The glycoprofile patterns of endothelial cells in usual interstitial pneumonia.

BACKGROUND: The pathological classification of cryptogenic fibrosing alveolitis has been a matter of debate and controversy for histopathologists. OBJECTIVE: To identify and specify the glycotypes of capillary endothelial cells in usual interstitial pneumonia (UIP) compared to those found in normal tissue. METHODS: Sections of formalin-fixed, paraffin-embedded blocks from 16 cases of UIP were studied by lectin histochemistry with a panel of 27 biotinylated lectins and an avidin-peroxidase revealing system. RESULTS: High expression of several classes of glycan was seen de novo in capillary endothelial cells from patients with UIP including small complex and bi/tri-antennary bisected complex N-linked sequences bolund by Concanavalin A and erythro-phytohemagglutinin, respectively, GalNAca1 residues bound by Helix pomatia and Maclura pomifera agglutinins, and L-fucosylated derivatives of type II glycan chains recognized by Ulex europaeus agglutinin-I. Glycans bound by agglutinins from Lycopersicon esculentum (ß1,4GlcNAc) and Wisteria floribunda (GalNAc) as well as GlcNAc oligomers bound by Phytolacca americana and succinylated Wheat Germ agglutinin were also seen in the capillary endothelial cells of UIP. In contrast, L-fucosylated derivatives of type I glycan chains were absent in cells from cases of UIP when Anguilla anguilla agglutinin was applied, unlike the situation in normal tissue. CONCLUSION: These results may indicate existence of two distinct populations of endothelial cell in UIP with markedly different patterns of glycosylation, reflecting a pattern of differentiation and angiogenesis, which is not detectable morphologically.

Glycophenotype of prostatic carcinomas.

The factors that affect the progression of prostatic carcinoma are poorly understood, but it is known that carbohydrate antigens on the tumour cell surface play a role in the transforming and metastatic processes. The present report aimed to perform a comparative, lectin-histochemical study of benign and carcinomatous prostates, using a battery of lectins, in combination with monoclonal antibodies against Lewis antigens, and a semi quantitative study, to investigate the changes in glycosylation patterns that occur in prostatic carcinoma. Blocks from 27 necropsy cases of prostatic carcinoma were sectioned and stained with H+E, fifteen biotinylated lectins chosen to probe for a wide range of oligosaccharide sequences within several categories of glycoprotein glycans, using a lectin-biotin avidin-peroxidase method, and monoclonal antibodies against Lewisa, sialyl Lewisa and sialyl Lewisx antigens. The glycophenotype of prostatic carcinoma differed from that of the noncancerous prostate in revealing more intense staining with the following lectins (AAA, UEA-1, DBA, WFA, VVA, HPA, BSA-1B4, MPA, ECA, AHA, and CTA), while the binding patterns of (GNA and NPA) were almost similar in both prostatic carcinoma and the noncancerous prostate. Lewis antigens are found to be expressed in prostatic carcinomas but not in the noncancerous prostate. The observations of this study suggest that the gylcophenotype of transformed prostatic cells was modified. It showed a moderate increase in, and changing patterns of, fucosylation and galactosylation, increased branching of side chains and sharp rise in 2 deoxy, 2 acetamido galactosylation and masking process by sialylation, especially by α2-3 and α2-6 linkages. All these changes in the glycosylation pattern of the transformed prostatic cells were observed on O-glycans, no changes were observed on N-glycans.

Lectin and other histochemical studies of the articular cartilage and the chondro-osseous junction of the normal human knee joint.

There are few studies on normal, adult diarthrodial joints which look in detail at the histochemical properties of the chondro-osseous junctional region. This study of the normal human knee joint was performed using lectin and other histochemical techniques. There were differences in the reactions of mineralised cartilage compared to those of hyaline cartilage with the former demonstrating more collagen and less glycosaminoglycans. Lectin histochemistry revealed more accessible terminal 2-deoxy,2-acetamido-alpha-D: -galactose and more N-acetyllactosamine but less fucosyl and alpha-2,6-linked-sialyl termini in the mineralised cartilage. The hyaline cartilage chondrocytes stained for N-glycans but those of mineralised cartilage did not. The staining patterns of prolongations and islands of uncalcified cartilage running through the calcified layer to abut bone and marrow spaces were distinct, resembling the patterns of the hyaline cartilage but with some unique features. A possible relationship was revealed between the presence of the Maclura pomifera ligand (Galbeta1,3GalNAcalpha1-) and mineralisation. Subchondral bone had a markedly restricted glycoprofile.

The tidemark of the chondro-osseous junction of the normal human knee joint.

The chondro-osseous junction includes the junction between calcified and non-calcified cartilage matrices often referred to as the tidemark. A detailed knowledge of the structure, function and pathophysiology of the chondro-osseous junction is essential for an understanding both of the normal elongation of bones and of the pathogenesis of osteoarthrosis. In this study the molecular anatomy of the tidemark was studied using histochemical techniques, including lectin histochemistry, on blocks of normal cartilage from human knee joints. The tidemark stained with H and E, picro-sirius red, toluidine blue, safranin O and methyl green, but not with alcian blue in the presence of magnesium chloride at 0.05 M or above. It stained with only four lectins, those from Datura stramonium, Maclura pomifera, Erythrina crystagalli and Helix pomatia, out of the 19 used. Therefore, it is rich in collagen and contains hyaluronan, but appears to lack the glycosaminoglycans of 'conventional' proteoglycans and it expresses a very limited and distinctive lectin staining glycoprofile, which is probably attributable to specific glycoproteins. In addition, the tidemark had a distinct microanatomical trilaminate appearance. From all of these results it is clear that this part of the chondro-osseous junctional region is chemically more complex and distinctive than has previously been described.

Application of solid-phase micro-extraction technology to drug screening and identification.

Benzodiazepines, tricyclic antidepressants and local anaesthetics are frequently involved in poisoning episodes and fatalities. A specific, sensitive and rapid procedure for identifying and quantifying such drugs in postmortem matrices has been developed using solid-phase micro-extraction (SPME) and gas chromatography-mass spectrometry. Very clean extracts were obtained in one step using SPME. The most commonly used fibre coatings were tested to select the best coating for SPME of the drugs. The appropriate fibre coating for most drugs was polyacrylate, followed by Carbowax-divinylbenzene. A Hewlett-Packard 5890 gas chromatograph in combination with a Trio 2000 mass spectrometer was used to analyse the samples. Temperature, time, pH and addition of sodium chLoride were optimized to obtain consistent extraction. The between-day and within-day coefficients of variation were less than 16% and less than 6%, respectively.

Magnetic resonance imaging results in patients with central electronystagmography findings.

The objective of this study was to determine the findings on magnetic resonance imaging (MRI) in patients identified as having central vestibular abnormalities on electronystagmography (ENG) testing, to discuss the issue of 'gold standard' in the investigation of central oculo-vestibular system diseases and to present a model for understanding this area. A retrospective review of the case notes of patients (n = 23) found to have central ENG findings at vestibular assessment and for whom MRI scanning data was available was undertaken. Each patient underwent a full ENG evaluation, including gaze, ocular-motor and caloric testing, and MRI. Only seven of the patients with central ENG findings had abnormal MRI scans. Thus, the incidence of the identification of structural abnormality on MRI in patients with central ENG findings is low. These investigations are complementary in the investigation of balance disorder patients.

An investigation of the audiologic handicap associated with unilateral sudden sensorineural hearing loss.

OBJECTIVE: To determine the incidence of tinnitus and associated handicap after unilateral sudden sensorineural hearing loss (SSNHL); in addition, to determine the hearing handicap experienced as a consequence of such a loss. STUDY DESIGN: Identification of patients and determination of demographic and audiologic data by retrospective case review; determination of handicap and distress by postal questionnaire. SETTING: Teaching hospital department of otolaryngology. PATIENTS: Thirty-eight patients were identified as having been treated for a unilateral sudden sensorineural hearing loss in the period 1988 through 1997. Of those, 21 (55.3%) replied to the questionnaire. MAIN OUTCOME MEASURES: Audiometric data at admission and at 4-week follow-up, Tinnitus Handicap Inventory (THI), visual analogue scales of tinnitus loudness and distress, Hearing Handicap Inventory in Adults (HHIA). RESULTS: The questionnaire responder group did not significantly differ from the questionnaire nonresponder group on demographic nor audiometric variables, and hence were considered to be a representative sample. Tinnitus was present in 14 patients (67%). Hearing handicap was found in 86% of patients (of the 21 questionnaire responders) and tinnitus handicap in 57% (of the 14 with tinnitus). Correlations were found between tinnitus loudness, distress, and handicap. There was no correlation between time elapsed since SSNHL and tinnitus or hearing handicap, nor was there a correlation between the extent of audiometric loss and hearing or tinnitus handicap. A strong negative correlation was, however, found between recovery in audiometry in the first 4 weeks after onset and tinnitus and hearing handicap. The audiometric status of the contralateral ear correlated with hearing handicap. CONCLUSIONS: A majority of patients after unilateral SSNHL have a perceived handicap associated with tinnitus and hearing. Although this condition is an otologic emergency, careful thought should be given to the audiologic rehabilitation of this patient group.

Glycosylation in the near-term epitheliochorial placenta of the horse, donkey and camel: a comparative study of interbreeding and non-interbreeding species.

Studies from this laboratory have shown great diversity in the glycosylation of tissues comprising the interhaemal barrier of species with different placental types. This diversity may be one of the factors preventing interbreeding between species. Glycan expression within the uterine epithelium and trophoblast of the interhaemal barrier was examined to test this proposition in three species with similar diffuse, microcotyledonary, epitheliochorial allantochorionic types of placenta: the horse (Equus caballus) and donkey (Equus asinus), which can interbreed with each other, and the camel (Camelus dromedarius), which cannot interbreed with either of the other two species. A panel of 14 lectins was used and it was found that glycosylation patterns were generally similar between placental tissues of the horse and donkey, except for the expression of non-bisected complex N-glycan and some sialic acids, whereas those of the camel showed striking differences in the binding of lectins to many structures carrying terminal residues of fucose, N-acetyl galactosamine and beta-galactose, as well as to complex N-glycans and sialic acids. These results are consistent with the proposition that interbreeding species carry similar glycans in tissues forming the interhaemal barrier whereas glycodiversity is one of the factors preventing implantation and subsequent placental development in interspecific hybrids.

Mycophenolic acid does not inhibit protein glycosylation in T lymphocytes.

BACKGROUND: Mycophenolic acid inhibits guanosine nucleotide synthesis and has been shown to be a potent inhibitor of lymphocyte proliferation as well as being effective at decreasing the incidence of graft rejection. Guanosine nucleosides are essential for protein glycosylation and many cell surface proteins including adhesion molecules, which are important for graft infiltration and rejection, are glycoproteins. There have been conflicting reports concerning the ability of MPA to interfere with glycosylation in lymphoid cells. Therefore, the purpose of this study was to investigate the effects of MPA on cell surface protein glycosylation in lymphoid cells. METHODS: Cells were cultured in the presence of increasing concentrations of MPA for different lengths of time and stained with fluorescent-labelled lectins specific for either mannose or fucose residues on glycoproteins. Analysis was then performed by flow cytometry. RESULTS: MPA treatment had no effect on the binding of either fucose or mannose-specific lectins to Con A stimulated human PBLs and rat lymph node lymphocytes or to a CEMC7a T cell line. CONCLUSION: The results show that, contrary to previous reports, MPA does not affect cell surface glycosylation in T cells using T cells from different sources of both human and non-human origin.

Equine placental cup cells show glycan expression distinct from that of both chorionic girdle progenitor cells and early allantochorionic trophoblast of the placenta.

Using lectin histochemistry on plastic-embedded material, the glycosylation patterns of equine girdle and cup cells, and associated endometrial glands, have been investigated from 37 to 67 days gestation. Results were compared with the glycosylation of the 50-day allantochorionic trophoblast of the established equine placenta that will later form the microcotyledons. The differentiated cup cells, which secrete equine chorionic gonadotropin (eCG), showed a pattern of glycosylation that was distinct both from the progenitor girdle cells and the allantochorionic trophoblast, with granules that bound lectins indicating high levels of alpha2,6 and alpha2,3-linked sialic acid, N-acetyllactosamine and bi/tri antennary non-bisected and bisected complex N-glycan. This is consistent with the known carbohydrate content of eCG. In contrast, the allantochorionic trophoblast at 50 days lacked detectable amounts of sialic acid and showed high levels of tri/tetra-antennary non-bisected complex N-glycan and N-acetyl galactosamine which was absent in the cup cells. During the process of girdle cell migration into maternal tissues, the uterine glands became greatly enlarged and dilated basally, with increased amounts of glycosylated secretory products revealed by lectins, which often seeped out into the extracellular space via ruptures in the apical regions of the gland wall.

A lectin binding analysis of glycosylation patterns during development of the equine placenta.

The glycosylation of the equine interhaemal barrier and areola was studied throughout the period of gestation. Placentae of 35, 37, 50, 119, 152, 200, 280 and 300 days gestation were investigated, using semithin plastic embedded sections and a panel of 15 biotinylated lectins with an avidin-peroxidase revealing system. Glycosylation of the trophoblast and maternal epithelium showed the most change during the first 50 days of gestation, being associated with the initial stages of adhesion and attachment. In the trophoblast, non-bisected tri/tetraantennary complex N-glycan was only evident after day 37 and terminal N-acetyl galactosamine, alpha2,3- and alpha2,6-linked sialic acids disappeared at the same time. The areolar trophoblast exhibited some differences from microcotyledonary areas, especially with respect to 2-deoxy, 2-acetamido alpha-galactose and tri/tetraantennary, non-bisected complex N-glycan, suggesting that the differences in function between microcotyledonary and areolar trophoblast are reflected at both the morphological and the biochemical level. Granules of the maternal uterine epithelium bound many lectins, particularly those with specificity for bisected and non-bisected bi/triantennary N-linked glycan, 2-deoxy, 2-acetamido alpha-galactosyl, beta-galactosyl and some fucosylated termini. Binding to sialic acids in alpha2,3- and alpha2,6-linkage was sparse. Maternal and fetal capillaries showed little change in glycan expression over the period studied, being rich in bisected and non-bisected bi/triantennary N-linked glycan and sialic acids, with some terminal N-acetyl galactosamine and no detectable terminal fucosyl residues.

Outcomes from adult implantation, the first 100 patients.

We present the outcome of implantation in the first 100 adult patients treated under the Midland Cochlear Implant Programme. All patients were post-lingually deaf with profound or total hearing loss. Performance was tested in lip-reading, implant only and combined lip-reading and implant modes using BKB sentences, connected discourse tracking (CDT) and environmental sound recognition. Assessments were made at nine and 18 months post-implant. The dominant aetiologies were idiopathic and meningitis. Meningitis was associated with the greatest numbers of ossified cochleas. Forty-three per cent of cases of partial ossification were identified only at surgery. Four per cent of patients became non-users of their devices, however the majority used their implants for more than 12 hours each day. The mean scores at nine months post-implant, in the implant only mode, were for environmental sound recognition 56.7 per cent, for BKB sentences 46.6 per cent (80 per cent of patients scored above 0 per cent) and for CDT 31.2 words per minute (w.p.m.) (62 per cent scored above zero per cent). In the combined lip reading and implant mode the mean scores, at nine months, were for BKB sentences 81.5 per cent and for CDT 65.8 w.p.m. All results were sustained at 18 months. Patients reported that implantation significantly reduced their hearing handicap. Pre-operative measures of depression were also significantly reduced at nine months post-implant. Results were sustained at 18 months. Post-operative audiological outcomes in the electrical stimulation only mode correlated significantly with length of profound deafness. Results suggest that performance outcome is also related to the number of active electrodes.

Medical, surgical and audiological complications of the first 100 adult cochlear implant patients in Birmingham.

Of the first 100 patients implanted on the Midland Cochlear Implant Programme the commonest aetiologies of deafness were idiopathic 31 per cent, meningitis 28 per cent and cochlear otosclerosis 16 per cent. The major complication rate was three per cent. The most severe was one individual who post-operatively developed a cerebral infarct and subsequently died. The minor complication rate was 39 per cent, all of which successfully resolved, and included 11 cases of wound infection, nine cases of vertigo, three transient facial palsies and two post-operative bleeds. Older patients and men were most likely to have a post-operative medical complication. Women were more likely to have an abnormal electrode insertion. Meningitis and otosclerosis were the most complicated aetiologies in terms of cochlear ossification and electrode insertion. A non-patient cochlea was associated with fewer active electrodes. In six cases which had been reported pre-operatively as showing patent cochleas, some form of obstructional ossification was encountered. Patients functioning with greater than 15 active electrodes performed better on auditory tests than patients with fewer than 15 active electrodes.

Electrode complications in 100 adults with multichannel cochlear implants.

At switch-on (first post-operative stimulation of the implant) and during subsequent reprogramming, electrodes can, in some patients, be found to be non-functional or to be performing sub-optimally for a number of reasons. This paper examines the reasons for the poor performance of these electrodes by means of a retrospective analysis of 100 patient records. All of these patients received the Nucleus multichannel device. The most common reason for an electrode to require de-activation was found to be facial nerve stimulation, with poor sound quality and pain also being very common. Other reasons included absence of auditory stimulation, vibration, reduced dynamic range, throat sensations, absence of loudness growth or dizziness. The occurrence of these reasons along the electrode array was examined, more basal electrodes being found to be non-functional as a result of having a small dynamic range or poor sound quality. Pain and vibration were found to occur throughout the array and the more apical electrodes were found to be non-functional as a result of facial nerve stimulation. It is suggested that the electrodes at the basal end of the array are likely to be extra-cochlear or are at the site of the most cochlear damage, whereas the more apical electrodes lie in closer proximity to the facial nerve.

Tinnitus, cochlear implants and how they affect patients.

The relationship between tinnitus and cochlear implantation is an important issue that needs to be established because it may affect implant use. In this study 99 patients over 15 years of age completed pre- and post-cochlear implantation questionnaires, and underwent performance testing. The findings show that after implantation, there was marked suppression of tinnitus in both implanted and contralateral ears whilst the implant was off, and this was further enhanced when the implant was switched on. These effects are probably a combination of local and central factors. Presence of tinnitus, before or after implantation, had no detrimental effects on performance. In conclusion, providing all other factors permit, this study recommends implanting the ear with the worst tinnitus.

Expression of proteoglycans by cultured chick sternal chondrocytes.

Chondrocytes isolated from the caudal and cephalic ends of the sterna of embryonic chicks were cultured in collagen gels. Differences were found, histochemically, between the proteoglycans produced by the "caudal" and "cephalic" cultures and with length of time in culture. The cultures were labelled with [14C]galactose and [35S]sulphate at 7 and 21 days in culture and labelled compounds from media, and cell and matrix extracts analysed with Sepharose CL-2B. A large aggrecan-like proteoglycan was detected in the media with some aggregated proteoglycans found in the cell extracts even under the dissociating conditions used. One group of 14C-labelled compounds, found in the cell and matrix extracts, was equivalent in size to chick aggrecan core protein. Smaller proteoglycans and glycoprotein glycans were present. The types and proportions of these proteoglycans varied between the two cell types demonstrating biosynthetic commitment.

Lectin histochemistry and cytochemistry-light microscopy : avidin-biotin amplification on resin-embedded sections.

Within the last 25 yr, the study of glycans, their structures, and their drstribution in tissues has emerged from relative obscurity to become a major theme of molecular and cellular biology: "glycobiology" (1). Glycans are major components of cellular surfaces (2,3), extracellular matrices (4,5), and secretions (6), and play important roles in cell-cell and cell-matrix recognition and adhesion (7,8). They regulate the surface environment of cells by influencing the structure of water (6,9), by modulating diffusion, by sequestering metabohtes such as metal ions, by presenting various growth factors to their receptors (10,11), by acting as ligands in recognition-adhesion systems (11-13), and by making major contributions to cell surface charge (2,14). In secretions, they are variously determinants of molecular folding (15), hydration, interaction, and targeting, and they serve in the mechanisms that monitor the aging of glycoconjugates in circulation (6,9,16,17). They are now implicated in mechanisms of molecular targeting and segregation within the endomembrane systems of cells (18-20), in calcium transport in mitochondria (21), the handling of mRNA and its export from the nucleus, the regulation of transcription (19), and in then classical role as energy stores. In all of this, the association of anatomical or ultrastructural localization with biological function is of paramount importance. specific glycans occur in specific places (22,23). The challenge for the histochemist is to reconcile the achievement of sufficiently precise anatomical localization of glycans with the maximum of chemical information about their nature. The lectins have been the main means of accompllshing this, since their first application as fluorochrome-labeled probes to paraffin sections in the early 1970s (2,24). More recently, fluorescence has been largely supplanted by nonfluorescent disclosing systems, such as biotin-avidin-peroxidase and, though paraffin sections are still widely used, resin-embedding of specimens has proved advantageous in terms of resolution and economy of material at the light microscopic level.

Environmental pollutants as aetiological agents in female reproductive pathology: placental glycan expression in normal and polychlorinated biphenyl (PCB)-exposed mink (Mustela vison).

Polychlorinated biphenyls (PCB) may cause growth retardation or fetal death in mink. Pathological changes in endotheliochorial mink placentae were examined following exposure to PCB during gestation. Placentae from six animals with average fetal crown-rump (C-R) lengths between 16 and 53 mm given 0.65 mg/day Clophen A50 (low dose), and one from five animals with an average fetal C-R length of 14 mm given 1.3 mg/day (high dose), were examined. Mink were treated from 9 to 24 days before mating until killed at day 53. Placentae were formalin-fixed with four size-matched controls and embedded in resin. Sections were stained with five biotinylated lectins to detect specific glycans. Both control and treated (low dose) mink showed degenerative changes in maternal endothelium from 13-16 mm, revealed by increased lectin binding, caused probably by high cell turnover during tissue remodelling. Controls of 47 and 50 mm exhibited fewer degenerate maternal endothelial cells. The 31-mm PCB-treated tissue showed separation of the trophoblast from the interstitial layer and, at 53 mm, loss of its normal architecture, increased damage to maternal endothelium and infarction. High-dose PCB was extremely toxic, producing fetal death or extensive placental infarction by 14 mm C-R length. Lectin staining thus revealed the effects of PCB toxicity, shown by increased injury to maternal endothelium and severe trophoblastic damage.

Localisation of glycans in the placenta: a comparative study of epitheliochorial, endotheliochorial, and haemomonochorial placentation.

Specimens of mid-term (horse), near-term (pig, cow, sheep, mink) and term (human) placentae and associated tissues have been examined with a panel of 15 biotinylated lectins combined with an avidin-peroxidase revealing system. The aim of this study has been to analyse the expression of glycans at the materno-fetal interface in order to establish whether the morphological diversity exhibited by these six species is reflected by accompanying biochemical diversity, or whether similar types of glycan are expressed in tissues performing similar functions. Lectin staining intensity was scored in the following elements of the interhaemal placental barrier: maternal capillaries, maternal uterine epithelium, the materno-fetal interdigitating microvillous membrane (brush border in the human), trophoblast, and fetal capillaries. A high degree of biochemical diversity was found in the glycan expression of the various placental components within and among placental types. Each layer showed widely differing patterns of lectin binding between species, with only a few findings in common: 1) the relative lack of simple fucosyl termini, 2) the presence of non-bisected bi/tri-antennary N-glycan in most layers, 3) an abundance of terminal N-acetyl galactosamine, and 4) the restriction of high mannose glycans to intracellular granules. This diversity may be a mechanism to avoid hybridisation, although glycan patterns may change between conception and placental development, or it may have evolved as a consequence of morphological changes. It is possible that it may also be part of the cause, rather than the result, of the structural diversity that is so characteristic of mammalian placentation.