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.

Proximity versus allostery: the role of regulated protein dimerization in biology.

Regulated dimerization of proteins is increasingly understood to be important in many cellular processes, including signaling, transciption and protein degradation. Organic molecules that induce dimerization may offer as much potential to regulate biological processes as those that allosterically induce conformational change.

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.

The transmission dynamics of antibiotic-resistant bacteria: the relationship between resistance in commensal organisms and antibiotic consumption.

We propose a mathematical model of the transmission dynamics of colonization by commensal bacteria within a human community subject to varying levels of antibiotic use designed to control morbidity induced by pathogenic strains of the normally commensal organisms. Colonization is assumed not to induce morbidity in the majority of cases, and antibiotic use is assumed to be related to the arrival and growth of pathogenic strains that give rise to infections including clinical symptoms of disease. In the absence of antibiotic resistance, the model shows how the pattern of antibiotic prescription and use can eliminate the non-pathogenic commensal strains from the host community if the fraction of people taking antibiotics with a defined efficacy exceeds some critical level. The model is extended to take account of the evolution of antibiotic resistance in the commensal population. We assume resistance may be either plasmid-mediated or conferred by selection of low-level pre-existing mutants, and that resistant organisms may experience reduced reproductive fitness. Invasion of the host community by drug-resistant commensals is possible if certain antibiotic prescribing patterns pertain. We calculate these conditions in terms of the transmission parameter of the organism and the level of antibiotic prescription and use. The model is employed to address the issues of how best to use antibiotics in populations harbouring resistant organisms, and when resistant bacteria will out-compete sensitive strains.

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.

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.

RU486 suppresses prolactin production in explant cultures of leiomyoma and myometrium.

OBJECTIVE: To assess the action of RU486 (mifepristone), in the presence and absence of P, on PRL production by explant cultures of leiomyoma and myometrium. DESIGN: Explant cultures using tissue from nine premenopausal women undergoing hysterectomy in the proliferative phase of the menstrual cycle; immunohistochemical staining of tissue sections from five patients for P receptor (PR) subtype. MAIN OUTCOME MEASURES: Prolactin secretion (measured by RIA), lactate dehydrogenase secretion (measured by quantitative colorimetric assay), and immunohistochemistry for PR subtype. RESULTS: Prolactin secretion was decreased in leiomyomas by RU486 at concentrations of 10(-7) M and 10(-5)M when normal serum-containing medium was used. In experiments with all detectable P removed from serum, PRL secretion was suppressed in both leiomyomas and myometrium at an RU486 concentration of 10(-7)M. Immunohistochemistry results suggest that the A form of the PR is the dominant form in both leiomyomas and myometrium. CONCLUSIONS: Prolactin production is suppressed in both leiomyomas and myometrium after treatment with RU486 in vitro, and this suppression may serve as a marker for the clinical effectiveness of agents used in the treatment of leiomyomas.

PIP: Uterine leiomyomas are the most common benign neoplasms of the uterus. They appear to be estrogen dependent. The role of progestins in regulating leiomyoma growth is, however, less clear. Studies have suggested that RU486 may provide long-term medical treatment for women with leiomyomas. The authors investigated the mechanism of action of RU486 on uterine leiomyomas and myometrium, using prolactin production as a marker. Explant cultures using tissue from nine premenopausal women undergoing hysterectomy in the proliferative phase of the menstrual cycle were used, along with the immunohistochemical staining of tissue sections from five patients for P receptor (PR) subtype. Prolactin production was suppressed in both leiomyomas and myometrium after treatment with RU486 in vitro. This suppression may be a marker for the clinical effectiveness of agents used to treat leiomyomas.

Treatment of chalazions with injection of a steroid suspension.

A trial was conducted to compare the efficacy of the treatment of chalazions by injection of triamcinolone acetonide with conventional incision and curettage. Of the 39 injected cases 77% resolved completely, though 54% of the injected cases required a second injection. Of the 30 surgically treated cases 90% resolved, but 27% required a second operation. Injection of chalazions with a steroid suspension is a convenient and reasonably effective alternative to the standard surgical management of this common condition.

Treatment of radiation retinopathy.

A case of radiation induced retinopathy with neovascularisation arising from the optic disc and retina was treated with panretinal argon laser photocoagulation. Complete regression of the new vessels was observed within 2 weeks of therapy. The pathogenesis of neovascularisation in this condition is discussed.

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.

An analysis of referral routes and diagnostic accuracy in cases of suspected glaucoma.

Over a nine-month period two hundred and thirteen referrals to the Department of Ophthalmology of the Leicester Royal Infirmary, England, for suspicion of glaucoma were examined by a single ophthalmologist. Ninety-nine percent of referrals resulted from the findings at an optometric visit. Despite this, less than 32% were confirmed as having glaucoma and less than 23% had ocular hypertension. Twenty-nine percent showed no abnormality. Of those with glaucoma 19% showed advanced field loss in their worse eye. The guidelines relating to referral practices as a result of optometric examinations need to be reviewed and agreed upon by ophthalmologists, optometrists and general practitioners.

Carotid endarterectomy for cerebrovascular insufficiency: long-term results in 592 patients followed up to thirteen years.

Seven hundred forty-eight carotid endarterectomies were performed on 592 patients with cerebrovascular insufficiency during a 13-year period. Overall operative procedure mortality was 2.7%. In the last 6 years, using a shunt routinely and avoiding operation on acute strokes, mortality was 1.47%. In frank strokes it was 3.7%; in transient ischemia, 0.77%; and zero for chronic ischemia and asymptomatic bruits. Incidence of operation-related deficits among transient ischemia and asymptomatic bruit patients was 0.9% for transient weakness and 2% for permanent deficits. Of 172 long-term deaths, 23 were due to cerebral causes, or 3.9% of the entire series. Among frank stroke survivors, 30.2% are normal and 58.7% improved. In transient ischemia survivors 81% are normal and 15.7% improved. In 65 asymptomatic bruit patients operated upon electively, two had strokes during follow-up, one mild and one severe. Among 37 asymptomatic bruit control patients, 24 or 65% developed symptoms of transient ischemia or frank strokes. Of 118 totally occluded carotid arteries explored, flow was restored in 48 (40.7%) but could not be restored in 70 (59.3%). For cerebral protection during carotid endarterectomy the routine use of a temporary inlying bypass shunt with general anesthesia is advocated for all partial occlusions. Endarterectomy is most useful for transient ischemia and selected patients with mild frank strokes and asymptomatic bruits. Acute profound and rapidly progressing strokes should not be operated upon as an emergency, but allowed to stabilize for several weeks and then be considered for possible operation.

Primary hypothyroidism presenting as severe vaginal bleeding in a prepubertal girl.

We describe a case of acquired primary hypothyroidism in a prepubertal girl who presented with profound bradycardia, precocious puberty, and severe vaginal bleeding. A pelvic ultrasound revealed markedly enlarged ovaries with numerous cysts. Cystic aspiration, in concert with L-thyroxine therapy, appeared to halt vaginal bleeding, and led to resolution of cysts. This is the first reported case of percutaneous cyst aspiration to treat ovarian hyperstimulation caused by hypothyroidism in a child.

The effects of a program of systematic movement on COPD patients.

The intent of the study described in this article was to determine (a) if patients with chronic obstructive pulmonary disease (COPD) who regularly participate in less vigorous exercise programs, such as the program of systematic movement sponsored by the American Lung Association, differ from those who do not participate; and (b) whether these differences, if they exist, persist over time. Two convenience samples of COPD patients--those who practice systematic movement by participating in a pulmonary exercise program (PEP) and those who do not--were obtained through the American Lung Association of Maryland. Demographic information and health history were obtained from the participants and their physicians. They all completed the vertical visual analogue dyspnea scale (VADS) and the Beck Depression Inventory (BDI) on two occasions 8 weeks apart and described the strategies they used in dealing with their illness. Differences were found between the groups on the BDI and VADS at both measurement times. Those who participated in the program had lower depression (5.9 versus 17.75) and lower dyspnea (58.9 versus 78.8). These findings persisted 8 weeks later, with exercisers having depression levels of 5.5 versus 19 for the nonexercisers and dyspnea levels of 55.2 versus 79.4 for the nonexercisers. Exercisers used fewer emotion-focused coping strategies than nonexercisers. These findings have implications for the rehabilitation of COPD patients.