In vitro diagnostics in diabetes: meeting the challenge.

Diabetes is one of the leading causes of death and disability in the world. There is a large population in the world suffering from this disease, and the healthcare costs increase every year. It is a chronic disorder resulting from insulin deficiency and hyperglycemia and has a high risk of development of complications for the eyes, kidneys, peripheral nerves, heart, and blood vessels. Quick diagnosis and early prevention are critical for the control of the disease status. Traditional biosensors such as glucose meters and glycohemoglobin test kits are widely used in vitro for this purpose because they are the two major indicators directly involved in diabetes diagnosis and long-term management. The market size and huge demand for these tests make it a model disease to develop new approaches to biosensors. In this review, we briefly summarize the principles of biosensors, the current commercial devices available for glucose and glycohemoglobin measurements, and the recent work in the area of artificial receptors and the potential for the development of new devices for diabetes specifically connected with in vitro monitoring of glucose and glycohemoglobin HbA(1c).     

In vitro and in vivo validation of time domain velocity and flow measurement technique.

This study was undertaken to validate the time domain processing method for measuring (1) the peak velocity in comparison to pulsed-wave spectral Doppler findings in an in vitro system; (2) the volumetric flow in comparison to the actual flow measured by a graduated cylinder in an in vitro circulation; and (3) the volumetric flow in comparison to a transit time flowmeter in a permanently instrumented neonatal lamb model. A prototype implementation of time domain processing in a commercial ultrasound device was used. For velocimetry, both time domain processing and Doppler methods showed low variance, low intrarater variability (0.03 and 0.09%, respectively), high reliability coefficients (97% and 96%, respectively), and a significant correlation (r = 0.96; P < 0.001). For in vitro flow quantification, time domain processing and graduated cylinder methods showed low variance, low intrarater variability (0.09 and 0.01%, respectively), high reliability coefficients (99.60% and 99.96%, respectively), and a significant correlation (r = 0.98, P < 0.001). For in vivo flow quantification, time domain processing and transit time flowmeter showed a significant correlation (r = 0.96; P < 0.001). Within the limits of the in vitro and in vivo experimental conditions, this study proves the validity of the time domain processing sonographic technique for measuring peak flow velocity and volumetric flow.     

In vitro diagnostics in the development and use of cardiovascular medicines

The list of potential cardiovascular biomarkers has expanded dramatically in recent years; however, the number of regulatory agency-approved diagnostic tests that guide treatment has been relatively unchanged compared with this growth in the discovery of putative biomarkers. Surrogate biochemical endpoints such as LDL and HDL are included in the current guidelines of various regulatory agencies for the management of cardiovascular diseases, as a result of many years of research. Inclusion of tests for these markers, as well as any future tests, in treatment guidelines requires data obtained from large-scale clinical trials comparing these endpoints with 'hard' clinical endpoints, such as morbidity and mortality. Consequently, current guidelines are limited to conventional in vitro tests and incorporate few novel tests for guiding or modifying treatment. Despite the failure to include newer in vitro tests in cardiovascular treatment and prevention paradigms, ongoing biomarker discovery and assay optimization has provided many improvements in drug discovery and development, and has afforded opportunities for the optimized medical treatment of patients with cardiovascular disease.     

Whistleblowing: it's time to overcome the negative image

The media perpetuates the stereotype of the whistleblower forced to go outside his or her organization and risk his or her career to bring wrongdoing to public awareness. In fact, NHS trusts should all have a whistleblowing policy in place which encourages people to raise genuine concerns about wrongdoing, and offer protection to those who do this. This article aims to help health workers understand the whistleblowing process and so assist them in raising concerns within their organization.     

Microbubble formation: In vitro and in vivo observation

Injection of liquid through a catheter into the circulation is known to produce clouds of signals detected by sonography. Blood forced through a stenotic conduit produced sonographic clouding, and bubbles of 10–100 μm were observed by light microscopy. The microbubbles persisted up to three and a half minutes. Microbubbles were observed in the microcirculation of the rat by placing the catheter tip into the descending aorta of 15 animals, viewing the mesentery at 400X magnification, and recording the results on videotape. Following injection of the rats' own blood, numerous microbubbles lodged promptly at the arteriolar level and obstructed blood flow for up to 200 sec before shrinking sufficiently to pass downstream and allow restitution of flow.     

In vitro and in vivo Phlebovirus inhibition by ribavirin.

Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) was markedly inhibitory in vitro to Adames and Balliet strains of Punta Toro virus (PTV), a Phlebovirus related to Rift Valley fever and sandfly fever viruses. By using inhibition of viral cytopathic effect in LLC-MK2 cells with both virus strains, the 50% effective dose was 4 to 10 micrograms/ml and the virus rating was 1.3. The Adames strain of PTV infection in mice was established for evaluation of the in vivo antiviral efficacy of ribavirin. The drug was administered subcutaneously (s.c.) twice daily for 5 to 7 days beginning 4 h pre-virus inoculation, 24 h post-virus inoculation, or 36 h post-virus inoculation, with increased survivors, reduced hepatic icterus, reduction of serum glutamic oxalic acid transaminase and serum glutamic pyruvic acid transaminase, and inhibition of infectious virus from sera and livers of infected mice. The minimum effective dose was 4.7 mg/kg per day, with a maximum tolerated dose of 75 mg/kg per day. When the same treatment schedule beginning 4 h pre-virus inoculation, 4 h post-virus inoculation, or 24 h post-virus inoculation was used, orally administered ribavirin was effective at doses as low as 6.3 mg/kg per day. Single s.c. ribavirin treatments at doses of 175 to 700 mg/kg administered from 4 to 48 h post-virus inoculation were also effective. No effect was seen when ribavirin was administered s.c. to mice infected intracerebrally with the PTV strain Balliet, even though treatment was begun 36 h before virus exposure.     

In vitro and in vivo activities of verteporfin-loaded nanoparticles.

The main goal of this study was to develop a dispersed polymeric drug delivery system for verteporfin, suitable for intravenous administration and capable of improving its phototherapeutic index and minimizing the side effects. To achieve this objective, two types of verteporfin-loaded nanoparticles (167 and 370 nm in diameter) based on poly(D,L-lactide-co-glycolide) were prepared using the salting-out technique and were first tested on EMT-6 mammary tumor cells in comparison with an aqueous solution (DMSO/PBS). It was observed that small nanoparticles exhibited greater photocytotoxicity compared to large nanoparticles or DMSO/PBS, and the photocytotoxic efficiency was graded as small nanoparticles>DMSO/PBS>large nanoparticles. Furthermore, verteporfin, entrapped into small nanoparticles transferred to serum proteins more rapidly than when dissolved in DMSO/PBS. Drug clearance, measured by skin phototoxicity investigated in mice exposed to simulated sunlight 15 to 150 min after the injection of small nanoparticles was modest at early light exposure times with the small nanoparticles and diminished rapidly with later exposure times. Tumor bioassay results indicated that verteporfin incorporated into small nanoparticles effectively controlled tumor growth for 20 days in mice with early light irradiation times following drug administration.     

Luminescent nanoparticles and their use for in vitro and in vivo diagnostics

Fluorescence spectroscopy has been shown to be a useful tool for a broad variety of biological and medical applications. Many of the analytical methods, as used for tumor marker and gene mutation detection, recognition of pathogens or monitoring of cell-related processes, are based on the labeling of the investigating object with luminescent nanoparticles. Owing to their size, which is comparable to that of biomolecules, and to their extraordinary optical properties, luminescent nanoparticles could well improve the sensitivity and flexibility of current detection techniques. This article provides a general overview of the synthesis, properties and application of luminescent semiconductor, metal and inorganic nanoparticles for in vitro and in vivo diagnostics, also reflecting the aspect of biocompatibility.     

In vivo expression of in vitro anticoccidial activity.

Large-scale screening has led to the identification of several experimental compounds that have very potent intrinsic activity against coccidia, but the lack of translation to in vivo efficacy has been a major hurdle in developing such leads into effective new drugs. We developed methods to explore the impact of oral availability and appropriate distribution in tissue, both of which are potentially important factors in the expression of activity in vivo. For the compounds that we examined, neither oral absorption nor distribution to the site of infection appeared to be the critical barrier to in vivo expression of intrinsic anticoccidial activity. Elucidation of the nature of additional factors that might be involved could assist greatly in the identification of useful new anticoccidial agents.     

In vitro and in vivo ciprofloxacin pharmacokinetics in human neutrophils.

Early in vitro investigations have shown that ciprofloxacin is concentrated within human neutrophils (polymorphonuclear leukocytes [PMNs]) at between 3 and 11 times the extracellular concentration. The elution of ciprofloxacin from cells is relatively rapid when the extracellular concentration is reduced. In order to estimate the in vivo intracellular penetration of ciprofloxacin and to determine its intracellular pharmacokinetics, PMNs were recovered from blood samples drawn from healthy volunteers at different times during a 24-h period after they were given a 750-mg oral dose. High-performance liquid chromatographic determination of ciprofloxacin in serum and cells showed that the intracellular/serum ratio was 3.7 at 1.5 h (maximum concentration of drug in serum), 5.7 at 12 h, and 20 at 24 h. The area under the curve ratio was 3.73. The mean elimination half-lives of ciprofloxacin were 3.7 and 6.2 h in serum and PMNs, respectively. These data show that in vivo findings are in agreement with in vitro findings. The large uptake of ciprofloxacin by PMNs combined with a prolonged intracellular half-life described under the conditions of human therapy should provide the basis for the use of ciprofloxacin in infections caused by susceptible intracellular bacteria.     

In vitro and in vivo evaluation of an oral system for time and/or site-specific drug delivery.

Aim of this work was the evaluation of an oral system (Chronotopic) designed to achieve time and/or site-specific release. The system consists in a drug-containing core, coated by a hydrophilic swellable polymer which is responsible for a lag phase in the onset of release. In addition, through the application of an outer gastroresistant film, the variability in gastric emptying time can be overcome and a colon-specific release can be sought relying on the relative reproducibility of small intestinal transit time. For this study, cores containing antipyrine as the model drug were prepared by tableting and both the retarding and enteric coatings were applied in fluid bed. The release tests were carried out in a USP 24 paddle apparatus. The in vivo testing, performed on healthy volunteers, envisaged the HPLC determination of antipyrine salivary concentration and a gamma-scintigraphic investigation. The in vitro release curves presented a lag phase preceding drug release and the in vivo pharmacokinetic data showed a lag time prior to the detection of model drug in saliva. Both in vitro and in vivo lag times correlate well with the applied amount of the hydrophilic retarding polymer. The gamma-scintigraphic study pointed out that the break-up of the units occurred in the colon. The obtained results showed the capability of the system in delaying drug release for a programmable period of time and the possibility of exploiting such delay to attain colon-targeted delivery according to a time-dependent approach.     

蛋白质组学技术及其在临床检验诊断学中的应用

人类基因组计划的完成加速了蛋白质组学的发展,使之成为当今生命科学的热点之一。疾病的发生,往往伴随着蛋白质数量、结构和性质的异常,蛋白质组学技术的应用,为疾病的诊断提供了新手段。临床捡验诊断学的根本目的是将识别疾病并诊断和治疗的生物标识物,包括血液、尿液、脑脊液(CSF)、乳头抽吸液(NAF)等各种体液都被用于研究与某些疾病的关系。采集有价值的样本,应用合理的蛋白质组学研究策略,进行蛋白质分离、鉴定,有望发现新的有意义的标志物,从而有助于疾病的早期诊断。本文着重对蛋白质组学技术及其在临床检验诊断学中的应用作一综述。1蛋白质组学研究的新技术1.1分离Righetti等[1]报道了一种用固相配体库(solid-phase ligandlibraries)缩小血浆蛋白丰度动态范围的样本处理方法。该配体库中的配体数目众多(约10亿),理论上血浆中的每一个蛋白都能在库中找到其相应的配体,并与之特异的结合。首先,固相配体先与样品血清孵育,高丰度蛋白很快就达到饱和,未结合部分被洗掉;由于各种低、高丰度蛋白配体数目相近,这样,保留下来的配体-蛋白数目也相近,从而达到稀释高丰度蛋白和浓缩低丰度蛋白的目的。同一条件下,未用固相配体...