Size and nature of emboli produced during carotid artery angioplasty and stenting: in vivo study.

AIM: Microembolization continues to be a major risk for patients undergoing carotid artery stenting (CAS) of high-grade atherosclerotic carotid stenoses. Further insight into the characteristics and significance of these embolized particles was deemed necessary. We aimed to assess the size and composition of debris captured by filters during CAS and to determine if this could be predicted using standard imaging techniques. METHODS: 20 patients (10 symptomatic, 15 men, mean age 64.6 years) undergoing CAS for high-grade ICA stenosis were recruited. All underwent pre-operative CT angiography and calcium scoring. All underwent CAS using the same protocol. A filter-type embolic protection device (EPD) was used and retrieved post-operatively and captured particles underwent analysis using a Scanning Electron Microscope (SEM) for counting, sizing, and composition. RESULTS: Clinical. Debris was found on 100% of filters when analysed with SEM. There were non-significant trends for CAS in asymptomatic patients to produce a greater number of smaller, calcified particles while in symptomatic patients we observed larger, lipid-rich particles. When stratified according to pre-operative calcium scores, 'calcium-rich' plaques produced significantly greater numbers of emboli captured on the EPD (p = 0.02). CONCLUSIONS: Filter-type EPDs collect debris of significant quantity and size during the CAS procedure as performed in our institution. The collected material was likely dislocated from the atherosclerotic plaque. CT calcium scoring allows us to predict the nature of material captured by the EPD. These data may allow the clinician to individualise care during CAS and thus reduce peri-operative risk.

The expression pattern of MUC1 glycoforms and other biomarkers of endometrial receptivity in fertile and infertile women.

Changes in the surface epithelium of the endometrium, characterized in part by alterations in cell-surface molecules, sex steroid receptors and the appearance of pinopodes, coincide with the window of endometrial receptivity in the menstrual cycle. This study was performed to evaluate the usefulness of hematoxylin and eosin staining, scanning and transmission microscopy, and MUC1 glycoform, sex steroid receptor, and interleukin receptor (type 1) expression as biomarkers of endometrial receptivity using carefully characterized clinical fertile and infertile groups of women. Using a combination of immunohistochemistry and scanning electron microscopy (SEM) called scanning immunoelectron microscopy (SIM), we confirmed that MUC1 mucin was not associated with the endometrial pinopodes, which have been linked with embryo adhesion. We also showed that failure of embryo implantation was associated with an abnormal endometrial expression of MUC1 mucin, and retention of nuclear progesterone receptor (PR) particularly in epithelial cells. Hematoxylin and eosin staining, transmission electron microscopy (TEM), SEM in isolation and immunohistochemistry for interleukin receptor were not shown to be useful markers. Progesterone-dependent regulation of MUC1 appears to be an important factor in determining endometrial receptivity.

Ultrastructure of the early human feto-maternal interface co-cultured in vitro.

BACKGROUND: The study was designed to investigate the ultrastructural features of the early human feto-maternal interface when generated by in-vitro co-culture, and compare these with findings reported previously from human pregnancies. METHODS: Placental villi and decidua parietalis tissues from 8-12 week pregnancies were co-cultured in vitro over a 4-day period. The co-incubations were ended at 24 h intervals and processed for electron microscopical studies, and for immunocytochemistry using anti-cytokeratin antibody (CAM 5.2) for trophoblast. RESULTS: Loss of the syncytium at points of contact with the decidual stroma, cytotrophoblast column formation, differentiation and invasion of extravillous trophoblast (EVT) cells into the decidual stroma over the 4-day period of co-culture were observed. Cellular components, such as actin filaments, microtubules, glycogen granules and lamellipodic processes found in EVT cells were consistent with active cellular locomotion. CONCLUSIONS: These ultrastructural studies emphasize the usefulness of this model in investigating the formation of the feto-maternal interface of human pregnancy. The recruitment of cytotrophoblast to the syncytium by a process involving fusion of the intervening plasma membranes, and the migration of EVT cells causing little or no damage to the surrounding decidual cells, resemble in-vivo data.

Salmonella maintains the integrity of its intracellular vacuole through the action of SifA.

A method based on the Competitive Index was used to identify Salmonella typhimurium virulence gene interactions during systemic infections of mice. Analysis of mixed infections involving single and double mutant strains showed that OmpR, the type III secretion system of Salmonella pathogenicity island 2 (SPI-2) and SifA [required for the formation in epithelial cells of lysosomal glycoprotein (lgp)-containing structures, termed Sifs] are all involved in the same virulence function. sifA gene expression was induced after Salmonella entry into host cells and was dependent on the SPI-2 regulator ssrA. A sifA(-) mutant strain had a replication defect in macrophages, similar to that of SPI-2 and ompR(-) mutant strains. Whereas wild-type and SPI-2 mutant strains reside in vacuoles that progressively acquire lgps and the vacuolar ATPase, the majority of sifA(-) bacteria lost their vacuolar membrane and were released into the host cell cytosol. We propose that the wild-type strain, through the action of SPI-2 effectors (including SpiC), diverts the Salmonella-containing vacuole from the endocytic pathway, and subsequent recruitment and maintenance of vacuolar ATPase/lgp-containing membranes that enclose replicating bacteria is mediated by translocation of SifA.

Characterization of human placental explants: morphological, biochemical and physiological studies using first and third trimester placenta.

The primary objective of this study was to characterize an in-vitro model of the human placenta using morphological, biochemical and physiological parameters. Placental villi were obtained from normal first trimester and term pregnancies. The villi were incubated with Dulbecco's modified Eagle's medium: Ham's F12 nutrient mixture in a shaking water bath at 37 degrees C for up to 310 min. The viability was determined by the production of beta human chorionic gonadotrophin (HCG) and lactic dehydrogenase (LDH) and the incorporation of [3H]thymidine, [3H]L-leucine and L-[U14C]arginine, while ultrastructure was assessed by transmission electron microscopy. In the first and third trimester group, the release into the medium of the intracellular enzyme LDH remained unaltered throughout the experiment. By contrast, beta-HCG concentrations increased linearly and concentrations were higher in the first trimester than term villi (354.5 +/- 37.8 versus 107 +/- 8.1 IU/g villi protein; P < 0.001). Electron microscopy confirmed preservation of tissue viability for up to 4 h of incubation. The incorporation of thymidine (12.2 +/- 2.9 versus 5.2 +/- 0.5 nmol/g villi protein; P < 0.05), leucine (9.4 +/- 2.1 versus 1.9 +/- 0.4 nmol/g villi protein; P < 0.02) and arginine (17 +/- 4.4 versus 4.2 +/- 0.5 nmol/g villi protein; P < 0.05) were markedly higher in early than in term placenta. Furthermore, placental uptake of L-leucine by the first (9.4 +/- 2.1 versus 17 + 4.4 mol/g villi protein; P < 0.001) and third trimester placental villi (1.9 +/- 0.4 versus 4.2 + 0.5 mol/g villi protein; P < 0.001) was less than that of L-arginine. This study describes a simple technique using placental explants to determine relative rates of uptake of substrate amino acids throughout gestation.

The myelotoxicity of chloramphenicol: in vitro and in vivo studies: I. In vitro effects on cells in culture.

1 Chloramphenicol is used extensively in non-industrialized countries for the treatment of life-threatening infections because it is cheap and effective, despite its known hemotoxicity and linkage to fatal aplastic anaemia. It is important to define the mechanism of toxicity so that means can be devised to ameliorate the toxic effects in order to produce safer usage. 2 Chloramphenicol, at concentrations from 5 mM to 2 mM initiated apoptosis in dividing cells from a monkey kidney-derived cell line and in haematopoietic progenitor cells from human neonatal cord blood. 3 Growth of progenitor cells was suppressed at concentrations of chloramphenicol which would be considered less than therapeutic during patient treatment. 4 These effects could be ameliorated in progenitor cells by co-culture with the antioxidant mercaptoethylamine and in monkey kidney cells by co-culture with vitamin C. 5 This is the first report of apoptosis in chloramphenicol toxicity and suggests a possible link between a metabolic event i.e. the production of free radicals; a morphological effect, apoptosis; and a clinical effect, bone marrow suppression and aplastic anaemia.

Transport and metabolism of thyrotrophin-releasing hormone across the fetal membrane.

To determine the transfer and metabolism of TRH by human fetal membranes, the bidirectional transport and uptake of TRH was investigated by adding 125I-labeled TRH (100,000 cpm) or commercial TRH either to the maternal or the fetal compartment of an in vitro model of cultured human fetal membranes obtained from term and preterm placenta. Transmembrane transfer was also studied in the presence of 200 microM p-hydroxy-mercuriphenyl-sulphonic acid (p-HMSA), a dipeptidase enzyme inhibitor. Creatinine and heparin were used as an internal markers. Metabolites of TRH were separated from intact molecules by gel filtration on Sephadex G-10. The structural integrity of the membrane was confirmed by electron microscopy. The transmembrane transfer of radiolabeled and commercial TRH were comparable across both preterm and term placenta. When transport was studied from the maternal to fetal side, the maternal concentration of TRH declined rapidly from 100% at time 0 to 19.31 +/- 2.26% at 8 h with a concomitant increase in the fetal concentration from undetectable to a maximum of 2.56 +/- 0.38% with a fetomaternal ratio of 0.16 +/- 0.01. Transfer of TRH from the fetal to maternal compartment was similar to that of maternal to fetal. Chromatography of maternal and fetal media showed that TRH was metabolized by the membrane into small molecular weight fragments. Treatment of the membrane with p-HMSA increased TRH transport from the maternal to fetal compartment to 18.12 +/- 0.91 (P < 0.001) with an fetomaternal ratio of 0.35 +/- 0.02 (P < 0.001). Although transmembrane transfer of TRH from the fetal to maternal side was also increased by p-HMSA, levels achieved were less than that from maternal to fetal (12.26 +/- 1.50%; P < 0.05). These results suggest that the human fetal membrane acts as an enzymatic barrier to the bidirectional transfer of TRH from 24 weeks gestation.

The endometrial nucleolar channel system as an indicator of progestin potency in HRT.

A quantitative assessment has been made of nucleolar channel systems (NCS) in the endometrial glands of postmenopausal women receiving hormone replacement therapy. The women were taking conjugated equine oestrogen and one of five progestins. The number of NCS induced was related to the dose of progestin administered. The minimum doses of progestin inducing a comparable response to premenopausal secretory phase endometria were found to be 1-2.5 mg norethindrone, 150 micrograms norgestrel and 20 mg dydrogesterone. Progesterone and medroxyprogesterone acetate were inadequate at the doses tested. The results show that the quantification of endometrial gland NCS would be a useful addition to the biochemical and morphological assessments made of any new progestin treatment.

Asthenozoospermia and the human sperm mid-piece.

This study provides a quantitative comparison between surface and ultrastructural features of motile spermatozoa in asthenozoospermic and fertile men. The study group consisted of 10 individuals with persistent asthenozoospermia and the controls were 10 fertile donors to a sperm bank. Scanning electron microscopy and image analysis were used to objectively measure sperm mid-piece and tail dimensions. Sperm mid-piece length was significantly shorter (P < 0.01) in asthenozoospermic subjects compared with the controls, with mid-piece width and tail length being comparable. Mid-piece ultrastructure was then examined with the transmission electron microscope and the number of mitochondrial gyres and their configuration recorded. At the ultrastructural level the asthenozoospermic subjects demonstrated significantly fewer mitochondrial gyres (P < 0.001) than their fertile counterparts. Energy for sperm movement is provided by mitochondria and a deficit in these organelles in the sub-fertile cohort provides an explanation for poor sperm function in these subjects.

Morphometric characteristics of motile spermatozoa in subfertile men with an excess of non-sperm cells in the ejaculate.

A comparison has been made between the morphological dimensions of motile spermatozoa in subfertile men demonstrating a persistent excess of non-sperm cells in their semen and those found in spermatozoa from fertile controls. Scanning electron microscopy and image analysis were used to make a morphometric assessment of defined sperm parameters in the study and control subjects. Half of the study cohort had an excess (> 5 x 10(6)/ml) of seminal leukocytes and the remainder an excess of sperm precursors in the ejaculate. In subjects with a sperm precursor excess, the motile spermatozoa had several significantly larger head parameters than those from both the fertile controls and those men with a leukocyte excess. Mid-piece and tail measurements did not differ significantly between groups. These findings suggest that, where there are large numbers of immature germinal elements present in semen, there is aberrant morphological development of the motile, apparently mature, spermatozoa which may represent disordered spermatogenesis.

Fertility in men with primary ciliary dyskinesia presenting with respiratory infection.

BACKGROUND: Primary ciliary dyskinesia is characterised by chronic rhinosinusitis, chronic bronchial sepsis (usually with bronchiectasis), dextrocardia in approximately 50% of cases, and male infertility. The latter, described in patients attending infertility clinics, results from immotile but viable spermatozoa. Experience in a respiratory clinic suggests that infertility in men is not invariable. METHODS: The seminal fluid of 12 men with primary ciliary dyskinesia, six with dextrocardia, who presented consecutively with upper and lower respiratory tract sepsis was examined. Nasal ciliary beating was dyskinetic or absent in all cases, and nasal ciliary ultrastructure was abnormal in those 11 patients examined. RESULTS: Viable but immotile spermatozoa with abnormal tail ultrastructure were found in the ejaculate of only two patients. Two other patients had apparently fathered children; seminology in both these cases showed a normal spermatozoa count, one with normal spermatozoal motility and normal ultrastructure, the other with moderately reduced spermatozoal motility and abnormal ultrastructure (dynein arm deficiency on the peripheral microtubule doublets). A further two patients had normal spermatozoa counts, normal spermatozoa tail ultrastructure, and normal or only moderately reduced motility of spermatozoa. The spermatozoa of one patient were normally motile but there was severe oligozoospermia, and five patients were azoospermic. CONCLUSIONS: Not all men with primary ciliary dyskinesia have immotile spermatozoa. Seminal analysis is recommended in men with primary ciliary dyskinesia so that accurate counselling about reproductive capability may be given.

A quantitative study of sperm head ultrastructure in subfertile males with excess sperm precursors.

OBJECTIVE: To make an objective comparison between sperm head ultrastructure in fertile subjects and a subfertile cohort with an excess of immature germinal elements in the ejaculate. DESIGN: A quantitative analysis of ultrastructural features of the sperm head using transmission electron microscopy in the defined groups. PATIENTS: Ten men of proven fertility as controls and 10 subfertile subjects with a persistent excess of sperm precursors in the ejaculate were investigated. SETTING: The Infertility Clinic at Queen Charlotte's and Chelsea Hospital, London. MAIN OUTCOME MEASURES: Each individual in the study achieved a score for a range of previously defined features of sperm head ultrastructure. These scores provided the basis for comparison between fertile and subfertile subjects. RESULTS: Subfertile individuals were found to have motile sperm with significantly more hypoplastic, detached, and abnormally shaped acrosomes than fertile controls. Sperm nuclei in these subjects also contained significantly more intranuclear vacuoles and immature chromatin and were associated more commonly with cytoplasmic droplets than fertile controls. CONCLUSION: Men with an excess of sperm precursors in the ejaculate have motile sperm with a range of abnormalities involving the nucleus and acrosome to account for reduced functional competence.

Two techniques for electron opaque staining of elastic fibres using tannic acid in fresh and formalin fixed tissue.

Two electron microscopic staining techniques, one using tannic acid-glutaraldehyde as a fixative, and the other using tannic acid-uranyl acetate solution as a stain on ultra-thin sections of glutaraldehyde fixed material, were directly compared for elastic fibre staining on several human and animal tissues. Various concentrations of tannic acid were compared using both techniques. The two techniques were also compared on formalin fixed tissues. The use of tannic acid-uranyl acetate solution as a stain on processed tissue is by far the more consistent technique and achieves equally good results on glutaraldehyde or formalin fixed tissue. It is suggested that the use of the term tannic acid technique/method should be reserved for this particular method to achieve a meaningful interpretation of results in scientific papers.

Epithelial cell morphology and airspace size in hypoplastic human fetal lungs associated with oligohydramnios.

The relative frequency of different types of respiratory epithelial cells in normal fetal lungs (control, CON) and hypoplastic lungs associated with oligohydramnios (OH) was determined at the electron microscopic level and airspace size was measured. At 24+ weeks CON lungs had 82.4 +/- 1.2% undifferentiated cells, 15.9 +/- 1.2% type II cells, and 1.7 +/- 0.4% type I cells (n = 3), whereas OH lungs had 94.6 +/- 2.1% undifferentiated cells, 5.4 +/- 2.1% type II cells, and no type I cells (n = 3). At 36+ weeks CON lungs had 7.8 +/- 3.4% undifferentiated cells, 46.1 +/- 3.1% type II cells, and 46.1 +/- 1.4% type I cells (n = 3), whereas OH lungs had 37.7 +/- 1.2% undifferentiated cells, 42.5 +/- 1.7% type II cells, and 19.8 +/- 0.8% type I cells (n = 3). Differences between CON and OH lungs in the proportions of undifferentiated and type I cells at 36+ weeks were highly significant (p less than .001), whereas type II cell proportions did not differ significantly in either age group. The proportion of lung occupied by airspaces increased from 38.3% at 24+ weeks to 68.7% at 36+ weeks in CON lungs but only from 26.7% to 35.7% in OH lungs. The differences between the groups were significant at both 24+ weeks (p less than .01) and 36+ weeks (p less than .001). Mean airspace size in CON lungs varied from 2.8 x 10(-6) mm2 at 24+ weeks to 4.4 x 10(-6) mm2 at 36+ weeks and in OH lungs from 1.7 x 10(-6) mm2 at 24+ weeks to 2.7 x 10(-6) mm2 at 36+ weeks. These results give quantitative expression to the severity of impaired morphologic maturation in OH lungs.

Failure of elastin development in hypoplastic lungs associated with oligohydramnios: an electronmicroscopic study.

Pulmonary hypoplasia associated with oligohydramnios results in delayed maturation of fetal lung as assessed at light microscope level. Using an elastin-specific electron-microscopic staining technique, we report absent elastic tissue development in the septal crests of these lungs even at term.

The inhibitory activity of a peptide derivative against the growth of simian immunodeficiency virus in C8166 cells.

The peptide derivative Ro 31-8959 is a potent and selective inhibitor of the aspartic proteinases encoded by HIV-1 and HIV-2 and it arrests the growth of both viruses in cell culture. We have demonstrated similar effects against the simian immunodeficiency virus SIVmac251 in the human T-cell line, C8166 (ED50 = 6nM) with a therapeutic index of 4,500. The antiviral activity of Ro 31-8959 was 250 and 22 times greater than that of ddI and ddC, respectively. The mode of action was confirmed by accumulation of the polyprotein p55 with concomitant reduction of the cleavage product, p27, and by the production of immature virions.

Two-sided culture of human placental trophoblast. Morphology, immunocytochemistry and permeability properties.

We describe the culture of human term placental trophoblast cells on cell-free amniotic membrane, with medium on both sides. Over the course of 2 days, the isolated cells, initially simple, mononucleated and probably cytotrophoblast, form a confluent layer of multinucleated syncytial cells with morphological and immunocytochemical properties of syncytiotrophoblast. This layer becomes polarized with respect to morphology, alkaline phosphatase distribution and hCG secretion. Contamination with amnion cells, and with other cell types that are present in placental tissue, was less than 1 per cent. A preliminary investigation of the permeability properties of the preparation showed that the trophoblast cell layer, rather than the amniotic membrane, was rate-limiting to transtrophoblast transfer, but that possible effects of the supporting membrane should be considered. The transtrophoblast transfer of D-glucose and the non-metabolisable analogue, 3-O-methyl-D-glucose (3OMG), had saturable and non-saturable/leak components in both directions, indicating that carrier-mediated processes were involved. The non-metabolisable amino acid 2-aminoisobutyrate (AIB) was both accumulated within the trophoblast cells, and transferred by saturable and non-saturable processes from the microvillous side, but no saturable accumulation or transfer was observed from the basal side, at the concentrations tried. The results suggest that this model may prove suitable for studies of transtrophoblast transfer.

A survey of the ultrastructural defects associated with absent or impaired human sperm motility.

The sperm tails of 400 patients having absent or impaired sperm motility were examined by electron microscopy. A wide variety of fine-structural defects were observed although all of the patients fell into clearly defined groups. Total or partial dynein arm deficiency was observed in 12 patients (3%). Ninety-one patients (23%) had sperm with a spectrum of fine-structural defects, whereas 90 patients (23%) were necrospermic. Subjects with low motility, but with at least a few tails of normal structure, had a 5% pregnancy rate, whereas those patients with similar overall motility, but in whom no normal sperm were seen, produced no pregnancies. The results confirm the importance of making an electron microscopical examination of the sperm of patients with asthenozoospermia.

Are fixed-dose oestrogen/progestogen combinations ideal for all HRT users?

Although progestogens protect the endometrium against excessive oestrogen-induced stimulation, they can cause adverse symptomatic and psychological effects and may have undesirable metabolic consequences. Thus, the minimum progestogen dose which results in consistent endometrial transformation should be prescribed. To define this dose for norethisterone and dl-norgestrel, 197 endometrial samples obtained from postmenopausal women receiving conjugated equine oestrogens (0.625 mg or 1.25 mg daily) with one of six doses of norethisterone (or the acetate), or one of three doses of dl-norgestrel added for the first 12 days of each calendar month were examined with the light microscope; 109 samples were also assessed by transmission electron microscopy. There was an inverse relation between the percentage of samples showing proliferative features and the progestogen dose. However, proliferative endometrium was observed in 6% of samples with the highest dose of dl-norgestrel (500 micrograms) and in 3% of samples with 2.5 mg norethisterone. Conversely, complete secretory transformation was observed in 25% of samples with the lowest dose of norethisterone (0.1 mg) and in 40% of samples with 75 micrograms dl-norgestrel. Mild atypical hyperplasia was diagnosed in four samples. There was a wide inter-patient variation in response and none of the nine progestogen dose regimens induced secretory change in every patient.

Ultrastructural changes and immunocytochemical analysis of human placental trophoblast during short-term culture.

Trophoblastic cells, of at least 95 per cent purity by immunofluorescence and morphological criteria, were obtained from human term placenta by a simple trypsinisation method without the additional purification steps or complex culture conditions used by others. The differentiation of these cells was followed over four days in culture by fluorescence immunocytochemistry, by scanning and transmission electron microscopy and by light microscopy. The results support the idea that the isolated cells are cytotrophoblast and that these differentiate during this time into cells with characteristics of villous syncytiotrophoblast. This process involved first the formation of a multicellular layer of mononucleated cells, then the development of a syncytium of multinucleated cells and, not necessarily concurrently, functional differentiation. This may be a useful model for the study of syncytiotrophoblast function.