Adaptation of the disector method to rare small organelles in TEM sections exemplified by counting synaptic bodies in the rat pineal gland

The disector is the only objective method for quantifying particles of variable size in a given volume. With this method, cell organelles are identified on adjacent sections, but only those present in one section are counted. When counting extremely rare structures in transmission electron microscope sections (physical disector), the usual procedure of counting on electron micrographs is limited for economic reasons (e.g. micrographs highly outnumbering the investigated structures). Hence, to apply this unbiased stereological method, a modification of the physical disector concerning 3 aspects has been developed. (1) The prerequisite of screening large corresponding tissue areas (here ∼65000 μm²) was fulfilled by examining tissue areas along the edges of ultrathin sections. (2) The size of the counting frame was determined by measuring the lengths of the section margins (minus a guard area) by means of a Morphomat. This value was multiplied by the width of the investigated tissue zone, corresponding to the diameter of the electron microscope viewing screen. (3) Disector counting was carried out simultaneously on both sections (bidirectional disector) to improve efficiency. In the present study tiny synaptic bodies (SBs) were quantitated by disector in a rat pineal gland, yielding ∼30 SBs/1000 μm³. By contrast, single section profile counts of SBs amounted to 90 SBs/20000 μm². Since the presently described adaptation of the disector is time-consuming, it is proposed to determine a proportion factor allowing to estimate number of structures per volume based on single section profile counts. This would decrease the evaluation time by more than 50%.     

The cold pressor test: Vascular and myocardial response patterns and their stability

The purposes of the present study were to compare the cardiovascular response patterns evoked by three versions of the cold pressor test (either forehead stimulation or hand or foot immersion) and to determine the reproducibility of the responses over a 2-week interval. Blood pressure, heart rate, stroke volume, cardiac output, total peripheral resistance, and systolic time intervals were obtained during rest and during the cold pressor test in 42 young men. Across conditions, the pressor response was supported by peripheral resistance increases with concomitant stroke volume decreases. Although the response panerns were generally similar across sites, exceptions were apparent for heart rate. Forehead stimulation was characterized by no significant change in heart rate, whereas limb (hand or foot) immersion was associated with significant heart rate acceleration. The responses elicited by the three cold pressor test conditions were reliable and showed little evidence of attenuation over the test-retest interval.     

Ki67 LABELLING INDEX: AN INDEPENDENT PREDICTOR OF PROGRESSION IN PROSTATE CANCER TREATED BY RADICAL PROSTATECTOMY

The clinical course of prostate cancer is highly variable and cannot satisfactorily be predicted by histological criteria alone. Both tumour cell proliferation and neuroendocrine differentiation have been suggested as additional prognostic parameters, neuroendocrine differentiation being considered to enhance tumour cell proliferation. This study investigated the prognostic value of tumour cell proliferation [Ki67 labelling index (LI), MIB 1] and neuroendocrine differentiation and their relationship to each other. One hundred and thirty-seven paraffin-embedded radical prostatectomy specimens were examined. Neuroendocrine differentiation was found in 58 per cent of cases, but was not associated with pTN stage, Gleason score, Ki67 LI, or tumour progression. Ki67 LI was not significantly associated with pTN stage or with Gleason score. High grade ( P =0·0005), advanced local stage ( P =0·0004), positive lymph nodes ( P =0·02), and high Ki67 LI ( P =0·0203) were predictors of tumour progression if univariate analysis was performed, but Cox stepwise regression showed that only advanced local stage ( P =0·0025) and Ki67 LI ( P =0·0105) were independent predictors of tumour progression, the relative risk being 3·6 and 2·5, respectively. It is concluded that Ki67 is an important prognostic marker in prostate cancer with a potential for routine application.     

EFFECT OF THE INTERACTION BETWEEN TRANSFORMING GROWTH FACTOR β AND ERYTHROPOIETIN ON THE PROLIFERATION OF NORMAL ERYTHROID PROGENITORS AND LEUKEMIC UT-7 CELLS: ACTION OF TRANSFORMING GROWTH FACTOR β ON THE ERYTHROPOIETIN RECEPTOR

The actions of transforming growth factor β (TGFβ) and erythropoietin (Epo) were studied using normal erythroid progenitors from fetal rat liver and spleen at 18, 19 and 20 days. rhTGFβ1 inhibited the growth of late BFUe colonies significantly at each age and in both organs in methylcellulose cultures containing 2 U/ml rhEpo. There was no significant inhibition of CFUe proliferation, except for spleen CFUe at 18 days, suggesting different CFUe sensitivities to growth factors at a given fetal age, 18 days, in liver and spleen. The colorimetric MTT assay was used to examine the inhibition of the growth of human leukemic UT-7 cells by TGFβ1. TGFβ1 inhibited the proliferation of UT-7 cells in cultures without Epo at 24 h and in cultures with Epo at 24 and 72 h. The specific binding of [¹²⁵I]Epo to UT-7 surface was decreased by TGFβ1 without any change in non-specific binding. TGFβ1 also inhibited the expression of Epo-receptors on UT-7 cells, without changing receptor affinity. The inhibition of hematopoietic progenitor cell growth by TGFβ could involve altering the cell surface expression of growth factor receptors.     

Tissue factor serum levels and the risk of future coronary artery disease in apparently healthy men and women: the EPIC-Norfolk prospective population study

INTRODUCTION: Tissue factor (TF) has been implicated in coronary artery disease (CAD). High levels of circulating TF are found in patients with acute atherothrombotic events. Whether high serum TF levels predict risk of future CAD independent of known risk factors remains unknown. METHODS: We conducted a prospective case-control study nested in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk population study. Cases (n=1037) were apparently healthy men and women, aged 45-79 years, who developed fatal or non-fatal CAD during follow-up. Controls (n=2005) were matched by age, sex, and enrolment time. Serum TF levels were measured using high-affinity antibodies. RESULTS: In men, median TF levels were not significant higher in cases than in controls (59.0 pg mL-1, range: 16.7-370.4 vs. 54.9 pg mL-1, range: 16.2-452.4). In women, median TF levels were not significant higher in controls than in cases (73.4 pg mL-1, range: 16.7-492.3 vs. 50.5 pg mL-1, range: 16.5-376.7). The incidence of smoking was about double in the lowest compared with the highest TF quartile. Correcting for sex, age, body mass index, smoking, diabetes, systolic blood pressure, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol and C-reactive protein levels, the risk of future CAD was 1.05 (95% CI: 0.81-1.36) for people in the highest TF quartile, compared with those in the lowest (P-value for linearity=0.8). CONCLUSION: High levels of serum TF were not independently associated with an increased risk of future CAD in apparently healthy individuals.     

A Fractally Coated, 1.3 mm2 High Impedance Pacing Electrode

Minimizing the geometric surface area of pacing electrodes increases impedance and reduces the current drain during stimulation, provided that voltage (pulse-width) thresholds remain unchanged. This may be feasible by coating the electrode surface to increase the capacity of the electrode tissue interface and to diminish polarization. Ten unipolar, tined leads with a surface area of 1.3 mm² and a “fractal” coating of Iridium (Biotronik SD-V137) were implanted in the ventricle, and electrogram amplitude (unfiltered), slew-rate, pacing threshold (0.5 ms), and impedance (2.5 V; 0.5 ms) were measured by the 5311 PSA (Medtronic). On days 0, 2, 5, 10, 28, 90, 180, 360 postimplant, sensing threshold (up to 7.0 mV, measuring range 1–14 mV on day 360 only) and the strength duration curve (0.5–4.0 V; 0.03–1.5 ms; steps: 0.5 V; 0.01 ms, respectively) were determined, the minimum charge delivered per pulse (charge threshold), and the impedance were taken from pacemaker telemetry (Intermedics 294–03). Data were compared with those of an earlier series of 20 unipolar, tined TIR-leads (Biotronik) with a surface area of 10 mm² and a “fractal” coating of titanium nitride. With the model SD-V137 versus TIR, intraoperative electrogram amplitudes were 15.1 ± 6.1 versus 14.4 ± 3.9 mV(NS), slew rates 3.45 ± 1.57 versus 1.94 ± 1.06 V/s (P < 0. 05), pacing thresholds 0.16 ± 0.05 versus 0.52 ± 0.15 V (P < 0.01) and impedance measurements 1,136 ±175 versus 441 ± 73 Ω (P < 0.0001), respectively. During follow-up, sensing thresholds were the same with both leads. Differences in pulse width thresholds lost its significance on day 28 but resumed on day 360 (SD-V137: 0.08 ± 0.04 ms; TIR: 0.16 ± 0.06 ms at 2.5 V; P < 0.01). With an electrode surface of 1.3 mm², charge per pulse and impedance consistently differed from control, beingO.15 ± 0.15 versus 0. 66 ± 0. 20 μC (P < 0.001) and 1,344 ± 376 versus 538 ± 79 Ω, respectively, one year after implantation (P < 0.0001). In summary, “fractally” coated small surface electrodes do not compromise sensing; by more than doubling impedance against controls they offer pacing thresholds (mainly in terms of charge) that are significantly lower than with the reference electrode.