an intervention study: experience from the DEFEND-2 otelixizumab trial.

AIMS: To demonstrate that analysis of urinary C-peptide across multiple study sites in the context of an intervention trial (DEFEND-2) is a viable alternative to mixed meal testing and delivers results that correlate to mixed meal testing estimation of endogenous insulin production. METHODS: Second morning void urine was collected for analysis and was available from 161 subjects at baseline (55 placebo, 106 otelixizumab), and 146 subjects (47 placebo, 99 otelixizumab) at month 12. Urinary C-peptide concentration was corrected for urinary creatinine [urinary C-peptide/creatinine ratio (UCPCR)] and serum C-peptide from the mixed meal tolerance test was calculated using area under the plasma concentration-time curve (AUC) normalized over 120 min. The correlation between mixed meal stimulated C-peptide AUC (mmol/l/min) and UCPCR (nmol/mmol), as well as the correlation between insulin use (IU/kg), and HbA1c (%) with UCPCR, was determined. RESULTS: UCPCR and mixed meal testing C-peptide AUC were correlated, with a correlation coefficient of 0.4172. UCPCR was not correlated with exogenous insulin use (r = -0.089) or with HbA1c (r = -0.032). CONCLUSIONS: Urinary C-peptide estimation should be considered as a measure of endogenous insulin production in future Type 1 diabetes mellitus outcome trials. A change in the timing for urine collection (to 120 min post standard meal) may provide a tighter correlation to C-peptide measured via a traditional mixed meal test.

Understanding the spread of antibiotic resistant pathogens in hospitals: mathematical models as tools for control.

As microorganisms become more resistant to antimicrobial agents, effective infection control measures will become increasingly important. However, despite multiple studies on infection prevention, few data exist on the quantitative effects of the individual aspects of infection control strategies. The combination of epidemiologic surveillance, molecular genotyping, observational studies on compliance, and mathematical modeling may improve our ability to determine the quantitative effects of individual infection control measures. This may help to design more effective infection control programs. In this study, we review several of the models that have been published and speculate on the usefulness of mathematical modeling for improving the prevention of infection.

The cloning of human genes using cDNA phage display and small-molecule chemical probes.

The cloning of genes based on protein function has become a powerful tool for protein discovery and should play an important role in proteomics in general. We have recently reported a technique for the functional identification of protein targets by combining traditional affinity chromatography with cDNA phage display. This procedure, referred to as display cloning, directly couples biologically active natural products to the gene of their protein cellular target. We now report the cloning of a human gene, the domain of F1 ATP synthase, using a synthetic scaffold molecule which serves as a prototype for a diverse chemical library. The ability to select genes from cDNA libraries using probes from combinatorial libraries would greatly increase the number of small molecule/protein interactions that can be identified. This method might prove valuable in furthering our understanding of biology and its application toward drug development.

Synthesis of a new class of 5'-functionalized adenosines using a rh(ii)-catalyzed 1,3-dipolar cycloaddition.

[reaction: see text] Chemically protected adenosine was functionalized at the 5' position to generate novel dipolarophiles and mesoionic dipoles. These species were found to undergo facile 1,3-dipolar cycloaddition to afford a new series of adenosine derivatives that contain a point of diversification at the 5' position of adenosine.

Combinatorial chemistry in cancer drug development.

The field of combinatorial chemistry has grown at an enormous rate in recent years, both in response to high-throughput capabilities and the discovery of a plethora of novel therapeutic targets. This review attempts to outlinethe recent developments of combinatorial chemistry in the search for novel cancer-related therapeutic agents.

Subcutaneous human menopausal gonadotropin administration for controlled ovarian hyperstimulation with intrauterine insemination cycles.

OBJECTIVE: This study was undertaken to determine the feasibility of administering human menopausal gonadotropin subcutaneously for controlled ovarian hyperstimulation with intrauterine insemination. STUDY DESIGN: This was a prospective nonrandomized matched-group comparison. Study patients (n = 25) undergoing controlled ovarian hyperstimulation with intrauterine insemination infertility treatment between June 1998 and March 1999 self-administered human menopausal gonadotropin subcutaneously for ovulation induction. Cycles (n = 39) were analyzed for duration of human menopausal gonadotropin treatment, total number of ampules of human menopausal gonadotropin used, peak serum estradiol level, number of mature follicles (> or =15 mm), cycle fecundity, and acceptability of the subcutaneous route of human menopausal gonadotropin administration. Age-matched historical control subjects who followed the same protocol except for the route of human menopausal gonadotropin administration, which was instead intramuscular, were used for comparison. RESULTS: Study and control cycles did not differ with respect to duration of human menopausal gonadotropin treatment (7.49 vs 8.18 d), total number of ampules of human menopausal gonadotropin used (17.44 vs 19.55), peak serum estradiol level (881 vs 769 pg/mL), number of mature follicles (>/=15 mm; 3.39 vs 2.92), or cycle fecundity rate (15.4% vs 17.9%). Two study patients were switched from subcutaneous to intramuscular administration because of minor local injection site reactions. CONCLUSION: Subcutaneous human menopausal gonadotropin administration for controlled ovarian hyperstimulation with intrauterine insemination treatment cycles was generally well tolerated and yielded stimulation parameters and pregnancy rates similar to those associated with the intramuscular route. Patients subjectively preferred subcutaneous human menopausal gonadotropin administration because of the ability to self-administer the injections, the use of a smaller injection needle, and reduced muscular pain at the injection site.

Phototherapeutic keratectomy for treatment of recurrent corneal erosion.

PURPOSE: To study the role of phototherapeutic keratectomy (PTK) in the management of recurrent corneal erosion (RCE) refractory to other forms of treatment. SETTING: The Eye Department of Leicester Royal Infirmary, a tertiary referral center, Leicester, England. METHOD: A retrospective analysis of all patients who had PTK for refractory RCE between July 1994 and October 1998 was performed. The patients were recalled to determine whether they had further symptoms and whether there had been a change in their refractive error or their best corrected visual acuity. RESULTS: Seventy-seven eyes of 68 patients were treated and divided into 3 etiologic groups: trauma, 40 eyes; corneal dystrophy, 24 eyes; and no established cause (idiopathic), 13 eyes. Phototherapeutic keratectomy was combined with photorefractive keratectomy (PRK) in 6 eyes with a good result. A single treatment was performed in 71 eyes (92.2%). No significant refractive change occurred in 67.5% of eyes; 22.1% developed hyperopia (range 0 to +2.0 diopters [D]), and 10.3% developed myopia (range 0 to -1.5 D). Best corrected visual acuity was unchanged in 72.7% of eyes; 11.7% lost 1 Snellen line and 15.5% gained 1 line. There were no symptoms in 68.8% of eyes; in 31.2%, minor symptoms were noted in the morning, and these patients continued to use ocular lubricants at night. CONCLUSION: Phototherapeutic keratectomy is a safe and effective treatment for refractory RCE and, where appropriate, can be combined with PRK.

Display cloning: functional identification of natural product receptors using cDNA-phage display.

BACKGROUND: The identification of cellular targets has traditionally been the starting point for natural product mode of action studies and has led to the understanding of many biological processes. Conventional experimental approaches have depended on cell-based screening and/or affinity chromatography. Although both of these techniques aid in the discovery of protein cellular targets, a method that couples protein identification with gene isolation would be extremely valuable. RESULTS: A procedure for the direct cloning of cellular proteins, based on their affinity for natural products, using cDNA phage display has been developed. The technique is referred to as display cloning because it involves the cloning of proteins displayed on the surface of a bacteriophage particle. The approach has been established by isolating a full-length gene clone of FKBP12 (FK506-binding protein) from a human brain cDNA library using a biotinylated FK506 probe molecule. During the affinity selection, the FKBP12 gene emerged as the dominant library member and was the only sequence identified after the second round of selection. CONCLUSIONS: The development of display cloning greatly facilitates the investigation of ligand-receptor interaction biology and natural product mode of action studies. This procedure utilizes heterologous protein display on infectious phage, which allows the amplification and repeated selection of putative sequences, leading to unambiguous target identification. In addition, the direct connection of a functional protein to its gene sequence eliminates the subsequent cloning step required with tissue homogenate or cell lysate affinity methods, allowing direct isolation of an expressible gene sequence.

Studies of antibiotic resistance within the patient, hospitals and the community using simple mathematical models.

The emergence of antibiotic resistance in a wide variety of important pathogens of humans presents a worldwide threat to public health. This paper describes recent work on the use of mathematical models of the emergence and spread of resistance bacteria, on scales ranging from within the patient, in hospitals and within communities of people. Model development starts within the treated patient, and pharmacokinetic and pharmacodynamic principles are melded within a framework that mirrors the interaction between bacterial population growth, drug treatment and the immunological responses targeted at the pathogen. The model helps identify areas in which more precise information is needed, particularly in the context of how drugs influence pathogen birth and death rates (pharmacodynamics). The next area addressed is the spread of multiply drug-resistant bacteria in hospital settings. Models of the transmission dynamics of the pathogen provide a framework for assessing the relative merits of different forms of intervention, and provide criteria for control or eradication. The model is applied to the spread of vancomycin-resistant enterococci in an intensive care setting. This model framework is generalized to consider the spread of resistant organisms between hospitals. The model framework allows for heterogeneity in hospital size and highlights the importance of large hospitals in the maintenance of resistant organisms within a defined country. The spread of methicillin resistant Staphylococcus aureus (MRSA) in England and Wales provides a template for model construction and analysis. The final section addresses the emergence and spread of resistant organisms in communities of people and the dependence on the intensity of selection as measured by the volume or rate of drug use. Model output is fitted to data for Finland and Iceland and conclusions drawn concerning the key factors determining the rate of spread and decay once drug pressure is relaxed.

Vancomycin-resistant enterococci in intensive-care hospital settings: transmission dynamics, persistence, and the impact of infection control programs.

Vancomycin-resistant enterococci (VRE) recently have emerged as a nosocomial pathogen especially in intensive-care units (ICUs) worldwide. Transmission via the hands of health-care workers is an important determinant of spread and persistence in a VRE-endemic ICU. We describe the transmission of nosocomial pathogens by using a micro-epidemiological framework based on the transmission dynamics of vector-borne diseases. By using the concept of a basic reproductive number, R0, defined as the average number of secondary cases generated by one primary case, we show quantitatively how infection control measures such as hand washing, cohorting, and antibiotic restriction affect nosocomial cross-transmission. By using detailed molecular epidemiological surveillance and compliance monitoring, we found that the estimated basic reproductive number for VRE during a study at the Cook County Hospital, Chicago, was approximately 3-4 without infection control and 0.7 when infection control measures were included. The impact of infection control was to reduce the prevalence from a predicted 79% to an observed 36%. Hand washing and staff cohorting are the most powerful control measures although their efficacy depends on the magnitude of R0. Under the circumstances tested, endemicity of VRE was stabilized despite infection control measures, by the constant introduction of colonized patients. Multiple stochastic simulations of the model revealed excellent agreement with observed pattern. In conjunction with detailed microbiological surveillance, a mathematical framework provides a precise template to describe the colonization dynamics of VRE in ICUs and impact of infection control measures. Our analyses suggest that compliance for hand washing significantly in excess of reported levels, or the cohorting of nursing staff, are needed to prevent nosocomial transmission of VRE in endemic settings.

Onapristone suppresses prolactin production in explant cultures of leiomyoma.

OBJECTIVE: To assess the action of onapristone, a type I antiprogestin, on prolactin (PRL) production by explant cultures of leiomyoma and myometrium. DESIGN: Explant cultures of myometrium and leiomyomas from 3 premenopausal women undergoing hysterectomy in the proliferative phase of the menstrual cycle. MAIN OUTCOME MEASURES: PRL secretion measured by radioimmunoassay. RESULTS: PRL secretion was decreased in leiomyomas by onapristone. There was no effect in the myometrium. There was no additional effect with the addition of the type II antiprogestin mifepristone (RU 486). CONCLUSION: PRL production is suppressed in leiomyomas but not in myometrium after treatment with onapristone in vitro. This suppression may serve as a marker for the clinical effectiveness of agents used in the treatment of leiomyomas.

Transmission dynamics of epidemic methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci in England and Wales.

A simple epidemiological framework for the analysis of the transmission dynamics of hospital outbreaks of epidemic methicillin-resistant Staphylococcus aureus (EMRSA) and vancomycin-resistant enterococci (VRE) in hospitals in England and Wales is presented. Epidemic strains EMRSA-15 and EMRSA-16 are becoming endemic in hospitals in the United Kingdom, and theory predicts that EMRSA-15 and EMRSA-16 will reach respective endemic levels of 158 (95% confidence interval [CI], 143-173) and 116 (95% CI, 109-123) affected hospitals with stochastic fluctuations of up to 30 hospitals in each case. An epidemic of VRE is still at an early stage, and the incidence of hospitals newly affected by VRE is growing exponentially at a rate r=0.51/year (95% CI, 0.48-0.54). The likely impact of introducing surveillance policies if action is taken sufficiently early is estimated. Finally, the role of heterogeneity in hospital size is considered: "Super-spreader hospitals" may increase transmission by 40%-132% above the expected mean.

The relationship between the volume of antimicrobial consumption in human communities and the frequency of resistance.

The threat to human health posed by antibiotic resistance is of growing concern. Many commensal and pathogenic organisms have developed resistance to well established and newer antibiotics. The major selection pressure driving changes in the frequency of antibiotic resistance is the volume of drug use. However, establishing a quantitative relationship between the frequency of resistance and volume of drug use has proved difficult. Using population genetic methods and epidemiological observations, we report an analysis of the influence of the selective pressure imposed by the volume of drug use on temporal changes in resistance. Analytical expressions are derived to delineate key relationships between resistance and drug consumption. The analyses indicate that the time scale for emergence of resistance under a constant selective pressure is typically much shorter than the decay time after cessation or decline in the volume of drug use and that significant reductions in resistance require equally significant reductions in drug consumption. These results highlight the need for early intervention once resistance is detected.

Recurrent symptoms following traumatic corneal abrasion: prevalence, severity, and the effect of a simple regimen of prophylaxis.

PURPOSE: (i) To describe the course of ocular symptoms and recurrent corneal erosion (RCE) following traumatic corneal abrasion (TCA). (ii) To assess the efficacy of a regimen of nightly eye ointment in preventing symptoms and RCE. METHODS: Patients presenting with TCA were treated with g. cyclopentolate 1% stat. and oc. chloramphenicol q.d.s. for 5 days. Eye pads were not used. For injuries caused by a fingernail, patients were randomised to receive either this 'standard regimen' alone, or to follow with a lubricating ointment (Lacrilube) nocte for 2 months. Follow-up was by telephone, using a symptom-based questionnaire. Recurrent symptoms were graded as: (i) none or minimal, (ii) mild, (iii) moderate (difficulty with some activities, or sought further opinion) and (iv) disabling (confirmed macroform RCE). After 2 years, case-notes were reviewed. The study is continuing, with further telephone follow-up taking place. RESULTS: Three-month follow-up was completed for 42 'fingernail' injuries and 32 'non-fingernail' TCAs. When treated with our standard regimen, 'mild' symptoms were reported in 10% of 'fingernail' and 10% of 'non-fingernail' injuries. Symptoms were 'moderate' in 0 and 12% respectively. Chisquared test confirmed a significantly higher prevalence of recurrent symptoms in the 'additional nightly ointment' group of 'fingernail' injuries (p = 0.016). Two macroform RCEs were confirmed by 2 years: one from each treatment group of 'fingernail' injury. CONCLUSIONS: When TCA is managed as above, there is a high prevalence of recurrent symptoms in the following 3 months. Additional nightly ointment appears to worsen prognosis. Macroform RCE is not common in the 2 years following TCA.

Vancomycin-resistant enterococci in intensive care hospital settings.

Vancomycin-resistant enterococci (VRE) have recently emerged as a nosocomial pathogen and present an increasing threat to the treatment of severely ill patients in intensive-care hospital settings. We outline results of a study of the epidemiology of VRE transmission in ICUs and define a reproductive number R0; the number of secondary colonization cases induced by a single VRE-colonized patient in a VRE-free ICU, for VRE transmission. For VRE to become endemic requires R0 > 1. We estimate that in the absence of infection control measures R0 lies in the range 3-4 in defined ICU settings. Once infection control measures are included R0 = 0.6, suggesting that admission of VRE-colonized patients can stabilize endemic VRE.

The dynamics of drug action on the within-host population growth of infectious agents: melding pharmacokinetics with pathogen population dynamics.

The use of simple mathematical models to study the kinetics of drug action and decay within vertebrate hosts has a long history with a major objective being to derive drug dosage regimens that optimize efficacy and minimize toxicity to the patient. Mathematical models of the relationship between dosage, route of delivery, drug concentration in defined sites and effect on a particular pathogen are widely used in the pharmacological literature. A more recent literature is that concerned with the population dynamics of pathogen replication within the host subjected to pressures exerted by the human immune system. In this paper we develop a theoretical framework to meld both approaches with the aim of identifying threshold criteria that dictate the optimum pattern of drug administration for pathogen clearance from the host. In particular we show how the percentage reduction in microparasite abundance is related to the pharmacokinetic parameter, AUC, recording the area under the drug concentration-time curve within the treated patient, in terms of the parameters that define the population dynamics of the pathogen and the properties of the drug. Two particular pathogens are examined to illustrate the principles underpinning the dynamics of the pharmacokinetic-population dynamic models, namely HIV and Plasmodium falciparum. Criteria for pathogen persistence or elimination are derived for these specific models based on the definition of a basic reproductive number, R0, which measures the average number of secondary infected target cells in a host generated by a single infected cell (CD4 lymphocyte for HIV, and erythrocyte for P. falciparum) within a population of susceptible cells. For the pathogen to invade the host and persist over time, R0

A metathetical cycloaddition-cycloreversion approach to the formation of furan scaffold libraries.

A general cycloaddition-cycloreversion metathesis procedure for the selective formation of a furan-based template-directed scaffold is described. In addition, features relative to library construction, such as the chemoselective nature of dipole formation, are discussed. Through the investigation of the temperature sensitive cleavage step, the furan synthesis was found to be accelerated by aqueous medium at physiological temperature leading to pure product from the solid-phase under biologically relevant conditions. The chemoselective nature of the rhodium(II) mediated cycloaddition allowed the selective formation of a key dipole intermediate, in the presence of a number of carbeneactive functional groups, to facilitate the split-pool combinatorial synthesis of a small library of compounds.