Assisted conception: current techniques and outcome

PURPOSE: To determine whether ingestion of a tooth whitener containing 6% hydrogen peroxide as bleaching agent affected the gastric mucosa of adult, female laboratory rats. MATERIALS AND METHODS: Thirty-six fasting rats were intubated with a single bolus (5 g/kg body weight) of the tooth whitener Natural White which contains 6% hydrogen peroxide. Thirty-two control rats received deionized water. Rats were necropsied 15 minutes, 2 hours, 1 and 2 weeks after whitener ingestion. The gastric mucosa was examined histologically and blood hematocrit, glucose, BUN, and bilirubin measured. RESULTS: Six of the 36 rats died within 2 hours of receiving whitener. Within 15 minutes of whitener ingestion, stomachs were grossly bloated with gas. Histological observation showed that gastric mucosal cells were vacuolated and gastric glands dilated. After 2 hours, gastric epithelial and glandular cells had sloughed off into the gastric lumen. Mean blood glucose and hematocrit were significantly (P < 0.05) elevated (233 +/- 41 mg/ml and 50.5 +/- 0.9%) over mean control values (129 +/- 8 mg/ml and 42.8 +/- 2.4%). Within 1 week, the stomachs of experimental rats were no longer bloated, the gastric mucosa appeared normal histologically, but mean blood hematocrit was significantly (P < 0.05) decreased (38.0 +/- 1.5%) from mean control value (43.3 +/- 0.5%). There were no significant differences in mean blood values 2 weeks after whitener ingestion.     

Blood substitutes: evolution and future applications

The development of oxygen-carrying blood substitutes has progressed significantly in the last decade with phase I and phase II clinical trials of both hemoglobin-based and perfluorocarbon-based oxygen carriers nearing completion. As these products approach clinical use it is important for the laboratory medicine community to be aware of their effects on routine laboratory testing and the settings in which they might be used. Here we review the forces driving the development of oxygen-carrying blood substitutes, the clinical settings in which they might be used, the major categories of oxygen carriers in clinical trials, and the challenges faced by these products as they approach clinical use.     

Fibrin sealant: current and potential clinical applications

There are few well-controlled studies of the clinical efficacy of fibrin sealant, defined by lives saved or reduced need for blood transfusions. Evaluation of fibrin sealant in trauma situations, e.g. liver laceration, has been difficult to perform. Only recently has fibrin sealant been actively promoted by US manufacturers as a commercially valuable alternative to the relatively inexpensive crude bovine thrombin and cryoprecipitate that are in current use. Regulatory agencies and manufacturers are aware that patients in the USA are receiving a suboptimal form of fibrin glue since cryoprecipitate is not virally inactivated and has a variable fibrinogen concentration. In addition, bovine thrombin is not regulated with respect to factor V content or any other impurities. During the past year regulatory agencies, together with manufacturers and clinicians, have begun to define clinically valid endpoints for efficacy of a commercially prepared fibrin sealant. These may include improvement in hemostasis compared with a placebo or agents considered to be 'standard of care'. Thus, the regulatory agencies may be willing to consider studies in animals that demonstrate efficacy as well as surrogate endpoints, such as reduced factor concentrate requirements in patients with severe hemophilia requiring dental extraction. As fibrin sealant becomes available in a liquid and potentially in a bandage form, it may also become an essential matrix for recombinant factors that can affect endothelial function.     

Pathophysiology of human spermatozoa

The effect of the addition of microbial phytase to a diet based on field beans (30%), wheat (28%), peas (25%), and barley (14%) was studied in a 2-week experiment with 3 x 8 castrated male, individually housed, hybrid piglets (live weight range 12-16 kg). All diets contained about 4.7 g Ca, 4.2 g P (77% present as phytate phosphorus), 1.0 g Mg, 60 mg Zn per kg diet, and 17% crude protein. Group I was fed the basal diet with a native phytase-activity of about 260 U per kg diet. In group II, 350 U, in group III, 700 U of microbial phytase per kg diet were added. The addition of microbial phytase improved the apparent P absorption (% of intake) from 48% (group I) to 66% (group II) and 71% (group III). Comparable positive effects from the phytase treatment were obtained for the calcium utilization. The phytase supplementation also enhanced plasma zinc concentration significantly. The concentration of inorganic phosphorus in plasma, the zinc digestibility, and the magnesium balance were improved in tendency. The utilization of nitrogen remained unchanged.     

Capillary electrophoresis and microdialysis: current technology and applications

Microdialysis sampling has become an important method for the continuous monitoring from an in vivo environment. This technique has been used to monitor many endogenous molecules, such as neurotransmitters, as well as exogenous species such as drug substances. Microdialysis samples have traditionally been analyzed by liquid chromatographic (LC) methods to gain resolution and quantification of the molecules of interest. However, LC separations have a relatively large injection volume requirement which, as a consequence, increases microdialysis sampling times. Capillary electrophoresis (CE), with its very small sample volume requirements and high resolving power, has therefore gained popularity as an alternative to LC. Reviewed here are many of the technologies currently available for CE and examples of how this technique has been effectively applied to the analysis of microdialysis samples.     

Cryopreservation of single human spermatozoa

A procedure is described that allows cryopreservation and efficient post-thaw recovery of either a single or a small group of human spermatozoa. This is achieved by injecting them into cell-free human, mouse or hamster zonae pellucidae before the addition of cryoprotectant. The method involves a combination of physical micromanipulation procedures and glycerol-mediated cryoprotection. Zonae were tracked by positioning them in straws between two small air bubbles prior to freezing. Spermatozoa from poor specimens were cryopreserved and their fertilizing ability after thawing was compared with that of fresh spermatozoa from fertile men. Human eggs used for fertilization testing were either 1 day old or in-vitro matured. Only 2% of the frozen zonae were lost and >75% of spermatozoa cryopreserved in this manner were recovered and prepared for intracytoplasmic sperm injection. The feasibility of cryopreserving a single spermatozoon was assessed. Fifteen motile spermatozoa were frozen in 15 zonae, of which 14 were recovered after thawing. Ten were injected into spare eggs, of which eight became fertilized. Spermatozoa recovered mechanically from human zonae fertilized the same proportion of oocytes as fresh fertile control spermatozoa. The recovery and fertilization rates with spermatozoa frozen in animal zonae were 87 and 78% respectively. The fertilization rate was marginally higher (P < 0.05) than that for spermatozoa frozen in human zonae, perhaps because the latter may have acrosome reacted more frequently. The zona pellucida appears to be an ideally suited sterile vehicle for storage of single spermatozoa.     

Ampullary carcinoma: demonstration by current MR techniques

Rheumatoid arthritis (RA) is characterised by chronic inflammation of synovial tissue with aggressive proliferation of synovial cells causing destruction of cartilage and bone. Immunopathological mechanisms, infectious causes and genetic factors have been discussed, but the etiology of the disease has not been understood until now. Especially, the mechanisms driving tumourlike growth and invasive behaviour of fibroblastoid synovial cells have not been identified yet. Our aim is to find cellular factors which are mediators for such pathways. One possibility to approach this, is searching for disease-relevant genes. We applied the mRNA-differential display technique to compare mRNA expression patterns of normal and rheumatic synovial fibroblasts. We identified an upregulation of the human semaphorin E gene in rheumatoid synovial fibroblastoid cells. Interestingly semaphorin E is a member of a protein-family described to play an immunosuppressive role via inhibition cytokines. A relevance of this finding towards the pathogenesis of RA is discussed.     

Adult intussusception: demonstration by current MR techniques

We describe magnetic resonance findings in three patients with small bowel intussusception from different etiologies including idiopathic, adenomatous polyps, and hamartomatous polyps. Magnetic resonance findings showed a bowel-within-bowel appearance in two patients and a coiled-spring appearance in one patient. These findings were best shown on T2-weighted images, and clear definition was present on breathing independent T2-weighted images using half fourier acquisition snap shot turbo spin echo T2-weighted images.     

Visually evoked potentials: theory, techniques and clinical applications

The visually evoked potential (VEP), the recording of which has recently been made possible by the development of computer averaging techniques, is a gross electrical signal generated by the occipital region of the cortex in response to visual stimulation. It is more specific than the electroencephalogram (EEG) and more sensitive to changes in the visual stimulus; thus, it can provide ophthalmologists and vision researchers with information about the human visual system that is unavailable by other methods. Clinically, the VEP is of special value in the areas of refraction, infant acuity, diseases of the optic nerve, color blindness, amblyopia and field defects. Theory, techniques and instrumentation are described, and applications of the VEP to clinical situations and to vision research are discussed.     

Internal pH of human spermatozoa: effect of ions, human follicular fluid and progesterone

The internal pH (pHi) of human spermatozoa was measured by the fluorescent indicator 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and the distribution of the radioactive [14C]-methylamine under different external ionic conditions. The effect of the addition of progesterone and human follicular fluid (HFF) on the spermatozoa pHi was also analysed. The pHi values obtained were almost identical with the two probes used. In sodium (NaM) and potassium (KM) media, a linear relationship between the internal and external pH was observed. In NaM, the pHi values were approximately 0.4 pH unit less than the external pH. In KM, the pHi measured was higher than in NaM and only slightly inferior to the external pH (0.1-0.2 pH unit). Addition of 10 microM progesterone, oestradiol 17 beta or 20% HFF to spermatozoa incubated at pH 7.2 in NaM did not induce any rapid variation of the BCECF fluorescence or change in the accumulation of methylamine. A slight change in pH (approximately 0.5 units) occurred with progesterone after 15 min. As a control, addition of 10 mM of NH4Cl induced a rapid alkalinization (0.4 pH unit) of the cell interior while 10 mM lactate produced only a slight acidification (approximately 0.2 pH unit). Under the same conditions (NaM, pH 7.2), the pHi of the spermatozoa prepared by Percoll gradient was found more acidic by 0.2 pH unit than washed unfractionated spermatozoa. Progesterone, oestradiol 17 beta and HFF had no effect on the pHi of these spermatozoa. The results obtained in this study show that it is possible to measure accurately the internal pH of human spermatozoa. Internal pH was found to be dependent upon the pH of the external medium and a quasi-linear relationship exists between the internal and external pH, suggesting no specific pH regulatory mechanisms. Our data suggest instead that the protons, under our experimental conditions, are passively distributed. Progesterone, oestradiol 17 beta and HFF, known to promote both capacitation and the acrosome reaction, do not act through a rapid pHi change.     

Oncologic applications of diagnostic imaging techniques

Before appropriate therapy can be instituted for a cancer patient, the presence and extent of tumor must be evaluated. Deciding which imaging technique to use depends on tumor location, type, and biologic behavior. Conventional radiography provides important information at a relatively low cost compared with other imaging modalities. Ultrasound is a valuable adjunct to radiography, but does not replace it because both imaging modalities provide unique information. Nuclear medicine procedures contribute additional, unique data by providing physiological information, but specificity is lacking. Both CT and MRI provide images with exquisite anatomic detail, but availability and cost prohibit their general use.     

Selection and characterization of human acrosome reacted spermatozoa

In order to study the characteristics of human acrosome reacted spermatozoa, we developed a method to select them. Spermatozoa, obtained from 20 fertile volunteers, were selected by using GB24 antibody fixed on magnetic immunobeads, after acrosome reaction induced by 50% follicular fluid. Bead-bound spermatozoa were then detached using sheep anti-mouse IGG F(ab')2 antibody. This method allowed recovery of 170 +/- 48 x 10(3) spermatozoa (n = 20), free of GB24 antibody, as assessed by incubation with FITC-rabbit anti-mouse antibody. The percentage of acrosome reacted spermatozoa in the selected population was 88 +/- 3% versus 32 +/- 6% in the whole sperm population. Concerning sperm morphology, the percentage of head abnormalities was lowered (15 +/- 3% versus 20 +/- 3%). The motility of selected spermatozoa was dramatically reduced (7 +/- 3% versus 53 +/- 7% in the whole population) despite no difference in viability (84 +/- 3% versus 80 +/- 4%). However, the viability after an 18 h incubation was very low (1 +/- 0.5% versus 46 +/- 5%). These results show that acrosome reaction occurs in the most morphologically normal spermatozoa and is followed by a loss in motility and a decrease in longevity.     

Detection of the nasal cycle with acoustic rhinometry: techniques and applications

Acoustic rhinometry is an appropriate method for detecting and recording the nasal cycle in normal subjects in terms of the cross-sectional areas and volume of the nasal cavity. In this study, we tried to detect and to define the nasal cycle in normal subjects so that we might develop a reliable and reproducible technique to be used in conjunction with studies on the physiology and pathology of nasal disease. We used normal volunteer adult subjects and performed bilateral acoustic rhinometry measurements every 15 minutes over 4 hours, along with the use of a visual analog scale for assessment of the subjective feeling of congestion (or patency) just before each acoustic rhinometry measurement. Volume and cross-sectional area changes were observed along with subjective patency-score changes in each subject. The subjective feeling of patency was not related to the volume and cross-sectional area changes measured simultaneously. The technique of recording the nasal cycle with acoustic rhinometry in nasal research is presented.     

Homografts in congenital heart disease: current applications and future directions

The first clinical use of homograft tissue in cardiovascular surgery was in 1948, when Gross used cadaveric arterial grafts to construct systemic to pulmonary artery shunts in patients with tetralogy of Fallot, and to repair coarctation of the aorta. Eighteen years later, a valved homograft was used for the first time in the treatment of congenital heart disease for reconstruction of the right ventricular outflow tract in a child with pulmonary atresia. Since these pioneering advances, valve and vascular homografts have become central to the management of congenital anomalies of the heart and great vessels. The primary use for homografts in congenital heart surgery today is establishment of a valved connection between the right ventricle and pulmonary arteries in children with tetralogy of Fallot with pulmonary atresia or other complicating factors, truncus arteriosus, transposition complexes, and double-outlet right ventricle, and in patients undergoing the Ross procedure. Homograft reconstruction of the left ventricular outflow tract has also been performed for many years in children with aortic insufficiency or recurrent aortic stenosis, but early homograft degeneration in young children has been a significant problem. Many surgeons are turning away from homografts in the systemic circulation in favor of the pulmonary autograft. Homograft is also widely used as a vascular patch material. In the present report, we discuss the various uses of homografts in congenital heart surgery, the benefits and drawbacks of homografts in young patients, and some of the future possibilities for homograft development and application in patients with congenital heart disease.     

Cholangiocarcinoma: spectrum of appearances on MR images using current techniques

This study describes the spectrum of appearances of cholangiocarcinoma on magnetic resonance (MR) sequences, including gadolinium-enhanced, fat-suppressed spoiled gradient echo images and MR cholangiography. Fifteen patients were included in the study. Histologic diagnosis was established in 11 patients by surgical resection (6 patients), percutaneous biopsy (4 patients), and open liver biopsy (1 patient). The final diagnosis was determined by correlation of the MR findings with cholangiographic studies and laboratory studies in 4 patients. MR studies were performed at 1.5 T, and the following sequences were obtained: T1-weighted spoiled gradient echo (SGE), T1-weighted fat-suppressed spin echo or SGE, T2-weighted fat-suppressed conventional or turbo spin echo, MR cholangiography, and gadolinium-enhanced T1-weighted fat-suppressed SGE images. The following determinations were made: tumor location, tumor extent, ductal dilatation, ductal wall thickness, signal intensity, enhancement pattern, and associated findings. Mass-like neoplasms were peripheral (6 patients), hilar (1 patient), and extrahepatic (2 patients). Circumferential tumors were hilar (2 patients) and extrahepatic (4 patients). All peripheral tumors were multifocal. Mass-like tumors were well-defined, rounded, and ranged from 1 to 14 cm in diameter. Circumferential tumors had less well-defined margins and measured from 3 to 15 mm in thickness. All mass-like tumors were moderately hypointense on T1-weighted images and mildly to moderately hyperintense on T2-weighted images. The circumferential tumors were iso- to moderately hypointense on T1-weighted images and iso- to mildly hyperintense on T2-weighted images. Mass-like tumors were generally well shown on non-contrast and immediate gadolinium-enhanced images, whereas circumferential tumors were poorly seen on non-contrast images and best shown on gadolinium-enhanced T1-weighted fat-suppressed images. The degree of enhancement ranged from minimal to intense on immediate gadolinium-enhanced images, with all tumors becoming more homogeneous in signal intensity on images obtained between 1 and 5 min following contrast administration. Tumor-containing lymph nodes greater than or equal to 1 cm in diameter were demonstrated in 11 out of 15 patients (73.3%). These were best shown on T2-weighted fat-suppressed images and gadolinium-enhanced fat-suppressed SGE images. MR cholangiography demonstrated the level of obstruction and degree of dilatation of the proximal biliary system in 5 out of 6 patients who underwent MR cholangiography. The spectrum of appearances of cholangiocarcinoma is demonstrable on MR images. Mass-like tumors are well shown on both pre- and post-gadolinium sequences. Circumferential tumors may cause minimally increased duct wall thickness and are most clearly shown on gadolinium-enhanced fat-suppressed SGE images obtained 1 to 5 min following gadolinium administration.     

Protein structure prediction by threading methods: evaluation of current techniques

This paper evaluates the results of a protein structure prediction contest. The predictions were made using threading procedures, which employ techniques for aligning sequences with 3D structures to select the correct fold of a given sequence from a set of alternatives. Nine different teams submitted 86 predictions, on a total of 21 target proteins with little or no sequence homology to proteins of known structure. The 3D structures of these proteins were newly determined by experimental methods, but not yet published or otherwise available to the predictors. The predictions, made from the amino acid sequence alone, thus represent a genuine test of the current performance of threading methods. Only a subset of all the predictions is evaluated here. It corresponds to the 44 predictions submitted for the 11 target proteins seen to adopt known folds. The predictions for the remaining 10 proteins were not analyzed, although weak similarities with known folds may also exist in these proteins. We find that threading methods are capable of identifying the correct fold in many cases, but not reliably enough as yet. Every team predicts correctly a different set of targets, with virtually all targets predicted correctly by at least one team. Also, common folds such as TIM barrels are recognized more readily than folds with only a few known examples. However, quite surprisingly, the quality of the sequence-structure alignments, corresponding to correctly recognized folds, is generally very poor, as judged by comparison with the corresponding 3D structure alignments. Thus, threading can presently not be relied upon to derive a detailed 3D model from the amino acid sequence. This raises a very intriguing question: how is fold recognition achieved? Our analysis suggests that it may be achieved because threading procedures maximize hydrophobic interactions in the protein core, and are reasonably good at recognizing local secondary structure.     

超重力冶金:科学原理、实验方法、技术基础、应用设计

超重力显著增大两相间的重力差,可用于加速固?液、液?液、液?气高温黏稠混和体的相分离速度;超重力具有定向性,避免搅拌等技术产生的熔体湍流返混,可用于深度脱除金属液中细小夹杂物;超重力条件下固?液界面张力微不足道,可容易实现微孔渗流;超重力条件下进行结晶凝固,按结晶顺序实现固?液分离,可用于制备梯度材料;超重力加速固?液分离,可细化凝固组织晶粒,但对非共晶熔体也易产生宏观偏析。将超重力技术应用于冶金及材料生产过程中,有望解决高温冶金和材料制备的一些难题,如复杂矿冶金渣有价组分的分离提取、冶炼渣中金属液的分离回收、多金属的熔析结晶分离、复杂矿直接还原铁的渣?金分离;在高端金属材料方面,应用超重力技术,有望解决近零夹物金属材料的精炼除杂难题,提高梯度功能材料、金属?陶瓷复合材料、多孔金属材料、器件材料表面电沉积修饰的制造水平。此外,在材料科学研究方面,超重力凝固可作为一种材料基因组高通量制备方法。      

Occurrence of GABA and GABA receptors in human spermatozoa

Acute graft-versus-host disease (GVHD) is effected by donor T lymphocytes which have been stimulated by host antigens. Activated donor T lymphocytes express interleukin-2 receptor (IL-2R), which is comprised of three subunits (alpha, beta, gamma). During activation, the a IL-2R subunit (CD25) is shed from the receptor complex and can be measured in the circulation. Soluble IL-2Ralpha (sIL-2R) levels are increased in states of immune activation including GVHD, and could theoretically be used as a guide to therapy. Since IL-2Ralpha expression is an early marker of T cell activation, we investigated: (1) if an increase in sIL-2R is specific for acute GVHD; and (2) if serial sIL-2R levels can identify patients with early GVHD, prior to the onset of clinical tissue damage (effector function). Weekly sIL-2R levels were monitored in 36 patients undergoing matched related (n=23) or matched unrelated (n=13) allogeneic bone marrow transplantation (BMT). There was no significant difference in sIL-2R levels between matched related and matched unrelated recipients. Patients with acute GVHD (n=19, 53%) demonstrated higher sIL-2R levels, than those without during weeks 2 and 3 post-BMT (P=0.02 and 0.04, Mann-Whitney U test, two-tailed). In patients with acute GVHD, the rise in sIL-2R preceded the clinical signs of GVHD (16/19 patients). However, patients with sepsis demonstrated a trend towards higher sIL-2R levels at week 1 and significantly greater levels by week 4 (P=0.02). Furthermore, patients with veno-occlusive disease (VOD) (25%) also had significantly higher sIL-2R levels at week 2 (P=0.03). We conclude that although sIL-2R levels increase in patients with acute GVHD, similar increases are seen in patients with VOD and/or sepsis and therefore, as a single biochemical marker, we find that serial measurements of sIL-2R lacks sufficient specificity to guide GVHD therapy.