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BACKGROUND: Keratinocyte cancers (KC) are common and pose a significant financial burden globally. Ultraviolet radiation is a significant factor in their development, through mutagenesis promotion but also through local and systemic immunosuppression. Although systemic immunosuppression is well understood, cutaneous immunity has been more difficult to evaluate. OBJECTIVES: This study used a contact sensitizer, diphencyprone (DPCP), which elicits a contact hypersensitivity reaction in skin, to compare the degree of reactivity to DPCP in patients with a high KC burden versus those with a low KC burden. METHODS: A prospective study was performed in immunocompetent patients aged 70 ± 5 years of age, comparing patients with a high KC burden (>10 previous KC) with those with a low KC burden (<2 previous KC). All patients were sensitized with 2% DPCP and then patch tested two weeks later with eight different concentrations of DPCP with the threshold concentration and total degree of reaction recorded. RESULTS: Nine patients were recruited, 5 in the 'high cancer' group and 4 in the 'low cancer' group. All patients were Fitzpatrick skin type 1 or 2. All patients developed a reaction to DPCP. Patients in the low cancer group developed a reaction at a significantly lower threshold DPCP concentration than the high cancer group (P = 0.039). The cumulative intensity of reaction was higher in the low cancer group (P = 0.087). CONCLUSION: Patients with a high KC burden required a higher threshold concentration of DPCP to elicit a hypersensitivity reaction, supporting the concept of a lower skin immunity in these patients. DPCP reactivity threshold could be a useful tool in the evaluation of skin immunity and propensity to develop keratinocyte cancers.

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PURPOSE: T50 is a novel serum-based marker that assesses the propensity of calcification in serum. Shorter T50 indicates greater propensity to calcify and it has been associated to cardiovascular disease (CVD) and mortality among patients with kidney disease. In the general population, neither the correlates of T50 nor the relationships of T50 with bone mineral density (BMD) are known. METHODS: We performed a nested cross-sectional study selecting 150 individuals at random among participants from the Osteoporotic Fractures in Men (MrOS) Study, a study of community-living older men. We categorized individuals into tertiles of T50 and compared demographics and disease indicators across tertiles. We utilized linear regression to evaluate the cross-sectional association between T50 and hip and spine BMD in multivariable models. RESULTS: Older age was associated with shorter T50. Kidney function tended to be lower in those with shorter T50 and the prevalence of CVD and peripheral arterial disease in those with shorter T50, albeit these findings did not achieve statistical significance. We found no statistically significant associations between T50 and total hip or total spine BMD in either unadjusted or multivariable adjusted models. CONCLUSIONS: T50, a novel indicator of serum calcification propensity, is not associated with BMD in community-living older men. Future larger studies should determine if T50 may give insights to CVD in the general population above and beyond traditional risk factors.

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In the preservation of tissues in as 'close to life' state as possible, rapid freeze fixation has many benefits over conventional chemical fixation. One technique by which rapid freeze-fixation can be achieved, high pressure freezing (HPF), has been shown to enable ice crystal artefact-free freezing and tissue preservation to greater depths (more than 40 µm) than other quick-freezing methods. Despite increasingly becoming routine in electron microscopy, the use of HPF for the fixation of inner ear tissue has been limited. Assessment of the quality of preservation showed routine HPF techniques were suitable for preparation of inner ear tissues in a variety of species. Good preservation throughout the depth of sensory epithelia was achievable. Comparison to chemically fixed tissue indicated that fresh frozen preparations exhibited overall superior structural preservation of cells. However, HPF fixation caused characteristic artefacts in stereocilia that suggested poor quality freezing of the actin bundles. The hybrid technique of pre-fixation and high pressure freezing was shown to produce cellular preservation throughout the tissue, similar to that seen in HPF alone. Pre-fixation HPF produced consistent high quality preservation of stereociliary actin bundles. Optimising the preparation of samples with minimal artefact formation allows analysis of the links between ultrastructure and function in inner ear tissues.

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An improved method for making fast quantitative determinations of membrane potential with voltage-sensitive dyes is presented. This method incorporates a high-speed, random-access, laser-scanning scheme (Bullen et al., 1997. Biophys. J. 73:477-491) with simultaneous detection at two emission wavelengths. The basis of this ratiometric approach is the voltage-dependent shift in the emission spectrum of the voltage-sensitive dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS). Optical measurements are made at two emission wavelengths, using secondary dichroic beamsplitting and dual photodetectors (<570 nm and >570 nm). Calibration of the ratiometric measurements between signals at these wavelengths was achieved using simultaneous optical and patch-clamp measurements from adjacent points. Data demonstrating the linearity, precision, and accuracy of this technique are presented. Records obtained with this method exhibited a voltage resolution of approximately 5 mV, without any need for temporal or spatial averaging. Ratiometric recordings of action potentials from isolated hippocampal neurons are used to illustrate the usefulness of this approach. This method is unique in that it is the first to allow quantitative determination of dynamic membrane potential changes in a manner optimized for both high spatiotemporal resolution (2 micrometers and <0.5 ms) and voltage discrimination.

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A novel approach to laser scanning microscopy is presented that utilizes diffraction-based scanning principles to achieve fast random-access positioning of a focused laser beam. This non-imaging approach overcomes the speed limitation of present reflection-based scanning microscopes while maintaining high spatial resolution. The presented system combines conventional video microscopy with fast non-imaging scanning microscopy. Together with readily available optical indicators of neuronal activity, this system permits multi-site optical recording from living brain tissue. In this paper, we will review the underlying principles of laser scanning microscopy and the steps in development that led to the current acousto-optic scanning system. We will present typical signals recorded with the current system, and we will outline ongoing extensions of the system. We will also discuss the present limitation of this instrumentation and look into directions of future development.

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The instrumental design and experimental conditions for high-speed, simultaneous optical recording of membrane potential and intracellular Ca2+ with subcellular resolution are presented. This method employs an extended version of a high-speed, random-access, laser-scanning fluorescence microscope designed to record fast physiological signals from small neuronal structures with high spatiotemporal resolution (Bullen, Patel, Saggau, Biophys J 73:477-491, 1997). With this instrument, imaging and optical recording functions are conducted separately allowing frame rates up to 3 kHz. Individual scanning points are selected interactively from a reference image collected with differential interference contrast (DIC) optics. At each recording site, fluorescence from two indicators is measured simultaneously by independent photodetectors. To optimize signal strength, spectral separation and the achievable signal-to-noise ratio, several combinations of voltage-sensitive dye, Ca2+ indicator and optical elements (dichroic mirrors, filters, etc.) were considered. The best results were achieved from the combination of the intracellular voltage-sensitive dye Di-2-ANEPEQ and the Ca2+ indicator Calcium Green-1. These indicators have overlapping absorption spectra allowing simultaneous excitation with a single laser line (488 nm). Spectral separation of the fluorescence from these two indicators was accomplished using a secondary dichroic mirror (DCLP580) and emission filters (535/45 and OG590). Representative records obtained with this instrument and this combination of indicators demonstrate the feasibility of simultaneous high fidelity measurements of membrane potential and intracellular Ca2+ from the same point at high spatial (2 micrometer) and temporal (

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The design and implementation of a high-speed, random-access, laser-scanning fluorescence microscope configured to record fast physiological signals from small neuronal structures with high spatiotemporal resolution is presented. The laser-scanning capability of this nonimaging microscope is provided by two orthogonal acousto-optic deflectors under computer control. Each scanning point can be randomly accessed and has a positioning time of 3-5 microseconds. Sampling time is also computer-controlled and can be varied to maximize the signal-to-noise ratio. Acquisition rates up to 200k samples/s at 16-bit digitizing resolution are possible. The spatial resolution of this instrument is determined by the minimal spot size at the level of the preparation (i.e., 2-7 microns). Scanning points are selected interactively from a reference image collected with differential interference contrast optics and a video camera. Frame rates up to 5 kHz are easily attainable. Intrinsic variations in laser light intensity and scanning spot brightness are overcome by an on-line signal-processing scheme. Representative records obtained with this instrument by using voltage-sensitive dyes and calcium indicators demonstrate the ability to make fast, high-fidelity measurements of membrane potential and intracellular calcium at high spatial resolution (2 microns) without any temporal averaging.

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Vibration-induced changes in target accuracy were examined to determine the influence of afferent information on unimanual and bimanual movements. Normal adult male subjects (N = 4) undertook a 90 degrees curvilinear positioning task (flexion and extension of the elbow) in the absence of vision. Each trial required the subject to reproduce a criterion target position while one of three vibration treatments was applied to the distal aspect of the triceps brachii tendon of the dominant arm. Vibration (100 Hz) was administered manually either prior to or concurrently with movement. Subsequent movement outcome was compared with a control or no-vibration condition. In the unimanual condition application of vibration to an antagonist muscle produced consistent undershoot of approximately 10 to 12% of the total movement amplitude. In contrast, vibration of the agonist produced minimal error in movement. A similar pattern was seen in the vibrated dominant arm of the bimanual condition; however, the results exhibited no interaction between hands. Target accuracy during prior vibration did not differ significantly from control trials and we concluded that vibration was an inappropriate stimulus to distort initial condition information. The observed results are consistent with vibration-induced activation of muscle spindle receptors in the lengthening muscle during movement. We concluded that afferent information from an antagonist muscle is critically important in the final movement outcome, but there was no evidence of contralateral effects.

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We describe a patient with intestinal lymphangiectasia who developed hyposplenism and speculate that it resulted from chronic loss of lymphocytes into the gut.

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The enhanced proliferative response of peripheral blood lymphocytes (P.B.L.) to Concanavalin A (Con A) occurring after a 24 hr period of incubation was used as the basis of a suppressor cell assay and the results expressed as a suppressor index (S.I.) which if low implies impaired suppressor cell activity. S.I. was measured on P.B.L. from 26 patients with coeliac disease and defined splenic function, 8 patients with splenic atrophy not associated with coeliac disease, 30 patients after surgical splenectomy, and 52 controls. S.I. was lower than control in the splenectomy group (P less than 0.01) and in the coeliac group, but coeliacs with splenic atrophy were no different from coeliacs with apparently normal splenic function, nor were the non-coeliac splenic atrophy group different from control. Coeliacs with the HLA B8 gene had lower S.I. than those without, as did controls. Suspensions of cells from spleens removed at laparotomy demonstrated suppressor activity but this was no more marked than in P.B.L. It is concluded that the intact spleen is required for normal activation of suppressor cells in man, but this function is retained in splenic atrophy, and that the HLA B8 gene is associated with low suppressor cell activity in coeliac disease and in health.

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Jejunal biopsies from controls and coeliac patients were maintained in organ culture in the presence of gluten fraction III. The culture media were assayed for evidence of lymphokine activity in a migration inhibition test using normal peripheral blood leucocytes. Significant inhibition of migration was produced by media from untreated coeliac patients compared with controls (P less than 0.005) or treated coeliac patients (P less than 0.001), indicating the production of a leucocyte migration inhibition factor (LIF) by untreated coeliac mucosa in response to gluten fraction III. The degree of inhibition correlated with the preculture interepithelial lymphocyte count in the coeliac biopsies (P less than 0.02). In six coeliac patients studied when on a normal diet and on a gluten-free diet, LIF was produced while on a normal diet, but not while on a gluten-free diet. These results suggest that a local cell-mediated immune reaction to gluten is present in the mucosa of patients with untreated coeliac disease but that this is reversed by treatment with a gluten-free diet.

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Jejunal biopsies from controls and coeliac patients were maintained in organ culture for up to 48 hours. The in vitro effect of gluten fraction III during the period of culture was assessed by measurement of the activity of the brush border enzymes alkaline phosphatase and alpha-glucosidase. Mucosa from controls and treated and untreated coeliacs behaved similarly and no reproducible in vitro effect of gluten was demonstrated. These results cast doubt on the in vitro diagnosis of coeliac disease by monitoring brush border enzyme activity.

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The leucocyte migration inhibition (LMI) test was used as an indicator of cell mediated immunity to gluten fraction III in 30 healthy controls and 58 patients with adult coeliac disease and the results related to HLA status and duration of treatment with a gluten-free diet. HLA-B8 controls showed significantly lower leucocyte migration indices, indicating greater immune response, than non-HLA B8 controls. Untreated coeliacs showed no difference from HLA-B8 controls. There was no difference between results from HLA-B8 and non-HLA-B8 coeliacs. Leucocyte migration was even lower in coeliacs early in treatment but rose after treatment for over one year. These results may reflect an immune response gene for gluten in linkage disequilibrium with HLA-B8. The increased immune response to gluten as measured in this test cannot be the sole factor in aetiology of coeliac disease. Furthermore, it is necessary to re-evaluate earlier results of cell-mediated immunity in coeliac disease with reference to HLA status of the controls.

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The levels of serum beta 2-m in hepato-biliary disease have been examined. Significant increase has been observed in various benign and malignant diseases of the liver, but not in jaundice due to acute pancreatitis. The age-dependent relationship of beta 2-m is present in controls, weaker in the malignant diseases and absent in the benign diseases. The level of beta 2-m is not influenced by jaundice. Serum beta 2-m levels have no role as a discriminant in liver disease.

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In view of the importance to the patient of demonstrating mucosal sensitivity to gluten we have quantitated morphologic change during organ culture of small bowel mucosa by measuring enterocyte height. In 27 normal controls the mean preculture enterocyte height was 28.3 micrometer +/- 1.8 (+/- 1 SD), decreasing to 25.6 micrometer +/- 1.9 and 26.1 micrometer +/- 2.0 after 24 h culture in the absence and presence of gluten, respectively. Both these decreases were significant (p < 0.001), but there was no difference between culture with or without gluten. In 9 abnormal controls, the mean preculture enterocyte height was 27.0 micrometer +/- 2.7, decreasing significantly (p < 0.05) to 24.5 micrometer +/- 1.1 after 24 h culture without gluten, and to 25.4 micrometer +/- 2.3 with gluten (NS). In 17 untreated celiac patients, the mean preculture enterocyte height was 19.6 micrometer +/- 1.9 increasing significantly to 23.8 micrometer +/- 2.0 (p < 0.001) after 24 h culture without gluten but decreasing to 18.7 micrometer +/- 3.1 after 24 h culture with gluten (NS). There was a significant difference between culture with or without gluten (p < 0.001). In 21 treated celiac patients, the mean preculture enterocyte height was 28.1 micrometer +/- 1.9 decreasing to 25.4 micrometer +/- 2.0 and 24.2 +/- 1.9 in the absence and presence of gluten, respectively. Both these decreases were significant (p < 0.001), but more importantly there was a significant decrease (p < 0.001) after culture with gluten compared with culture without gluten. These data indicate that it is possible to quantify morphologic change during organ culture and to demonstrate gluten-sensitivity not only in untreated, but also in treated celiac mucosa. These studies have important implications as regards the diagnosis and further investigation of the etiology of celiac disease.

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Functional hyposplenism is associated with a variety of disorders including coeliac disease. The aims of this study were to estimate the need for a small intestinal biopsy in the investigation of hyposplenism, and to assess the relationship of autoimmunity to hyposplenism and coeliac disease. During one year, the features of hyposplenism were found in blood films of 27 patients who had not had a splenectomy. Ten patients were already known to have coeliac disease. Intestinal biopsy was performed in another 13 patients; coeliac disease was diagnosed in six. Of the 23 patients biopsied, coeliac disease was present in 16 (70%). Autoantibodies were detected in significantly more patients with hyposplenism than in healthy controls (P less than 0.05), and in significantly more coeliacs with hyposplenism than coeliacs with normal blood films (P less than 0.01). The increased incidence of autoantibodies in coeliacs with hyposplenism compared with other coeliacs was not associated with a difference in the incidence of HLA-B8. Small bowel biopsy should be carried out in the investigation of unexplained hyposplenism. There may be a link between hyposplenism and the autoimmune manifestations of coeliac disease.

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