V. Lo Cascio

The Acute-Phase Response after Bisphosphonate Administration

SummaryIn patients who have never previously received bisphosphonate therapy, the intravenous administration of 4-amino-1-hydroxybuthilidene-1,1-bisphosphonate (AHButBP), 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP), or 6-amino-1-hydroxyhexylidene-1,1-bisphosphonate (AHHexBP) induced an acute-phase response (APR) irrespective of the underlying disease, manifested by a fall in circulating lymphocyte number and serum zinc concentration and in a rise in C-reactive protein (CRP); a febrile reaction occurred in 30% of the patients. The APR was maximally expressed within 28–36 hours of i.v. administration of the bisphosphonates and disappeared 2–3 days later despite continuous treatment. These effects were dose dependent and the lowest doses necessary for an APR were 10 mg of AHButBP and AHPrBP and 75 mg of AHHexBP. Doses up to 1,000 mg/day i.v. of dichloromethanebisphosphonate (Cl2MBP) were devoid of these side effects. In patients treated with either a single i.v. dose of amino-bisphosphonates which resulted in an APR or with a suboptimal dose, a subsequent challenge 12–160 days later of the high dose failed to cause a rise in CRP or a fall in the lymphocyte count. The desensitization to AHButBP or AHPrBP was also seen following pretreatment with Cl2MBP. These findings suggest that bisphosphonates interact with macrophage-like cells resident in the skeleton and stimulate interleukin-1 release which is responsible for the appearance of the APR. At the same time, however, the bisphosphonates render these cells insensitive to further stimulation for several months. This latter observation might be relevant to the long-lasting suppression of bone resorption observed after bisphosphonate therapy.

Elevated levels of soluble E-selectin in patients with IDDM and NIDDM: relation to metabolic control

SummaryThe adhesion of leucocytes to the endothelium, an early step in atherogenesis, is mediated by cell adhesion molecules. In this study we evaluated the concentration of soluble adhesion molecules in patients with insulin-dependent (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) and studied its relation to glycaemic control. Soluble adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) were measured in 31 diabetic patients (18 with IDDM and 13 with NIDDM), 20 hyperlipoproteinaemic patients (10 with type IIa and 10 with type IIb) and 20 healthy subjects. Increased E-selectin concentrations were found in the patients with IDDM and NIDDM and in the hyperlipoproteinaemic patients when compared to the control subjects (p<0.01 for all the groups). ICAM-1 was found to be elevated only in the patients with NIDDM (p<0.01). No significant differences in VCAM-1 concentration were found in the different groups of subjects. The concentration of plasma E-selectin was positively correlated with the glycated haemoglobin (r=0.54, p<0.01) in patients with IDDM and NIDDM. In the same patients E-selectin was not related to the concentrations of plasma lipids in spite of the fact that it was found to be elevated in hyperlipoproteinaemic subjects. The results though preliminary suggest that in diabetic patients the concentration of soluble adhesion molecules and especially of E-selectin may be related to metabolic control.

Predisposition to LDL oxidation in patients with and without angiographically established coronary artery disease

Oxidative modification of low density lipoprotein (LDL) may play an important role in the mechanism of atherosclerotic damage to blood vessels. In the present study the LDL isolated from the plasmas of 73 coronary artery disease (CAD) patients, 28 valvular heart disease (VHD) patients, 59 subjects affected by type IIa hyperlipoproteinemia and 71 controls was oxidatively modified by incubation with copper ions. In 15 CAD and 15 Type IIa patients and 15 controls the LDL chemical composition and polyunsaturated fatty acid (PUFA) content were also measured. Differences in the LDL susceptibilities to lipid peroxidation were studied by measuring the changes of fluorescence intensity. The lag phase in the CAD patients was found to be significantly lower than in the VHD and controls (P < 0.001). The lag phase in the type IIa patients was significantly higher than in the CAD patients (P < 0.01), and significantly lower than the VHD and controls (P < 0.01). The LDL isolated from the type IIa patients had an increase in the relative content of free and esterified cholesterol (P < 0.05), while the CAD patients had a decrease in the relative content of free cholesterol (P < 0.05), and an increase in the relative content of protein (P < 0.05). The lowest value of the LDL cholesterol to protein ratio and LDL size, was found in the CAD patients (P < 0.05). When expressed in micrograms/mg LDL cholesterol, the concentration of the LDL PUFAs was significantly higher in the CAD group than in the others (P < 0.05). The LDL alpha-tocopherol concentration was quite similar in the different groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Dichloromethylene-Diphosphonate in Patients with Prostatic Carcinoma Metastatic to the Skeleton

A total of 17 patients with multiple osteoblastic bone metastases owing to prostatic carcinoma was treated with 2-dichloromethylene-diphosphonate, a powerful inhibitor of bone resorption. The drug was given intravenously (300 mg.) for 2 weeks and then orally (3,200 mg.) or intramuscularly (100 mg.) for 4 to 11 weeks. A definite improvement in pain, assessed by daily consumption of analgesic drugs and by an analogic scale, was observed within 10 days in 16 of the 17 patients. Four patients confined to bed rest for pain were able to walk after 2 weeks and reversal of paralysis also was noted in 1 patient. Transient changes in serum calcium (decreasing) and alkaline phosphatase (increasing) were observed in most patients. In the 3 patients in whom it was performed, repeated bone scanning showed a partial regression of pathological areas in 2 and the complete disappearance of most pathological areas in 1. Our results suggest that 2-dichloromethylene-diphosphonate may represent an important supportive treatment in patients with bone metastases owing to prostatic carcinoma, providing sustained relief of pain and regression of bone destruction without undesirable side effects.

Oxidized low-density lipoprotein increases the production of intracellular reactive oxygen species in endothelial cells : inhibitory effect of lacidipine

Objective The mechanisms by which oxidized low-density lipoprotein (ox-LDL) induces the expression of adhesion molecules on endothelial cells (HUVECs) are still not clear. The signal transduction pathways for these binding molecules include the translocation of the transcription factor NF-kB and the intracellular reactive oxygen species (ROS) are said to play a key role in this process. Aim of this study was (1) to evaluate the effect of ox-LDL on intracellular production of ROS in culture of HUVECs; (2) to evaluate if the intracellular increase of ROS induced by ox-LDL is mediated by the binding to a specific endothelial receptor; (3) to ascertain if lacidipine can decrease ox-LDL-induced ROS production in HUVECs. Methods Five *MCu2 + ox-LDL were incubated with HUVECs for 5 min. 2′,7′-Dichlorofluorescein (DCF) as an expression of intracellular ROS production, was measured by flow cytometry. Results ox-LDL induced a significant dose-dependent increase in DCF production (P < 0.001) through the binding to a specific receptor. The preincubation of HUVECs with radical scavengers compounds and lacidipine significantly reduced (P < 0.001) the ox-LDL-induced DCF production. Conclusions ox-LDL increased the intracellular formation of ROS through the ligation to a specific endothelial receptor. Preincubation of HUVECs with lacidipine, a calcium antagonist with antioxidant properties, significantly reduced the intracellular ROS formation induced by ox-LDL. We propose that the effect of lacidipine on adhesion molecule expression and on NF-kB activation can be explained by its effect on intracellular ROS formation.

A new questionnaire for the repeat of the first stage of the European Community Respiratory Health Survey: a pilot study

In the early 1990s a multicentre survey on asthma was performed on the young adult population (European Community Respiratory Health Survey - ECRHS). This study is to be repeated in order to estimate changes in the prevalence of asthma-like symptoms during the last decade and to assess the social and economic costs of the disease and their variations among countries. The self-administered questionnaire devised for this purpose is a two-page questionnaire. The first page contains the same items as those used in the first survey with four additional questions related to: 1) the frequency and severity of asthma attacks; 2) the presence of chronic bronchitis; 3) smoking habits; and 4) a visual analogue scale assessing perception of outdoor pollution. The second page aims to collect information regarding the direct and indirect costs of asthma. The influence of the length of the questionnaire on the response rate was assessed in a pilot study in Italy. Two random samples of 150 subjects received either the one-page questionnaire (first page) or the two-page questionnaire. The response rate was compared with that obtained from the first postal wave in the 1991-1992 survey. Although the response rate was unchanged when using the one-page questionnaire (45% versus 45%), it decreased by 7% when the two-page questionnaire was used (38% versus 45%). On the basis of these results, no problem should arise if four more questions are added to the one-page questionnaire. The slight reduction in the response rate of the two-page questionnaire is worrying but could be corrected by the use of telephone interviews.

Treatment of Paget's Disease of Bone with Intravenous 4-Amino- 1 -Hydroxybutylidene- 1,1-Bisphosphonate

Summary4-amino-1-hydroxybutylidene-1,1-bis-phosphonate (AHButBP) was given intravenously (2.5–25 mg/day for 4 days) to 14 patients with Pagets disease of bone, five of whom had been treated with dichloromethylidene bisphosphonate (Cl2MBP) 32 months earlier. In the nine patients who had not been treated previously with bisphosphonates, the short course of AHButBP induced a suppression of serum alkaline phosphatase and urinary hydroxyproline values down to 30% of initial values. The biochemical suppression of the disease was sustained for 2–18 months and the time to relapse did correlate to the logarithm of the dose (P<0.001). In the five patients previously treated for Pagets disease, an apparent resistence to treatment with AHButBP was observed. However, in these patients both serum alkaline phosphatase and urinary hydroxyproline fell to or even below the nadir values which had previously been achieved with Cl2MBP, irrespective of the degree of relapse. Thus the degree of suppression of Pagets disease of bone, achievable after treatment with bisphosphonates, seems to be constant for each patient, such that normal levels of serum alkaline phosphatase and urinary hydroxyproline cannot usually be attained in patients with extremely active disease.

Effects of two oral doses of alendronate in the treatment of Paget's disease of bone.

Twenty patients with mild Pagets disease of bone were given either 20 (10 patients) or 40 mg alendronate daily for 6 months. The 20-mg dose was well tolerated, but in 3 patients on 40 mg/d alendronate, the treatment was withdrawn after 3-5 months because of gastric and oesophageal disturbances. Urinary hydroxyproline excretion fell within the first month to 77 +/- 5% (SD) and to 47 +/- 5% of pretreatment values in the 20- and 40-mg dosing group, respectively (p < 0.001 between group comparison). The serum alkaline phosphatase fell more slowly with the maximum suppression of disease activity reached at 4 months, when it attained a plateau in both groups of patients. However, the decrease in serum alkaline phosphatase was significantly more pronounced in the patients treated with 40 mg/d tablets (50 +/- 10% of pretreatment values) than in those given 20 mg alendronate per day (76 +/- 9% of initial value), in none of whom a disease remission was observed. It appears, therefore, that while 20 mg/d oral doses of alendronate are insufficient, 40 mg/d are associated with a high incidence of side effects. Furthermore, the suppression of disease activity depends on the dose of bisphosphonate given daily or over a short period of time and lower doses cannot be compensated by a longer duration of the treatment course.

The effects of menopause and estrogen replacement therapy on the renal handling of calcium.

Mineral metabolism was studied in 99 premenopausal and 80 postmenopausal women both before and after 9–14 months of treatment with 50 µg/day transdermal estradiol. In estrogen-repleted subjects (premenopausal women and postmenopausal women on estrogen replacement therapy) total serum calcium was significantly lower (0.065 mmol/l;p<0.001) than in those who were estrogen-depleted (untreated postmenopausal women). This difference was smaller but still significant for calculated ultrafiltrable calcium (UFCa: 0.02–0.03 mmol/l;p<0.001). However, ionized calcium (both calculated and measured) was not different in the two groups of women. This finding explains why estrogen repletion does not induce changes in the serum level of intact parathyroid hormone (PTH), despite lower total or ultrafiltrable serum calcium. In a parallel study we have shown that intravenous administration of aminobutane bisphosphonate, a powerful inhibitor of bone resorption, produces similar decreases in serum calcium which were associated with significant increases in intact PTH.Estrogen-depleted women had, on the one hand, significantly higher serum levels of bicarbonate, anion gap, complexed calcium, pH, phosphate and alkaline phosphatase, and higher rates of tubular reabsorption of phosphate and urinary excretion of calcium and hydroxyproline. On the other hand they had lower serum chloride levels and lower rates of tubular reabsorption of calcium.Altogether these findings might indicate that estrogen deficiency decreases renal sensitivity to PTH. This is responsible for the higher serum phosphate and bicarbonate levels, the resulting mild metabolic alkalosis leading to higher serum levels of complexed ultrafiltrable calcium and higher rates of urinary excretion of calcium, but unchanged serum levels of ionized calcium and PTH.

Lacidipine inhibits the activation of the transcription factor NF-kappa B and the expression of adhesion molecules induced by pro-oxidant signals on endothelial cells

Objective The adhesion of monocytes to endothelium, an early event in atherosclerosis is mediated by cell adhesion molecules. Signal-transduction pathways for these binding molecules include the translocation of the transcription factor NF-κB; moreover, intracellularly generated oxygen-derived free radicals play a major role in this process. In this study we evaluated the extent to which lacidipine, a calcium antagonist with antioxidant properties, affects the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on human umbilical vein endothelial cells, induced by different pro-oxidant signals such as oxidized low density lipoprotein (LDL) and tumor necrosis factor-α (TNF-α). Methods We incubated 5 mmol/l Cu2+-oxidized LDL and TNF-α (2 ng/ml) with human umbilical vein endothelial cells for 48 and 6 h, respectively. ICAM-1, VCAM-1 and E-selectin were measured by flow cytometry. NF-κB was evaluated by electrophoretic mobility shift assay. Results The incubation of 5 μmol/l Cu2+-oxidized LDL not only caused a dose-dependent increase in ICAM-1, VCAM-1 and E-selectin (P <0.001), but also synergically increased their TNF-α–induced expression (P <0.001). The addition of lacidipine to human umbilical vein endothelial cells significantly reduced the expression of ICAM-1, VCAM-1 and E-selectin induced by TNF-α alone or with oxidized LDL (P <0.001). The reduction in adhesion molecule expression caused by lacidipine was paralleled by a significant fall in NF-κB translocation. Conclusions The results suggest that lacidipine may have prevented NF-κB-mediated adhesion molecule expression by exerting its effects on oxygen-derived free radicals. The results support previous observations that lacidipine may have therapeutic effects in atherosclerosis.