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Galectin-1 (Gal-1) is involved in tumoral angiogenesis, hypoxia and metastases. Actually the Gal-1 expression profile in multiple myeloma (MM) patients and its pathophysiological role in MM-induced angiogenesis and tumoral growth are unknown. In this study, we found that Gal-1 expression by MM cells was upregulated in hypoxic conditions and that stable knockdown of hypoxia inducible factor-1a significantly downregulated its expression. Therefore, we performed Gal-1 inhibition using lentivirus transfection of shRNA anti-Gal-1 in human myeloma cell lines (HMCLs), and showed that its suppression modified transcriptional profiles in both hypoxic and normoxic conditions. Interestingly, Gal-1 inhibition in MM cells downregulated proangiogenic genes, including MMP9and CCL2, and upregulated the antiangiogenic ones SEMA3Aand CXCL10. Consistently, Gal-1 suppression in MM cells significantly decreased their proangiogenic properties in vitro. This was confirmed in vivo, in two different mouse models injected with HMCLs transfected with anti-Gal-1 shRNA or the control vector. Gal-1 suppression in both models significantly reduced tumor burden and microvascular density as compared with the control mice. Moreover, Gal-1 suppression induced smaller lytic lesions on X-ray in the intratibial model. Overall, our data indicate that Gal-1 is a new potential therapeutic target in MM blocking angiogenesis.

The relationship between bone marrow (BM) cytokine and chemokine levels, cytogenetic profiles and skeletal involvement in multiple myeloma (MM) patients is not yet defined. This study investigated a cohort of 455 patients including monoclonal gammopathy of uncertain significance (MGUS), smoldering MM and symptomatic MM patients. Skeletal surveys, positron emission tomography (PET)/computerized tomography (CT) and magnetic resonance imaging (MRI) were used to identify myeloma bone disease. Significantly higher median BM levels of both C-C motif Ligand (CCL)3 and CCL20 were found in MM patients with radiographic evidence of osteolytic lesions as compared with those without, and in all MM patients with positive PET/CT scans. BM levels of CCL3, CCL20, Activin-A and Dickkopf-1 (DKK-1) were significantly higher in patients with high bone disease as compared with patients with low bone disease. Moreover, CCL20 BM levels were significant predictors of osteolysis on X-rays by multivariate logistic analysis. On the other hand, DKK-1 levels were related to the presence of MRI lesions independently of the osteolysis at the X-rays. Our data define the relationship between bone disease and the BM cytokine and chemokine patterns highlighting the tight relationship between CCL20 BM levels and osteolysis in MM.

OBJECTIVE: In the present study we have measured the concentrations of interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and IL-1 receptor antagonist (IL-1Ra) in the serum of patients with Graves' disease (GD). By multivariate analysis, we have evaluated the effect of antithyroid treatment, thyroid function, the presence or absence of active thyroid-associated ophthalmopathy (TAO), the patient's smoking habits and the relation to circulating anti-thyrotropin (TSH) receptor (TRAb) and anti-thyroperoxidase antibodies (TPOAb). SUBJECTS: We studied 84 GD patients, 51 untreated and 33 receiving methimazole (MMI) therapy. Twenty-three (45%) untreated patients and 18 (54%) patients on MMI had active TAO. We also studied 67 normal subjects as controls. Thirty-one GD patients (43%) and 16 controls (36%) were smokers. RESULTS: Serum IL-6 concentrations were significantly higher in both untreated patients (P<0.001) and treated patients (P<0.006), when compared with controls. Serum sIL-6R concentrations were significantly affected by treatment (P=0.001). Serum IL-1Ra concentrations were not different in GD patients, whether treated or untreated, compared with controls. Serum IL-6 concentrations were not influenced by thyroid function and there was a significant interaction between treatment and the presence of active TAO (P=0.003). In hyperthyroid patients with active TAO serum, sIL-6R concentrations were significantly higher than in those with inactive TAO (P=0.003). In untreated GD patients there was no significant effect of thyroid function and TAO activity on the serum concentrations of TNF-alpha and IL-1 beta. Serum IL-1Ra concentrations were not affected by the presence of TAO. Smoking had no effect on serum IL-6, sIL-6R, TNF-alpha, IL-1 beta and IL-1Ra concentrations, even in the presence of an active TAO. Serum concentrations of IL-6, sIL-6R, TNF-alpha and IL-1 beta and IL-1Ra were not different in patients with and without TRAb or TPOAb, in relation to either thyroid function, TAO activity or smoking. CONCLUSIONS: Our work shows that: (i) the proinflammatory cytokine pattern in GD is greatly influenced by antithyroid drug treatment; (ii) the increased circulating IL-6/sIL-6R concentrations observed in patients with active TAO may derive from the activation of humoral reactions in sites other than the thyroid; and, (iii) cigarette smoking has no effect on serum IL-1/IL-1Ra concentrations in TAO.

The present study was undertaken in order to establish the possible involvement of 5-HT3 serotonergic receptors in the control of basal and/or hypoglycemia-stimulated arginine vasopressin (AVP) and/or oxytocin (OT) secretion. For this purpose, 12 normal men were injected intravenously with a bolus of 4 mg ondansetron, a specific 5-HT3 receptor antagonist, under basal conditions (n= 6) or 30 min before insulin (0.15 lU/kg body weight) administration (n= 6) (insulin tolerance test (ITT)). Control experiments with normal saline instead of ondansetron treatment were performed. Furthermore, on a different occasion, the same subjects were tested in identical experimental conditions with 8 mg ondansetron. Our results showed that the hypoglycemic response to insulin was similar during the ITT and ondansetron plus ITT. Inhibition of 5-HT3 serotonergic receptors with ondansetron (4 or 8 mg) did not modify the basal secretion of AVP and OT and the OT response to insulin-induced hypoglycemia. In contrast, the administration of 4 or 8 mg ondansetron significantly reduced in a similar manner hypoglycemia-induced AVP rise. Mean peak level at 45 min after insulin injection was 2.25 times higher than baseline in the control ITT and 1.5 times higher than basal value in the ondansetron (4 or 8 mg) plus ITT. These data demonstrate that 5-HT3 serotonergic receptors at least partially mediate the AVP response to hypoglycemia, without modifying the simultaneous OT response. On the other hand, 5-HT3 receptors do not appear to be involved in the control of basal posterior pituitary hormone secretions.

The purpose of this study was to examine the serum levels of bone alkaline phosphatase (BALP) measured with a new assay in normal and in osteoporotic women, and to evaluate prospectively its responsiveness to changes of bone metabolism. The following groups of subjects were studied: (1) 95 healthy women (44–75 years) (22 pre- and 73 postmenopausal) and 35 osteoporotic women [vertebral bone mineral density (BMD) more than 2.5 SD below the normal adult mean]; (2) 10 women (44–50 years) ovariectomized (OVX) for benign uterine diseases, examined before and 12 months after surgery; (3) 16 OVX women (36–54 years), examined before and after 12 months of transdermal estrogen replacement therapy (50 μg/day); (4) 12 previously untreated pagetic patients (4 women and 8 men, 50–80 years), examined before and 3 months after the I.V. administration of clodronate (600 mg) or alendronate (5 mg) for 2 consecutive days. The median BALP value was 11.6 U/liter (25–75th percentiles: 10.5–12.7; range 7.7–19.3) in healthy premenopausal (PreMP) women and significantly higher (median: 16.8 U/liter; 25–75th percentile: 13.8–21.8; P< 0.01) in postmenopausal (PostMP) women. There was a clear age-related increase in normal subjects (r = 0.43; P< 0.001). In the osteoporotic group, BALP levels, as well as other biochemical parameters of bone turnover, were not significantly different from those of normal women when adjusted for age. In OVX women, BALP levels showed a marked increase 12 months after surgery (median: 113%; 25–75th percentile: 87–139%), significantly higher than the increase of total ALP (median: 43%; 25–75th percentile: 25–66%; P< 0.001), and similar to the increases of serum osteocalcin and urinary hydroxyproline. Transdermal estrogen treatment prevented the BALP increase, even if no reduction was observed; total ALP showed a similar behavior. The basal levels of BALP were significantly elevated in pagetic patients (median: 91 U/liter; range 18–610 U/liter) and correlated to the scintigraphic extent of the disease (r = 0.76; P< 0.01). Three months after the I.V. administration of bisphosphonates, the decrease of BALP was more marked than that of total ALP (median: −54% versus −41%; P< 0.05). In conclusion, these results suggest that BALP measurement with this immunoassay may be clinically useful, and more sensitive than total ALP, in the assessment of bone turnover during changes of the estrogen status as well as in monitoring the effects of treatments that modify the metabolic activity of the skeleton.

Summary.: In order to establish whether melatonin alters basal and/or stimulated oxytocin secretion, 18 normal men were treated (p.o.) with 6 or 12 mg melatonin or placebo in basal conditions (N-6 subjects) or concomitantly to the administration of insulin (0.15 IU/kg body weight in an I.V. bolus) (N-6 subjects) or angiotensin II (increasing doses of 4, 8 and 16 ng/kg/min, at intervals of 20 min). The administration of 6 or 12 mg melatonin did not change basal and angiotensin II-stimulated oxytocin secretion. In contrast, the oxytocin response to insulin-induced hypoglycemia was significantly reduced by melatonin treatment. In fact, the mean peak oxytocin response to hypoglycemia was 2.2 times higher than baseline in the absence of melatonin, whereas it was 1.6 times higher than basal value after administration of 6 or 12 mg melatonin. These data indicate an involvement of melatonin in the regulation of the oxytocin response to hypoglycemia in normal men. The lack of effects of melatonin on basal and angiotensin II-stimulated oxytocin secretion argues against the possibility that melatonin exerts an overall modulatory role on oxytocin secretion in humans.

Alcohol abuse is commonly associated with reduced bone mass and osteoporosis as a consequence of both systemic and direct cellular effects. To clarify some of the pathways by which alcohol exerts its actions directly on bone cells, we investigated the formation of early osteoblast progenitors (colony‐forming units for fibroblasts; CFU‐F) in long‐term murine and human bone marrow cultures exposed to ethanol and to its main metabolite, acetaldehyde. In murine bone marrow cultures, obtained from Swiss female mice, ethanol inhibited CFU‐F formation (maximal reduction ± SEM: 50 ± 2%; p< 0.01) at concentrations ranging from 0.04% to 0.6% that are similar to those reached in vivo in alcoholics. Acetaldehyde strongly reduced CFU‐F formation at concentrations of 0.004% and 0.02%, and completely abolished it at the dose of 0.06%. Similarly, ethanol (at concentrations ≥0.02%) and acetaldehyde (from 0.004% to 0.06%) significantly decreased the number of CFU‐F in human bone marrow cultures; the mean reduction observed with ethanol was 63 ± 12% (p < 0.05), whereas acetaldehyde completely prevented CFU‐F formation at the concentration of 0.06%. These in vitro observations were confirmed by the in vivo findings that the CFU‐F formation in bone marrow cultures from nine young, chronic, noncirrhotic alcoholics was significantly reduced (70 ± 15%), compared with seven age‐matched normal subjects (p< 0.01). In addition, acetaldehyde inhibited cell proliferation in human osteoblastic cells (MG‐63 and HOBIT cell lines), whereas ethanol reduced proliferation only in MG‐63 cells. Our results indicate that ethanol and acetaldehyde may directly inhibit the osteoblastogenic potential of the bone marrow, and this effect may contribute to the decreased bone formation observed in alcoholics.

ABSTRACTMultiple myeloma (MM) derives from malignant transformation of plasma cells (PC), which accumulate in the bone marrow (BM), where microenvironment supports tumor growth and inhibits anti-tumor immune responses. Adenosine (ADO), an immunosuppressive molecule, is produced within MM patients' BM by adenosinergic ectoenzymes, starting from ATP (CD39/CD73) or NAD+[CD38/CD203a(PC-1)/CD73]. These ectoenzymes form a discontinuous network expressed by different BM cells. We investigated the expression and function of ectoenzymes on microvesicles (MVs) isolated from BM plasma samples of patients with MM, using asymptomatic forms of monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM) as controls.The percentage of MVs expressing ectoenzymes at high levels was higher when derived from MM patients than controls. BM CD138+PC from MM patients expressed high levels of all ectoenzymes. Paired MVs samples confirmed a higher percentage of MVs with high ectoenzymes expression in MM patients than controls. Pooled MVs from MM patients or controls were tested for ADO production. The catabolism of ATP, NAD+, ADPR and AMP to ADO was higher in MVs from MM patients than in those from controls.In conclusion, our results confirmed the hypothesis that MVs in MM niche are main contributor of ADO production. The ability of MVs to reach biological fluids strongly support the view that MVs may assume diagnostic and pathogenetic roles.

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