Characterization of isolated liver sinusoidal endothelial cells for liver bioengineering.

BACKGROUND: The liver sinusoidal capillaries play a pivotal role in liver regeneration, suggesting they may be beneficial in liver bioengineering. This study isolated mouse liver sinusoidal endothelial cells (LSECs) and determined their ability to form capillary networks in vitro and in vivo for liver tissue engineering purposes. METHODS AND RESULTS: In vitro LSECs were isolated from adult C57BL/6 mouse livers. Immunofluorescence labelling indicated they were LYVE-1+/CD32b+/FactorVIII+/CD31-. Scanning electron microscopy of LSECs revealed the presence of characteristic sieve plates at 2 days. LSECs formed tubes and sprouts in the tubulogenesis assay, similar to human microvascular endothelial cells (HMEC); and formed capillaries with lumens when implanted in a porous collagen scaffold in vitro. LSECs were able to form spheroids, and in the spheroid gel sandwich assay produced significantly increased numbers (p = 0.0011) of capillary-like sprouts at 24 h compared to HMEC spheroids. Supernatant from LSEC spheroids demonstrated significantly greater levels of vascular endothelial growth factor-A and C (VEGF-A, VEGF-C) and hepatocyte growth factor (HGF) compared to LSEC monolayers (p = 0.0167; p = 0.0017; and p < 0.0001, respectively), at 2 days, which was maintained to 4 days for HGF (p = 0.0017) and VEGF-A (p = 0.0051). In vivo isolated mouse LSECs were prepared as single cell suspensions of 500,000 cells, or as spheroids of 5000 cells (100 spheroids) and implanted in SCID mouse bilateral vascularized tissue engineering chambers for 2 weeks. Immunohistochemistry identified implanted LSECs forming LYVE-1+/CD31- vessels. In LSEC implanted constructs, overall lymphatic vessel growth was increased (not significantly), whilst host-derived CD31+ blood vessel growth increased significantly (p = 0.0127) compared to non-implanted controls. LSEC labelled with the fluorescent tag DiI prior to implantation formed capillaries in vivo and maintained LYVE-1 and CD32b markers to 2 weeks. CONCLUSION: Isolated mouse LSECs express a panel of vascular-related cell markers and demonstrate substantial vascular capillary-forming ability in vitro and in vivo. Their production of liver growth factors VEGF-A, VEGF-C and HGF enable these cells to exert a growth stimulus post-transplantation on the in vivo host-derived capillary bed, reinforcing their pro-regenerative capabilities for liver tissue engineering studies.

Socioeconomic deprivation and the burden of head and neck cancer-Regional variations of incidence and mortality in Merseyside and Cheshire, North West, England.

OBJECTIVES: The aim of this longitudinal study was to examine the distribution of head and neck cancer (HANC) disease burden across the region comparing it to national trends. DESIGN: We undertook a retrospective study of routine data combining it with indicators of deprivation and lifestyle at small geographical areas within the 9 Local Authorities (LAs) of Merseyside and Cheshire Network (MCCN) for head and neck cancers. Data from the North West of England and England were used as comparator regions. SETTING: This research was undertaken by the Cheshire and Merseyside Public Health Collaborative, UK. PARTICIPANTS: The Merseyside and Cheshire region serves a population of 2.2 million. Routine data allowed us to identify HANC patients diagnosed with cancers coded ICD C00-C14 and C30-C32 within 3 cohorts 1998-2000, 2008-2010 and 2009-2011 for our analysis. MAIN OUTCOME MEASURES: Directly age-standardised incidence rates and directly age-standardised mortality rates in the LAs and comparator regions were measured. Lifestyle and deprivation indicators were plotted against them and measured by Pearson's correlation coefficients. RESULTS: The incidence of head and neck cancer has increased across the region from 1998-2000 to 2008-2010 with a peak incidence for Liverpool males at 35/100 000 population. Certain Middle Super Output Areas contribute disproportionately to the significant effect of incidence and mortality within LAs. Income deprivation had the strongest correlation with incidence (r = .59) and mortality (r = .53) of head and neck cancer. CONCLUSION: Our study emphasises notable geographical variations within the region which need to be addressed through public health measures.

Development of the flagellar apparatus of Naegleria.

Flagellates of Naegleria gruberi have an interconnected flagellar apparatus consisting of nucleus, rhizoplast and accessory filaments, basal bodies, and flagella. The structures of these components have been found to be similar to those in other flagellates. The development of methods for obtaining the relatively synchronous transformation of populations of Naegleria amebae into flagellates has permitted a study of the development of the flagellar apparatus. No indications of rhizoplast, basal body, or flagellum structures could be detected in amebae. A basal body appears and assumes a position at the cell surface with its filaments perpendicular to the cell membrane. Axoneme filaments extend from the basal body filaments into a progressive evagination of the cell membrane which becomes the flagellum sheath. Continued elongation of the axoneme filaments leads to differentiation of a fully formed flagellum with a typical "9 + 2" organization, within 10 min after the appearance of basal bodies.

Basal bodies, but not centrioles, in Naegleria.

Amebae of Naegleria gruberi transform into flagellates whose basal bodies have the typical centriole-like structure. The amebae appear to lack any homologous structure, even during mitosis. Basal bodies are constructed during transformation and, in cells transforming synchronously at 25 degrees C, they are first seen about 10 min before flagella are seen. No structural precursor for these basal bodies has been found. These observations are discussed in the light of hypotheses about the continuity of centrioles.

Isolation, ultrastructure, and protein composition of the flagellar rootlet of Naegleria gruberi.

Attached to the basal bodies of Naegleria gruberi flagellates is a striated rootlet or rhizoplast. The rootlet-basal body complex has been isolated by Triton X-100 lysis of deflagellated cells and differential centrifugation through a 25% glycerol medium. Rootlets isolated from mature flagellates are approximately 13 micrometers long but vary from 8 to 15 micrometers in length: they taper at both ends from a maximum width of approximately 0.25 micrometers in the vicinity of the basal bodies. They are highly stable during isolation but can be solubilized by urea, high salt, low pH, or detergent (Sarkosyl). Partial dissociation of rootlets with 1 M urea reveals that they are composed of filaments, approximately 5 nm diameter, associated in a linear fashion to yield the characteristic 21-nm cross-banded appearance. Differential solubilization of rootlets and their associated contaminants allowed identification of a major rootlet protein, comprising at least 50% of any purified rootlet preparation, with an apparent subunit molecular weight of 170,000. The localization of rootlets in situ by indirect immunofluorescence using a specific antibody directed against the purified rootlet protein demonstrated unequivocally that this 170,000-dalton protein is an organelle component.

Self-reported depression and suicide attempts among U.S. women physicians.

OBJECTIVE: Studies examining suicide rates for U.S. women physicians and other U.S. women have found odds ratios as high as 4 to 1. Although such reports are controversial and are based on small groups (N = 17 to 49 suicides), they are often cited as evidence of a high prevalence of psychopathology among women physicians. METHOD: The authors used the results of the Women Physicians' Health Study (N = 4,501), a large, nationally distributed questionnaire, to assess the lifetime prevalence of self-identified depression and suicide attempts among U.S. women physicians. RESULTS: An estimated 1.5% (N = 61) of U.S. women physicians have attempted suicide, and 19.5% (N = 808) have a history of depression. Those who were born in the United States, were not Asian, had histories of cigarette smoking, alcohol abuse or dependence, sexual abuse, domestic violence, poor current mental health, more severe harassment, or a family history of psychiatric disorders were significantly more likely to report suicide attempts or depression. Depression was more common among those who were not partnered, were childless, had a household gun, had more stress at home, drank alcohol, had worse health, or had a history of obesity, chronic fatigue syndrome, substance abuse, an eating disorder, or another psychiatric disorder and among those who reported working too much, career dissatisfaction, less control at work, and high job stress. Strata reporting higher rates of depression tended to show higher (although usually nonsignificant) rates of suicide attempts. CONCLUSIONS: Depression is approximately as common among U.S. women physicians as among other U.S. women, but suicide attempts may be fewer. A number of conditions may help identify women physicians at high risk for suicide attempts and depression.

Ponalrestat: a potent and specific inhibitor of aldose reductase.

Many of the complications of diabetes appear to be closely linked to increased conversion of tissue glucose to sorbitol which is catalysed by aldose reductase (aldehyde reductase 2, ALR2). Inhibition of ALR2 could, therefore, lead to a reduction in the development of diabetic complications. Ponalrestat ["Statil" (a trademark, the property of Imperical Chemical Industries PLC), "Prodiax" (a trademark, the property of Merck, Sharp and Dohme), ICI 128436, MK538] inhibits ALR2 from a number of sources. Until now, the mechanism of this inhibition has not been fully elucidated. In this paper, we present a detailed mechanism for inhibition of bovine lens ALR2 by ponalrestat. Treatment of humans with some ALR2 inhibitors leads to side-effects, some of which may result from interactions with other enzymes. Aldehyde reductase (ALR1) is probably the most closely related enzyme to ALR2. Inhibition of ALR1 from bovine kidney was, therefore, investigated in order to assess the specificity of ponalrestat. The values of Ki and Kies (apparent dissociation constants for inhibitor from enzyme-inhibitor and enzyme-inhibitor-substrate complexes, respectively) for the interactions of ponalrestat with ALR1 and ALR2 has been calculated by non-linear fitting of kinetic data. These values indicate that ponalrestat does not compete with binding of glucose of NADPH to ALR2, nor with binding of glucuronate or NADPH to ALR1. Lack of competition and the structural dissimilarity of substrates and inhibitor make it unlikely that ponalrestat will utilize substrate binding sites on other enzymes, and so produce undesirable side-effects via such a mechanism. Ponalrestat is a potent inhibitor (Ki = Kies = 7.7 nM) of ALR2 and follows a pure noncompetitive mechanism with respect to glucose. Efficacy, therefore, will not be decreased by development of hyperglycaemia. The compound is a mixed noncompetitive inhibitor of ALR1 when glucuronate is varied. The values of Ki and Kies are 60 microM and 3 microM, respectively, so that inhibition tends towards uncompetitive. The selectivity of ponalrestat in favour of ALR2, therefore, lies in the range 390 to 7,800-fold, being higher at lower concentrations of glucuronate. The high selectivity of ponalrestat in favour of ALR2 rather than ALR1 suggests that the compound is unlikely to inhibit other enzymes which have less homology with ALR2.

A multistage system to detect epileptiform activity in the EEG.

A PC-based system has been developed to automatically detect epileptiform activity in sixteen-channel bipolar EEG's. The system consists of three stages: data collection, feature extraction, and event detection. The feature extractor employs a mimetic approach to detect candidate epileptiform transients on individual channels, while an expert system is used to detect focal and nonfocal multichannel epileptiform events. Considerable use of spatial and temporal contextual information present in the EEG aids both in the detection of epileptiform events and in the rejection of artifacts and background activity as events. Classification of events as definite or probable overcomes, to some extent, the problem of maintaining high detection rates while eliminating false detections. So far, the system has only been evaluated on development data but, although this does not provide a true measure of performance, the results are nevertheless impressive. Data from 11 patients, totaling 180 minutes of sixteen-channel bipolar EEG's, have been analyzed. A total of 45-71% (average 58%) of epileptiform events reported by the human expert in any EEG were detected as definite with no false detections (i.e., 100% selectivity) and 60-100% (average 80%) as either definite or probable but at the expense of up to nine false detections per hour. Importantly, the highest detection rates were achieved on EEG's containing little epileptiform activity and no false detections were made on normal EEG's.

Real-time detection of epileptiform activity in the EEG: a blinded clinical trial.

The aim of this study was to determine the performance of a PC-based system for real-time detection and topographical mapping of epileptiform activity (EA) in the EEG during routine clinical recordings. The system incorporates a mimetic stage to locate candidate spikes (including sharp-waves) followed by two expert-system-based stages, which utilize spatial and wide-temporal contextual information in deciding whether candidate events are epileptiform or not. The data comprised 521 consecutive routine clinical EEG recordings (173 hours). Performance was evaluated by comparison with three independent electroencephalographers (EEGers-I). A second group of two EEGers (EEGers-II) separately interpreted the spike topographical maps and, for EEGs categorized as containing only questionable EA by the detection system, reviewed 6 sec segments of raw EEG centered on each questionable event. Thirty-eight of the EEGs were considered to contain definite EA by at least two of EEGers-I. The false detection rate of the system was 0.41 per hour. The system was found to have a sensitivity of 76% and a selectivity of 41% for EEGs containing definite EA. However, it only missed detection of EA in 5% of the recordings. EEGers-II agreed with EEGers-I on the distribution (generalized, lateralized, focal, multifocal) of EA in 79% of cases. This is by far the largest clinical evaluation of computerized spike detection reported in the literature and the only one to apply this in routine clinical recordings. The false detection rate is the lowest ever reported, suggesting that this multi-stage rule-based system is a powerful and practical tool in clinical electroencephalography and long-term EEG monitoring.

Structure and protein composition of the striated flagellar rootlets of some protists.

The striated rootlets of different protists are extremely diverse and, on the basis of structural organization, can be assigned to no fewer than four major types. In light of this extreme variation in fine-structure is it reasonable to expect that all striated rootlets may share common protein species? Using the flagellar rootlet of Naegleria gruberi strain NB-1 as reference, we compared rootlet size, structure, and protein composition to another Naegleria strain, another amoebo-flagellate, Tetramitus rostratus, and to the ciliate Tetrahymena pyriformis. Although differing in size and distribution in the cells, the rootlets of all three amoebo-flagellates appeared very similar in structure, periodicity, and in the presence of a common 170 000 Dalton subunit. Kinetodesmal fibres of Tetrahymena differed markedly in detailed fine-structure, in periodicity, and in the apparent absence of the 170 000 Dalton subunit as tested either by SDS gel electrophoresis or by indirect immunofluorescence staining using a specific antiserum directed against the NB-1 major rootlet protein. Consideration of literature describing striated rootlet structure in a wide variety of ciliated and flagellated cells led to the speculation that striated rootlets arose subsequent to primitive flagella and likely evolved along two major pathways: a narrow-period rootlet similar to those discussed above, which developed from the microtubule rootlets of algal flagellates, and a wide-band, contractile rootlet which originated from the primitive interbasal body connector prominent in both algal and protozoan species.

Fistula between the external auditory canal and the temporomandibular joint: a rare complication of otitis externa.

Otitis externa is a relatively common complication of ear syringing. Temporomandibular joint complications of otitis externa are rare. A case of otitis externa with communication between the external auditory canal and the temporomandibular joint is described.

The implementation of audit in an ENT unit.

ENT Departments in the United Kingdom are faced with increasing pressure from government, management and the Colleges responsible for surgical training to introduce formal medical self audit. The plethora of guidelines and publications on the subject rarely address the differences in clinical practice between general medicine or surgery and that of otolaryngology. Clearly the lead in the introduction of medical audit will come from the appropriate specialist associations; however, this paper aims to share the experience of the implementation of audit in one otolaryngology, head and neck surgery unit and offer suggestions for a programme elsewhere.

Airway obstruction with stridor due to nasal secretions.

Two cases of non-fatal airway obstruction by nasal secretions are described. The diagnosis was made after laryngoscopy and the features which suggested this previously undescribed aetiology were: depression of conscious level; poor oral hygiene; and irregular friable mass with mucoid areas obstructing the airway. The diagnosis was confirmed by histological examination of the obstructing material which was composed of laminated fragments of squamous epithelium, keratin debris and mucus. It is suggested that these masses form in the post-nasal space and then become dislodged descending into the larynx, where they cause partial laryngeal obstruction and stridor. In patients whose conscious level is depressed and who have poor oral hygiene, nasal secretions should be considered as a cause of sudden unexplained airway obstruction and stridor.

Inflammatory pseudotumour in the submandibular region.

We present a rare case of inflammatory pseudotumour in the submandibular region. A review of the literature revealed that this is only the second case of inflammatory pseudotumour in the submandibular region to be reported. Clinical presentation and management of this condition are discussed.

The mini-grommet and tympanosclerosis: results at two years.

One hundred and sixteen children with otitis media with effusion (OME) underwent surgery with grommet insertion. A conventional Shah grommet was used in one ear, and a Mini-Shah grommet in the other. Final review of the subjects two years after surgery revealed a significantly lesser degree of tympanosclerosis in the ear into which the Mini-Shah grommet had been inserted. This benefit might have resulted from the lesser mass of the mini-tube or its shorter duration in situ.

Distribution of ear drops using a non-aerosol spray delivery system.

The distribution of ear drops in normal ears is variable. A new product Otomize (Stafford-Miller) containing dexamethasone and neomycin has a non-aerosol spray mist delivery system. This has been applied to normal ears and found to be superior in its distribution to generally available ear drops previously assessed.

Distribution of ear drops in normal ears.

Ear drops are prescribed widely with little thought for the distribution inside the ear canal. This study compares the distribution of water and oil based drops. The results indicate that, under the test conditions, the penetration of ear drops is extremely variable. Water provided the greatest overall coverage, while the most viscous drops fared worst. It is suggested that in diseased ears even poorer penetration may result, which may explain the lack of response in some circumstances.

Retropharyngeal haematoma: a diagnosis for concern?

Three cases of retropharyngeal haematoma are described. In one case there was underlying aneurysm of the carotid artery which could have led to significant morbidity if undetected.