Essential, but at what risk? A prospective study on central venous access in patients with haematological malignancies.

AIMS: Central venous catheters (CVC) are integral to modern haematology practice; however, they are associated with a range of complications. This prospective study aimed to determine the rate of CVC-related complications and risk factors in haematology patients, who are vulnerable because of their underlying pathology and treatments. METHODS: All inpatients that had a non-tunnelled CVC inserted in a 14-month period in the haematology ward at St Vincent's Hospital were enrolled. Complications (immediate and late), demographics, type of device, insertion technique and duration of dwell, were examined using multivariate analysis. RESULTS: One hundred and seventy-four CVC in 84 patients were recorded, representing 3016 catheter-days. At least one complication was found in 43 (24.7%) patients. Immediate complications occurred in 13 (7.5%) insertions, with a higher rate in those inserted after ≥2 attempts compared with one (P = 0.02). Catheter-related bloodstream infection occurred at a rate of 7.6 per 1000 catheter-days, with acute lymphoblastic leukaemia associated with a higher rate (P = 0.02), and subclavian vein CVC had a lower rate compared with other locations (P < 0.01). Thrombosis was found in seven (4.0%) patients, with subclavian CVC carrying an increased risk (P = 0.02). CONCLUSIONS: This prospective observational study found almost a quarter of haematology patients experience a CVC-related complication. An association was found with a number of attempts at insertion and immediate complications; other risk factors included anatomical location, underlying disease and duration of catheterisation. The relatively high complication rate, compared with reports of non-haematology patients, highlights the need to improve CVC management, a vital part of care for this population.

Modulation of rat uterine steroid hormone receptors by estrogen and antiestrogen.

The effect of sc injections (25 microgram) of estradiol benzoate, monohydroxytamoxifen [1-(4 beta-dimethylaminoethoxyphenyl)1-(4-hydroxyphenyl)-2-phenylbut-1-ene] and tamoxifen [trans-1-(4 beta-dimethylaminoethoxyphenyl)1,2-diphenylbut-1-ene] every 12 h on uterine wet weight, DNA, and cytoplasmic estrogen and progesterone receptor levels has been studied in the immature rat for up to 90 h. Estradiol benzoate produced a 4-fold rise in uterine wet weight, with a doubling of uterine DNA within 48 h. Tamoxifen and monohydroxytamoxifen doubled uterine weight, with a small rise in uterine DNA. Cytoplasmic estrogen receptor levels were reduced within 24 h by all treatments. Estradiol benzoate and monohydroxytamoxifen produced a maximal rise in nuclear estrogen receptor levels within 8 h, followed by a rapid decline to control levels within 80 h. Tamoxifen produced a slower rise in nuclear estrogen receptor levels and never reached the levels achieved by estradiol or monohydroxytamoxifen. The level of nuclear tamoxifen-estrogen receptor complexes slowly decreased with time. In each case, the cytoplasmic progesterone receptor levels increased as nuclear estrogen receptor levels decreased. Cycloheximide (5 microgram/2 h for 8 h before and 20 h after the first estrogen or antiestrogen injection) was used to determine the effect of protein synthesis inhibition on the hormone receptor profiles. Progesterone receptor synthesis was inhibited by cycloheximide. Cycloheximide did not affect translocation, but produced a rapid decrease in nuclear estrogen receptor levels. The results suggest that without the continual translocation of estrogen receptors from the cytoplasm, the antiestrogen-estrogen receptor levels in the nuclear compartment decrease because of destruction or processing. The nuclear antiestrogen-estrogen receptor pool is therefore not static but dynamic. High affinity ligand-estrogen receptor complexes are readily processed in the nucleus to effect progesterone receptor synthesis in the cytoplasm; however, this series of biochemical reactions is only secondary to the fundamental events essential for cell division. After an initial increase in cytoplasmic steroid receptor synthesis by estrogen or antiestrogen, there is a gradual reduction in total cytoplasmic and nuclear estrogen receptor complexes in response to continual nuclear stimulation. This reduction, in turn, reduces progesterone receptor synthesis.

The increased sensitivity of platelets to prostacyclin in marathon runners.

Platelet-rich plasma was obtained 24 hr after the race ended from athletes who ran in the London marathon. The platelets were only marginally less sensitive to adrenaline than were those of non-runners using conventional aggregation tests. However, the runners' platelets were much more sensitive to inhibition by prostacyclin, a prostaglandin synthesized by endothelial cells. It appeared that this effect was due to a greater activity in the platelets of the membrane-bound adenylate cyclase enzyme which generates intracellular cyclic AMP. Cyclic AMP production is known to be stimulated by prostacyclin and to cause the inhibition of platelet aggregation. The results indicate another possible protective effect of exercise against cardiovascular disease which is independent of the known changes in lipoprotein concentrations previously observed in athletes.

Subcellular effects of monohydroxytamoxifen in the rat uterus: steroid receptors and mitosis.

The administration of either monohydroxytamoxifen (25 micrograms) or oestradiol benzoate (25 micrograms) to immature rats resulted in similar depletion of the cytoplasmic oestrogen-receptor pool, with a transient (approx. 48 h) increase in nuclear oestrogen-receptor levels. Oestradiol benzoate increased uterine wet weight with a corresponding increase in uterine cytoplasmic progesterone-receptor levels, DNA content and cell division at 48 h. In contrast, monohydroxytamoxifen produced only a partial increase in uterine wet weight. Although the increase in concentration of uterine progesterone receptors (per mg DNA) by monohydroxytamoxifen was comparable to that produced by oestradiol benzoate, the nuclear antioestrogen-oestrogen-receptor complexes were unable to stimulate a large rise in whole uterine DNA content and cell division. Examination of stromal and epithelial mitotic activity 48 h after administration of 0.25, 2.5 and 25 micrograms oestradiol benzoate, monohydroxytamoxifen or tamoxifen showed that the inability of antioestrogens to stimulate oestrogen-like mitotic activity was not related to the dose of antioestrogen that was administered. It is suggested that the inability of the antioestrogen-oestrogen-receptor complexes to initiate the nuclear events which lead to cell division should be exploited in the investigation of the nuclear mechanism of oestrogen action. The high potency of monohydroxytamoxifen and its inherent biological activity, in that it is not metabolically activated before exerting its effects, provide clear advantages for its future use as a pharmacological probe.

Dose-related effects of non-steroidal antioestrogens nad oestrogens on the measurement of cytoplasmic oestrogen receptors in the rat and mouse uterus.

The dose-related effects of non-steroidal antioestrogens and oestrogens on the measurement of cytoplasmic oestrogen receptors in the rat uterus have been determined. The simultaneous administration of tamoxifen or monohydroxytamoxifen and oestradiol on three consecutive days resulted in dose-dependent decreases in both the wet weight of the uterus and the number of available cytoplasmic oestrogen receptors. The oestrogenic triphenylethylenes ICI 47 699 and ICI 3188 both produced dose-dependent decreases in the number of available cytoplasmic oestrogen receptors. Increasing doses of ICI 47 699 resulted in increasing concentrations of oestrogen receptors within the nucleus. The effects of tamoxifen and oestradiol-17 beta were compared in the ovariectomized mouse; replenishment of uterine oestrogen receptors was less evident in tamoxifen-treated animals than in animals receiving oestradiol, although increases in uterine weight were similar. A single large dose of tamoxifen (50 microgram) produced a prolonged depletion of cytoplasmic oestrogen receptors whilst stimulating rises in uterine weight and DNA and protein content. The results demonstrate that the depletion of the uterine cytoplasmic oestrogen receptor pool is a function of the dose administered for any compound with the ability to translocate oestrogen receptors to the nucleus and as such is not an exclusive characteristic of non-steroidal antioestrogens.

The dynamics of LH-induced desensitization of adenylate cyclase and LH receptor internalization in rat Leydig cells at physiological levels of LH.

The LH-induced desensitization of adenylate cyclase and LH receptor internalization in rat Leydig cells in vitro has been investigated using sub-physiological to pharmacological concentrations of LH. Leydig cells pretreated with LH for 1 h in the presence of a phosphodiesterase inhibitor showed a dose-dependent decrease in the subsequent response to a high dose of LH; this was significant with concentrations of greater than 350 fmol/l. The maximum amount of desensitization was 60%. The time-course of LH-induced desensitization of the adenylate cyclase system was investigated; with 3 nmol LH/l cyclic AMP was increased in a linear manner up to 45 min, after which time there was either no further production or (with the higher concentrations) a decreased rate of production. In order to determine whether the 'non-desensitized' adenylate cyclase activity was dependent upon LH, the LH-treated cells were acid-washed to remove residual LH; cyclic AMP production still continued, albeit at a lower rate, thus indicating that this adenylate cyclase activity did not require the further presence of LH. The effect of various concentrations of LH on the level of surface-associated LH receptors was determined in the presence of monensin to prevent receptor recycling. A dose- and time-dependent decrease was found; this was significant after 2 h with 3.5 pmol LH/l and reached a maximum of 63% of the control with 3.5 nmol/l. A dose- and time-dependent reversal of desensitization occurred if the cells pretreated with LH were washed and reincubated; with 35 pmol LH/l and after 2 h the cells were fully responsive to a challenge with fresh LH. With higher concentrations of LH in the pretreatment, partial or no recovery was found. These studies demonstrate that physiological and sub-physiological concentrations of LH induce a rapid desensitization of Leydig cell adenylate cyclase. Internalization of occupied and unoccupied LH receptors also occurred. It is proposed that these two events are linked and may, paradoxically, and because of the low numbers of LH receptors, be necessary to maintain the normal response of Leydig cells to LH in vivo.

Effects of lutropin (LH) receptor internalization and recycling on the apparent numbers of LH receptors in rat testis Leydig cells at different temperatures.

The effect of different temperatures on the Scatchard analysis of binding studies with 125I-hCG and intact rat testis Leydig cells has been investigated. The results show that at temperatures greater than 4 degrees C an overestimation of the number of receptors/cell is likely to occur. A one-site analysis of the data gives values of 13,573, 61,924 and 3802 LH receptors/cell at 34 degrees C, 21 degrees C and 4 degrees C respectively. At 34 degrees C and 21 degrees C (but not at 4 degrees C) a two-site analysis of the data is possible, giving similar high affinity binding components at both temperatures (KD 1 X 10(-10) M) but with dissimilar low affinity binding components (34 degrees C, 2.17 X 10(-9) M and 21 degrees C, 6.3 X 10(-8) M). The calculated total number of LH receptors using a two-site analysis at 21 degrees C is equal to 114,766 receptors/cell, and at 34 degrees C it is 37,987 receptors/cell. It is proposed that the differences in the level of binding at different temperatures and the associated changes in the value of KD, reflect the temperature sensitivity of the endocytic pathway of the 125I-hCG/LH receptor complex within the rat testis Leydig cell. From previous studies it is known that at 34 degrees C and 21 degrees C, a large part of the cell-associated hormone is not receptor bound but rather it represents 'processed' hormone before it is released from the cell. At 4 degrees C the cell-associated 125I-hCG remains bound only to cell surface LH receptors and thus gives a more accurate measure of total receptor numbers/cell.

Catecholamine stimulation of testosterone production via cyclic AMP in mouse Leydig cells in monolayer culture.

The effects of the beta-adrenergic hormone agonist, isoproterenol, on testosterone and cyclic AMP production in mouse Leydig cells in culture have been investigated. It was found that isoproterenol increased testosterone production on days 1, 2 and 3 of culture but not in freshly cultured cells. Cyclic AMP production was however increased on all days of culture. In subsequent studies carried out on day 2 of culture the amounts of testosterone formed during incubation with isoproterenol were 20-90% of those obtained with maximum stimulating levels of luteinizing hormone. The amounts of cyclic AMP formed were extremely low compared with those obtained with luteinizing hormone (22 +/- 5.3 and 2320 +/- 100 pmoles/10(6) cells/2 h respectively). Isoproterenol (10(-8) -10(-7) M) gave a significant increase in testosterone production and reached a maximum with 10(-6) M. Similar dose-response curves for cyclic AMP production were obtained. The stimulation of cyclic AMP and testosterone by isoproterenol was highly dependent on the presence of the phosphodiesterase inhibitor, methylisobutylxanthine. Propranolol blocked, in a dose-dependent manner, both isoproterenol-stimulated cyclic AMP and testosterone production. In the presence of excess luteinizing hormone no additional effects of isoproterenol were detected. Epinephrine also stimulated testosterone production. It is concluded that catecholamines stimulate testosterone production in mouse Leydig cells in monolayer culture and that this effect if mediated by cyclic AMP.

Stimulation and inhibition by LHRH analogues of cultured rat Leydig cell function and lack of effect on mouse Leydig cells.

The effect of 2 luteinizing hormone-releasing hormone (LHRH) analogues(10-8-10-6 M) on the functional activity (testosterone and cyclic AMP production and [125I]hCG binding) of purified mouse Leydig cells in culture was examined. The analogues were found to have no significant effect on the cells over a period of 3 days. No specific binding of a labelled analogue to impure or pure mouse Leydig cells could be detected. In contrast high levels of specific binding to impure rat interstitial cells occurred. Centrifugation of the rat interstitial cells on 0-90% Percoll gradients showed that the LHRH analogue bound specifically to the active lutropin-responsive Leydig cells. The purified rat Leydig cells were cultured in the presence of LHRH analogue (ICI 118630) (10-7 M) and after an initial lag period (2h) a marked stimulation of testosterone production occurred over a 32-h period (up to 400 ng/10(6) cells). The response to LH alone increased with time in culture up to 10 h, and the LHRH analogue enhanced this LH-stimulated testosterone production. When the cells were cultured for longer time periods (24 h) the LHRH analogue was found to inhibit LH-stimulated testosterone production at all concentrations of LH used (p less than 0.01). The LHRH analogue had no consistent effect on LH-stimulated cyclic AMP production, although when added alone, cyclic AMP production was increased. These results show that LHRH analogues do not bind to or have any detectable effect on mouse Leydig cells in vitro. However, LHRH analogue does bind specifically to purified rat Leydig cells. After a short lag period the analogue stimulates testosterone production which turns to inhibition after 20 h in culture.

The functional activity of adult mouse Leydig cells in monolayer culture. Effect of lutropin and foetal calf serum.

Purified Leydig cells were obtained from adult mouse testes by mechanical dispersion followed by Percoll density-gradient centrifugation as described by Schumacher et al. (1978). The cells were then established in monolayer culture by maintaining them in medium and 10% serum at 32 degrees C in 95% O2, 5% CO2. The cells rapidly attached to the culture dishes, gradually flattened and became epitheloid in appearance. Testosterone production by the cells in response to maximum stimulating levels of LH (100 ng/ml) and dibutyryl cyclic AMP (1 mM) was maintained for at least 2 days (approximately 1 microgram/10(6) cells/2h) and then declined to lower levels by days 3-4. Cyclic AMP production in response to LH was higher on day 1 than day 0 and then declined to lower levels by days 3-4. Binding of [125I]hCG was similar on day 0 and day 1 (approximately 20 fmoles/10(6) cells) and then declined to lower levels by days 3-4. The functional activity of the cells cultured in 0, 1 and 10% foetal calf serum was also examined; no significant effect of the serum on LH-stimulated testosterone or cyclic AMP production was found; however, a decrease of up to 50% in the binding of [125I]hCG to the Leydig cells occurred in the presence of serum. These results demonstrate that the function of differentiated adult Leydig cells can be maintained for at least 2 days in culture.

Inhibition of cell division and stimulation of progesterone receptor synthesis in rat oestrogen target tissues by non-steroidal antioestrogens.

Recent studies from this laboratory have demonstrated oestradiol-oestrogen receptors associated with the nuclear compartment of the rat uterine cell will initiate protein synthesis, as evidenced by a rise in progesterone receptor concentrations, and cell division whereas the anti-oestrogen - oestrogen receptor complex causes protein synthesis and cellular hypertrophy rather than hyperplasia. It is probable that this separation of biological activities resides in the intrinsic activity of the respective receptor complexes. We have demonstrated that caution should be exercised in the interpretation of low affinity ligand-hormone receptor interactions undertaken in vitro. Simple tests for ligand specificity for a binding protein are clearly insufficient evidence to characterise a hormone receptor complex using a conventional 15 hr technique of sucrose density gradient analysis. Oestrogens and anti-oestrogens do not seem to disrupt the subunit integrity of the cytoplasmic oestrogen receptor and it appears likely that the ligand plays a fundamental role in confering the correct biological properties to the hormone receptor complex.