Preparation of primary monolayer cultures of mouse pancreatic epithelial cells

Summary We describe a technique using normal and diabetic (dbdb) mice to establish primary pancreatic cultures that spread and assume a characteristic epithelial morphology. These cultures contain 4 to 7% beta cells, secrete insulin in response to stimuli for 10 to 14 d, contain few fibroblasts, and have a cell viability that is greater than 95%. The cells attach firmly to glass cover slips and are ideal for the study of insulin secretory granules or contractile proteins using indirect immunofluorescence.     

Spinothalamic and propriospinal neurones in the upper cervical cord of the rat: terminations of primary afferent fibres on soma and primary dendrites

Summary Experiments were performed on rats to determine whether primary afferents from the upper cervical region terminate directly on Spinothalamic and propriospinal neurones. The central terminations of primary afferents from the upper cervical region were identified by diffusely filling their axons with horseradish peroxidase. Spinothalamic neurones or propriospinal neurones were identified in the same experimental animals by using retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase. Approximately 3–11 % of Spinothalamic cells in laminae 4–6 of spinal segments C2–4 received apparent synaptic contacts from primary afferents on the soma or primary dendrites. Approximately 18–36% of propriospinal neurones with axons descending to lower thoracic or lumbar levels received apparent synaptic contacts on the soma or primary dendrites. These data provide anatomical evidence that Spinothalamic and long propriospinal neurones in the upper cervical cord are excited directly by primary afferents. The data also help to clarify the neural circuitry underlying somatic sensation and reflex movements evoked by neck receptors.     

A comparison of microaerobic systems for the culture ofCampylobacter jejuni andCampylobacter coli

The relative ability of two commercial gas generating envelopes, the evacuation-replacement technique, and a candle jar, to produce a satisfactory microaerobic atmosphere for the culture of 45Campylobacter strains on non-selective medium and five selective media (Skirrow's, modified Butzler's, Blaser's, Campy-BAP and Preston medium) was investigated quantitatively. A candle jar, and modified Butzler's medium proved to be of limited use. The ability of four commercial gas generating envelopes to produce a satisfactory microaerobic atmosphere in four anaerobic jars of different volume was investigated using five referenceCampylobacter strains. Not all of the combinations worked. The oxygen and carbon dioxide concentrations produced in the microaerobic systems studied were measured with gas analysers. The evacuation-replacement technique produced far less variable concentrations of oxygen and carbon dioxide than did the envelopes.     

Blood-free selective medium for isolation of Campylobacter jejuni from feces

A blood-free selective agar is described which contains charcoal, ferrous sulfate, sodium pyruvate, casein hydrolysates, cefazolin, and sodium deoxycholate (CCD agar). CCD agar was compared with Preston medium for isolation of Campylobacter jejuni from human feces, and isolation rates were similar on both media, but CCD agar was less selective. Temperature studies at 37 and 42 degrees C confirmed that incubation of direct plates at 42 degrees C for 48 h was necessary for maximum isolation of C. jejuni.     

Non-Contractile Cells with Thin Processes Resembling Interstitial Cells of Cajal Found in the Wall of Guinea-Pig Mesenteric Arteries

Arterial interstitial cells of Cajal (ICC)-like cells (AIL cells) with a multipolar, irregular, elongated shape and with numerous thin (often less than 1 μm), sometimes branching, processes with lengths up to ≈60 μm were isolated enzymatically from 1st to 7th order branches of guinea-pig mesenteric artery. Some of the processes of AIL cells were growing (average speed ≈0.15 μm min⁻¹) and their growth was blocked by 10 μ M latrunculin B, an inhibitor of actin polymerisation. Staining with BODIPY phalloidin, a fluorescent dye selective for F-actin, showed the presence of F-actin in the processes of AIL cells. Voltage clamp of single AIL cells revealed an inward current that was four times more dense than in myocytes and was abolished by 10 μ M nicardipine, and an outward current carried exclusively by potassium ions that was reduced by 1 m M 4-aminopyridine and/or 100 n M iberiotoxin but unaffected by 10 n M dendrotoxin-K. Imaging of intracellular ionised calcium with fluo-4 using a laser scanning confocal microscope showed local or global calcium transients lasting several seconds in ≈28 % of AIL cells. When membrane current was recorded simultaneously, the calcium transients were found to correspond to long-lasting transient outward currents, which occurred at potentials positive to −40 mV. Unlike myocytes, AIL cells did not contract in response to 1 m M caffeine or 5 μ M noradrenaline, although they responded with a [Ca²⁺]_i increase. The segments of intact arteries did not stain for c-kit, a marker of ICCs. Single AIL cells stained positive for vimentin, desmin and smooth muscle myosin. The presence of ICC-like cells is demonstrated for the first time in the media of resistance arteries.     

The pathogenicity of long versus short fibre samples of amosite asbestos administered to rats by inhalation and intraperitoneal injection.

For many years it has been accepted that fibre dimensions are the most important factor in the development of asbestos related disease with long fibres being more dangerous than short for all types of asbestos. This information has been derived from in vitro experiments and injection or implantation experiments since the kilogramme quantities of specially prepared dusts that are necessary for long term inhalation have not been available. The present study has taken advantage of the availability of a sample of amosite produced so that almost all fibres were less than 5 micron in length. The effects of this dust were compared to dust prepared from raw amosite that contained a very high proportion of long fibres. Previous data from studies with UICC amosite, which was intermediate in length, were also available for comparison. At the end of 12 months of dust inhalation, significantly more short fibre amosite was present in the lung tissue compared to the long but while the long fibre dust caused the development of widespread pulmonary fibrosis, no fibrosis at all was found in animals treated with short fibre. One third of animals treated with long fibre dust developed pulmonary tumours or mesotheliomas but no pulmonary neoplasms were found in animals treated with short fibre dust. Following intraperitoneal injection, the long fibre amosite produced mesotheliomas in 95% of animals with a mean induction period of approximately 500 days. With short fibre dust, only a single mesothelioma developed after 837 days. In previous inhalation studies with UICC amosite, relatively little pulmonary fibrosis had developed and only two benign pulmonary tumours. This would suggest that to produce a significant carcinogenic response in rat lung tissue amosite fibres must be longer than those in the UICC preparation. Following the injection of UICC amosite, however, mesotheliomas developed in the same proportion of animals and with the same mean induction period as with long fibre dust. From this it would appear that while very short fibres exhibit little carcinogenicity to either lung or mesothelial tissues, mesotheliomas can be produced by dust preparations consisting of shorter fibres than are needed to produce tumours.     

Learning disability and epilepsy in an epidemiological sample of individuals with tuberous sclerosis complex

BACKGROUND: Intellectual impairments are a recognized feature of tuberous sclerosis complex (TSC), but the frequency and degree of intellectual impairments has not been systematically studied in large epidemiological samples using standardized measures. As such, the form of the IQ distribution (uni- or bi-modal) has not been established and the relationship between IQ and other features (e.g. epilepsy history) is poorly delineated. To address these shortcomings, we assessed the intellectual abilities of a large epidemiological sample of individuals with TSC, drawn from the 'Wessex' area of SW England and compared them with the abilities of their unaffected siblings. METHOD: Standardized tests were used to estimate the abilities of 108 (56 males, 52 females, median age = 25, range = 4-75) individuals with TSC and 29 unaffected siblings (14 males, 15 females, median age = 18, range = 6-55). Seizure history was obtained from informants and medical records. RESULTS: Estimated IQ was bi-modally distributed: 55.5% had an IQ in the normal range; 14% had mild to severe impairments: and 30.5% had profound disability (IQ < 21). Forty-four per cent of the individuals with TSC had an IQ < 70. In the subset of normally intelligent individuals with TSC, IQ was normally distributed with a mean of 93.6. This mean was significantly lower than the mean IQ of unaffected siblings (IQ = 105.6). All individuals with learning disability had a history of seizures that usually commenced before 12 months of age and that often presented as infantile spasms. Multivariate analyses indicated that a history of seizures as well as a history of infantile spasms was predictive of the degree of intellectual impairment. CONCLUSIONS: Intellectual abilities were bi-modally distributed in a representative sample of individuals with TSC. The likelihood of impairment was associated with a history of seizures, particularly infantile spasms. The genetic and brain basis of these findings requires further investigation.