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PubMed1973

Miniature cell formation in Chlamydia psittaci.

In an electron microscope study on the developmental cycle of the goat pneumonitis strain of Chlamydia psittaci in L cells, it was observed that miniature reticulate bodies, measuring approximately 0.2 mum in diameter and surrounded by double unit membranes, were produced infrequently from normal-sized reticulate bodies through a "budding"-like process. As little as 0.1 mug of penicillin per ml greatly increased the frequency of the miniature reticulate body formation.

Animals↗
PubMed2004

Miniature tissue microarrays for HercepTest standardisation and analysis.

AIMS: To assess the practicality of using a miniature tissue microarray (TMA) with several examples of each HercepTest score from 0 to 3+ as a control for routine HercepTest immunohistochemistry. METHODS: A TMA was constructed from in house cases of breast cancer where HercepTest on the whole sections showed scores 0, 1+, 2+, or 3+. The TMA, which measured 5 x 5 mm, was designed with four rows (each representing scores 0, 1+, 2+, and 3+), with five 0.6 mm cores from separate cases. In all, 20 individual cases were represented and the TMA took less than one hour to construct. Fifty sequential 4 microm sections were cut from the TMA to maximise the number of available sections. They were stored at 4 degrees C for 1-270 days and when a case needed HercepTest staining the section was added to the TMA tissue control slide. RESULTS: All slides contained tissue spots and immunohistochemical staining was consistent throughout the time period. CONCLUSIONS: The miniature TMA with examples of all HercepTest scores described here is an ideal tissue control and can be used as a visual reference for scoring a case. Slides stored at 4 degrees C could be used for up to 270 days.

Breast Neoplasms↗
PubMed1992

The public health management of tuberculosis among the single homeless: is mass miniature x ray screening effective?

STUDY OBJECTIVE: The aim was to test the assumption that mass miniature x ray screening of the single homeless (hostel residents) is a cost-effective means of controlling pulmonary tuberculosis. DESIGN: The study was a prospective experimental screening exercise to identify new cases of active tuberculosis completing treatment. SETTING: The setting was eight hostels in south London. A mobile x ray screening facility was set up outside the hostels. SUBJECTS: Subjects were 547 single homeless residents in the hostels. They were encouraged to attend for chest x ray, and for active follow up of abnormal x rays. MAIN RESULTS: No new cases of active tuberculosis were found. CONCLUSIONS: Mass miniature x ray is ineffective in controlling tuberculosis because of its unacceptability and increasing inaccessibility to this population.

Adolescent↗
PubMed2001

Scapulohumeral osteoarthritis in 20 Shetland ponies, miniature horses and falabella ponies.

This paper describes the clinical and diagnostic features of 20 cases of scapulohumeral osteoarthritis in Shetland ponies, miniature horses and falabella ponies. The history and clinical signs were similar in all the cases Radiographically they all had consistent changes which consisted predominantly of articular osteophytes and periarticular enthesiophytes. Six of the cases had radiographic evidence of dysplasia of the scapulohumeral joint, although it was uncertain whether this was a primary or a secondary finding. No specific treatment appeared to be advantageous. At follow up, six of the ponies had to be euthanased owing to continuing severe lameness; the other 14 ponies remained lame, but were maintained at pasture by the occasional use of oral non-steroidal anti-inflammatory drugs. No definitive aetiology for the condition was identified, but it is proposed that an underlying dysplasia, or lack of collateral support may predispose the scapulohumeral joint of miniature horse breeds to the disease.

Animals↗
PubMed1976

A miniaturized counting technique for anaerobic bacteria.

A miniaturized counting technique gave results as good as the pour-plate and Most Probable Number (MPN) techniques for enumeration of clostridia spp. and anaerobic isolates from the gut. Highest counts were obtained when ascorbic acid (1%) and dithiothreitol (0.015%) were added to the reinforced clostridial medium used for counting. This minimized the effect of exposure to air before incubation. The miniature technique allowed up to 40 samples to be plated and incubated in one McIntosh-Filde's-type anaerobic jar, compared with 3 or 4 by the normal pour plate.

Anaerobiosis↗
PubMed1989

Miniaturization of catheter systems for angiography.

Miniature techniques offer many advantages for performance of routine angiography, as well as invasive vascular procedures. On the basis of the authors' experience with over 16,000 diagnostic and interventional procedures, this report describes the equipment and methods used for miniangiography. Development of sturdy, high-flow 3-5-F catheters and 0.014-0.018-inch guide wires, combined with the use of small needle puncture techniques, has improved the safety of angiography and intervention such that outpatient procedures are now routine. Miniature catheter systems are now preferable to larger catheter systems in most instances.

Angiography↗
PubMed1980

Coronary blood flow in conscious miniature swine during +GZ acceleration stress.

One of the factors determining tolerance to +GZ acceleration may be the ability to maintain adequate coronary blood flow. Consequently, the purpose of these studies was to determine the effect of acute exposure (60 s) to several levels of positive acceleration (+GZ) on total and regional coronary blood flow in conscious adult miniature swine. Blood flow was measured with the radiolabeled microsphere technique in chronically instrumented miniature swine during 60-s exposures to accelerations of +3 GZ, +5 GZ, or +7 GZ with anti-G suit support. All levels of acceleration stress caused two- to threefold increases in coronary blood flow. The regional distribution of coronary blood flow during +GZ was similar to that under resting control conditions as long as aortic diastolic pressure was maintained. All left ventricular endocardial/epicardial flow ratios were significantly greater than one, except in two animals, during exposure to +7 GZ. These were the only animals to have aortic diastolic pressures less than 100 Torr during +GZ stress. These studies indicate that, if an animal is able to compensate and maintain a cardiovascular steady State, coronary blood flow will remain adequate for myocardial needs. However, if decompensation occurs and aortic diastolic pressure falls below a critical value, subendocardial blood flow may become inadequate.

Animals↗
PubMed1980

Renal blood flow in miniature swine during +GZ stress and anti-G suit inflation.

The effects of +GZ acceleration stress on renal blood flow were studied in chronically instrumented, conscious miniature swine. Renal blood flow was determined using the radiolabeled (46Sc, 85Sr, 141Ce, and 125I) microsphere technique (29 animals) and Doppler-principle flow probes (1 animal). The results obtained with the microsphere technique indicate that +GZ exposure with anti-G suit support caused decreases in renal blood flow throughout the kidney, and that renal blood flow remains decreased for 1-10 min after +GZ stress. Medullary blood flow appeared to recover after +GZ exposure more rapidly than cortical blood flow. The flow probe data also indicate that +GZ stress with or without anti-G suit support results in decreases in renal blood flow, and that anti-G suit inflation in the absence of +GZ can also cause significant decreases. We conclude that the reductions in renal blood flow observed in miniature swine, during +GZ stress with anti-G suit support, may be due to a combination of direct anti-G suit interference and +GZ-induced reflex increases in alpha-constrictor sympathetic tone to the renal vascular bed.

Animals↗
PubMed2002

Spontaneous miniature hyperpolarizations affect threshold for action potential generation in mudpuppy cardiac neurons.

Mudpuppy parasympathetic neurons exhibit spontaneous miniature hyperpolarizations (SMHs) that are generated by potassium currents, which are spontaneous miniature outward currents (SMOCs), flowing through clusters of large conductance voltage- and calcium (Ca(2+))-activated potassium (BK) channels. The underlying SMOCs are initiated by a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Perforated-patch whole cell voltage recordings were used to determine whether activation of SMHs contributed to action potential (AP) repolarization or affected the latency to AP generation. Blockade of BK channels by iberiotoxin (IBX, 100 nM) slowed AP repolarization and increased AP duration. Treatment with omega-conotoxin GVIA (3 microM) or nifedipine (10 microM) to inhibit Ca(2+) influx through N- or L-type voltage-dependent calcium channels (VDCCs), respectively, also decreased the rate of AP repolarization and increased AP duration. Elimination of CICR by treatment with either thapsigargin (1 microM) or ryanodine (10 microM) produced no significant change in AP repolarization or duration. Blockade of BK channels with IBX and inhibition of N-type VDCCs with omega-conotoxin GVIA, but not inhibition of L-type VDCCs with nifedipine, decreased the latency of AP generation. A decrease in latency to AP generation occurred with elimination of SMHs by inhibition of CICR following treatment with thapsigargin. Ryanodine treatment decreased AP latency in three of six cells. Apamin (100 nM) had no affect on AP repolarization, duration, or latency to AP generation, but did decrease the hyperpolarizing afterpotential (HAP). Inhibition of L-type VDCCs by nifedipine also decreased HAP amplitude. Inhibition of CICR by either thapsigargin or ryanodine treatment increased the number of APs generated with long depolarizing current pulses, whereas exposure to IBX or omega-conotoxin GVIA depressed excitability. We conclude that CICR, the process responsible for SMH generation, represents a unique mechanism to modulate the response to subthreshold depolarizing currents that drive the membrane potential toward the threshold for AP initiation but does not contribute to AP repolarization. Subthreshold depolarizations would not activate sufficient numbers of VDCCs to allow Ca(2+) influx to elevate [Ca(2+)](i) to the extent needed to directly activate nearby BK channels. However, the elevation in [Ca(2+)](i) is sufficient to trigger CICR from ryanodine-sensitive Ca(2+) stores. Thus CICR acts as an amplification mechanism to trigger a local elevation of [Ca(2+)](i) near a cluster of BK channels to activate these channels at negative levels of membrane potential.

Action Potentials↗
PubMedDate not supplied

D-methionine utilization in young miniature pigs, adult rabbits, and adult dogs.

Rats and chickens, unlike man, utilize D-methionine efficiently. We have studied urinary excretion of methionine isomers in young miniature pigs, adult rabbits, and adult dogs given D-methionine in an attempt to find an animal model that, like man, utilizes the D-isomer poorly. Six-week-old miniature pigs ingesting a protein-free diet were infused with 8.5% amino acid solutions differing only in methionine isomer content (L- vs DL-) to supply amino acid requirements. Each solution was infused for a 2- or 3-day period in a cross-over design. Plasma methionine levels were significantly higher (p = 0.01) during infusion of the solution providing DL-methionine, with 25% of total plasma methionine present as the D-configuration. However, urinary methionine excretion was similar with both solutions, with D-methionine utilization calculated as greater than 99%. Adult rabbits ingesting DL-methionine (0.14 g/100 g food) showed good utilization of D-methionine, excreting less than 1.3% D-isomer in the urine. Mixed breed dogs given 1.25 g D-methionine in their drinking water excreted less than 0.4% of ingested D-methionine in the urine. These data indicate that the pig, rabbit, and dog utilize D-methionine efficiently, behaving like the rat and chicken rather than man.

Amino Acids↗
PubMedDate not supplied

Effects of intravenous administration of Travenol 10% lipid emulsion to miniature swine for 28 consecutive days.

The potential toxicity of Travenol 10% lipid emulsion was studied using miniature swine. Physiological (0.9%) saline, USP, was used as the control, and Intralipid 10% fat emulsion as the reference article. The emulsions were administered intravenously at dosages of 40 milliliters (approximately 4 grams of lipid) and 60 milliliters (approximately 6 grams of lipid) per kilogram of body weight per day to eight animals (four males and four females) in each treatment group on each of 28 consecutive days. The saline was administered to eight animals at 60 milliliters per kilogram per day. On day 29, one half of the male and female animals in each group were necropsied. The remaining pigs were observed and necropsied on either day 56 or 57. Toxicity was assessed on the basis of animal survival; changes in body weight, urinalyses, and hematological, and serum biochemical analyses; ophthalmological examination; gross pathology; and histopathology. The results obtained for the Travenol emulsion correlated well with those for the Intralipid emulsion. The emulsions were well tolerated, and they did not produce any major clinical signs of toxicity. All Travenol emulsion-treated animals survived. In addition to demonstrating the similarity of Travenol and Intralipid emulsions, the results of this study indicate that the Travenol emulsion demonstrated an adequate margin of safety for prolonged administration. Travenol emulsion was well tolerated by miniature swine infused at about one and one-half (40 milliliters per kilogram per day) and two (60 milliliters per kilogram per day) times the proposed clinical dose, and at three and two times the anticipated clinical rate, respectively.

Animals↗
PubMed2000

Common variable immunodeficiency in miniature dachshunds affected with Pneumonocystis carinii pneumonia.

Seven miniature dachshunds, all under the age of 1 year, were presented with polypnea, tachypnea, and exercise intolerance as a result of Pneumocystis carinii pneumonia, which was diagnosed on transtracheal aspirate cytology. In all of the dogs, historical and clinical signs were suggestive of immune incompetence. Immunological studies undertaken were leukogram parameters, serum immunoglobulin fraction quantification, lymphocyte transformation assay. CD3 and CD79a lymphocyte markers on lymphoid tissue, and anti-canine immunoglobulin G immunoperoxidase staining. The immunological studies showed hypogammaglobulinemia, deficiency of serum immunoglobulins A, G, and M, decreased lymphocyte transformation response to phytohemagglutinin and pokeweed mitogens and absence of B lymphocytes with presence of T lymphocytes in the lymphoid tissue stained with CD3 and CD79a lymphocyte markers. The preceding findings suggest that P. carinii pneumonia occurring in the miniature dachshund is a result of both a T- and B-cell deficiency. This presentation is not the classic primary severe combined immunodeficiency syndrome but rather combined variable immunodeficiency, which has been well documented in humans but never in the dog.

Animals↗
PubMed2000

Ultra-high throughput screen of two-million-member combinatorial compound collection in a miniaturized, 1536-well assay format.

Results of a complete survey of the more than 2-million-member Pharmacopeia compound collection in a 1536-well microvolume screening assay format are reported. A complete technology platform, enabling the performance of ultra-high throughput screening in a miniaturized 1536-well assay format, has been assembled and utilized. The platform consists of tools for performing microvolume assays, including assay plates, liquid handlers, optical imagers, and data management software. A fluorogenic screening assay for inhibition of a protease enzyme target was designed and developed using this platform. The assay was used to perform a survey screen of the Pharmacopeia compound collection for active inhibitors of the target enzyme. The results from the survey demonstrate the successful implementation of the ultra-high throughout platform for routine screening purposes. Performance of the assay in the miniaturized format is equivalent to that of a standard 96-well assay, showing the same dependence on kinetic parameters and ability to measure enzyme inhibition. The survey screen identified an active class of compounds within the Pharmacopeia compound collection. These results were confirmed using a standard 96-well assay.

Chemistry, Pharmaceutical↗
PubMed2001

Identification of inhibitors of bacterial transcription/translation machinery utilizing a miniaturized 1536-well format screen.

This report presents the miniaturization of a HTS screen to identify inhibitors of prokaryotic transcription-translation in a 1536-well format. The in vitro assay design utilized the bacterial expression machinery to drive expression of a firefly luciferase reporter gene, which was read as an endpoint luminesence measurement. This multicomponent system permits identification of inhibitors at different steps in this pathway. Successful miniaturization required integration of homogeneous assay formats, robust liquid-handling workstations, and second-generation imaging systems. Comparison of data from a triplicate 1536-well screen of a subset of a target library that had been previously validated and followed up for hit confirmation in a 384-well plate format confirmed that triplicate screening yields data of higher confidence and quality, eliminates the time-consuming and potentially error-prone step of cherry-picking, and reduces the number of false positives and negatives. The substantial savings of reagents and reduction of the numbers of plates to process obtained in a 1536-well format as compared to a 384-well format allowed a full triplicate evaluation of the entire library of 183,000 compounds at lower cost and in less time. The triplicate-screen statistics are consistent with a highly reliable data set with a coefficient of variation of 14.8% and Z' and Z values of 0.57 and 0.25, respectively. This screen resulted in the identification of 1,149 hits (0.63% hit rate), representing a compound population at 2.5 standard deviations from the mean cutoff. Furthermore, the data demonstrate good agreement between IC(50) values derived for this assay in a 1536-well format and 384-well format.

Bacteria↗
PubMed2006

Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format.

This article discusses the development of homogeneous, miniaturized assays for the identification of novel kinase inhibitors from very large compound collections. In particular, the suitability of time-resolved fluorescence resonance energy transfer (TR-RET) based on phospho-specific antibodies, an antibody-independent fluorescence polarization (FP) approach using metal-coated beads (IMAP technology), and the determination of adenosine triphosphate consumption through chemiluminescence is evaluated. These readouts are compared with regard to assay sensitivity, compound interference, reagent consumption, and performance in a 1536-well format, and practical considerations for their application in primary screening or in the identification of kinase substrates are discussed. All of the tested technologies were found to be suitable for miniaturized high-throughput screening (HTS) in principle, but each of them has distinct limitations and advantages. Therefore, the target-specific selection of the most appropriate readout technology is recommended to ensure maximal relevance of HTS campaigns.

Biological Assay↗
PubMed1999

Miniaturization of a Mammalian Cell-Based Assay: Luciferase Reporter Gene Readout in a 3 Microliter 1536-Well Plate.

The combined efforts of the fields of combinatorial chemistry and genomics have significantly increased the number of compounds and therapeutic targets available for screening. The number of compounds will reach into the million range in the near future and provide vast chemical diversity for drug discovery. However, this reservoir of chemical diversity creates downstream hurdles for any screening effort. Properly examining this number of compounds increases investments dramatically, both in the number of dollars spent and amount of limited reagents depleted. Traditional HTS techniques, such as the use of 96-well microtiter plates, have paved the way for faster processing speeds, but are being rapidly overwhelmed by screening demands. Miniaturization of such assays will allow for greater throughput, while concurrently reducing cost. To date, miniaturization efforts have been most successfully applied to bacterial and soluble protein based assays. Questions about the ability to deliver microquantities of mammalian cells without disruption of the cell membrane and/or activation of stress responses have been raised. An assay has been developed in which a human T-cell screen has been adapted to a 1536-well plate format. Through the use of a luciferase reporter gene system, it is shown that a mammalian cell-based assay may be successfully performed in 3 µl and potent inhibitors of the target of interest identified.

Journal Article↗
PubMed1999

Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy.

Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule detection (SMD) techniques and related approaches, such as fluorescence correlation spectroscopy (FCS), stochastically sample the fluorescence properties of individual constituent molecules and only then average many such detection events to define the properties of the assay system as a whole. Analysis of single molecular events is accomplished using confocal optics with an illumination/detection volume of approximately 1 fl (10(-15) L) such that the signal is insensitive to miniaturization of HTS assays to 1 µl or below. In this report we demonstrate the general applicability of one SMD technique (FCS) to assay configuration for target classes typically encountered in HTS and confirm the equivalence of the rate/equilibrium constants determined by FCS and by macroscopic techniques. Advantages and limitations of the current FCS technology, as applied here, and potential solutions, particularly involving alternative SMD detection techniques, are also discussed.

Journal Article↗
PubMedDate not supplied

Delivery of intravenous treprostinil at low infusion rates using a miniaturized infusion pump in patients with pulmonary arterial hypertension.

PURPOSE: Treprostinil is approved for the treatment of pulmonary arterial hypertension (PAH) via continuous intravenous (IV) infusion. Treprostinil's anti-platelet aggregation characteristics and stability at room temperature may allow for low infusion rates (0.1-0.2 mL/hr) using a miniaturized infusion pump. METHODS: A 12-week, multi-center, open-label study in 12 adult PAH patients, evaluated the feasibility and safety of low-flow IV treprostinil administration via the 407C miniaturized pump. Patients receiving IV treprostinil at a stable dose were transitioned from their current CADD-Legacy pump to the 407C and were assessed for adverse events including catheter occlusions, pump alarms, and efficacy (six minute walk distance (6MWD), Borg Dyspnea Score (BDS), NYHA functional class, and PAH signs/symptoms). All patients were also maintained on therapeutic doses of warfarin, heparin or low molecular weight heparin throughout the study. RESULTS: Baseline mean (+/-SD) 6MWD was 477 +/- 76 m (n = 9) with mean BDS of 2.1 +/- 1.2 (n = 9). Week 12 mean 6MWD and BDS were 500 +/- 92 m and 2.3 +/- 1.7, respectively (n = 9). Four patients discontinued the study prematurely (3 AEs and 1 consent withdrawn). Adverse events included headache, flushing, and nausea. Pump complications occurred in 5 of 12 patients, and although no catheter occlusions occurred in any patient during the 12-week study, further study is needed regarding pump complications. CONCLUSION: This study demonstrates that treprostinil can be administered intravenously at infusion rates as low as 0.1 mL/hr for 12 weeks without catheter occlusions. Further studies are warranted because the potential for adverse events is of some concern.

Adult↗