Neutral endopeptidase-24.11 (NEP) deactivates PDGF- and TGF-beta-induced cell shape changes in invertebrate immunocytes.

Using a cytofluorimetric assay, we found that immunocytes of the mollusc, Mytilus galloprovincialis, express CD10, a surface antigen known to be identical to neutral endopeptidase-24.11 (NEP). The spectrofluorimetric analysis demonstrates that the growth factors PDGF-AB and TGF-beta1 provoke an increase in NEP-like activity in membrane preparations from the immunocytes, but have no effect on the soluble form in the serum. On the other hand, computer-assisted microscopic image analysis reveals that NEP deactivates the PDGF-AB- and TGF-beta1-induced shape changes in immunocytes. However, Western blots show that, in solution, NEP does not cleave PDGF-AB or TGF-beta1, indicating that the inactivation is not due to proteolysis. These results suggest a functional interplay in invertebrate immunocytes between growth factors and NEP, as previously shown in vertebrate cells.

Neutral endopeptidase-24.11 (NEP) activity in human fibroblasts during development and ageing.

Neutral endopeptidase-24.11 (NEP, EC 3.4.24.11) is a cell surface Zn metallopeptidase that hydrolyzes bioactive regulatory peptides. Using a spectrofluorimetric procedure, we assessed NEP activity in plasma membranes of normal human skin and lung fibroblasts. We found a considerable increase in NEP activity during fetal-to-adult transition. Adult skin fibroblasts from an old donor exhibited significantly higher levels of NEP activity than cells from young donors. Interestingly, however, the NEP activity of fibroblasts from a centenarian donor was similar to that of cells from young donors. Increased levels of NEP activity were also found in in vitro aged lung fibroblasts. Finally, adrenocorticotropin hormone (ACTH (1-24)), a regulatory peptide that can be cleaved by NEP, provoked an increase in enzymic activity in fetal and young adult donor fibroblasts and a decrease in this activity in fibroblasts from adult and old donors. This finding suggests that ageing may affect NEP activity.

The CRH-ACTH-biogenic amine axis in invertebrate immunocytes activated by PDGF and TGF-beta.

In immunocytes from the mollusc Mytilus galloprovincialis, the major pathway followed by platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 in provoking the release of norepinephrine, epinephrine and dopamine into cell-free hemolymph (serum) is mediated by a corticotropin-releasing hormone-adrenocorticotropin hormone (CRH-ACTH) biogenic amine axis. This axis not only annulled the inhibiting properties of PDGF-AB, it even reversed the latter's effect, while the inducing effect of TGF-beta1 was amplified. These findings show that non-classical immune-neuroendocrine molecules, such as PDGF-AB and TGF-beta1, are involved in building stress response, using the same conserved mechanisms present from invertebrates to vertebrates.

Inter-individual variability of benzene metabolism to trans,trans-muconic acid and its implications in the biological monitoring of occupational exposure.

Unmodified benzene (UBz) and trans,trans-muconic acid (t,t-MA) were measured in urine samples collected at the end of the first half-shift in 80 bus drivers from a large city in Northern Italy. Mean UBz was 1155 ng/l (S.D. = 494), range 85-1980 ng/l; these values roughly correspond to 10-1000 micrograms/m3 of benzene in air. Mean t,t-MA was 297 micrograms/g creatinine; the range was large (20-1295 micrograms/g creatinine), and the distribution of values was bimodal. At further analysis of t,t-MA data, two subgroups of 59 and 18 subjects were identified (3 outliers were excluded): mean values of the index were 108 (S.D. = 65) and 916 (S.D. = 264) micrograms/g creatinine respectively, and the values within each subgroup were normally distributed. The mean ratio between t,t-MA and UBz in the subgroups were 0.15 and 0.85, respectively; the difference was significant. The first subgroup was defined as 'poor t,t-MA metabolizers', the other as 'efficient t,t-MA metabolizers'. No inter-subgroup differences were observed regarding the main characteristics (age, dietary and smoking habits, etc.). As the parent compound of t,t-MA, trans,trans-muconaldehyde is myelotoxic, and its production has been implicated in benzene-induced leukemia. 'efficient' t,t-MA metabolizers may be at higher risk of developing benzene toxicity. If confirmed in further studies, the inter-individual variability rate of metabolizing benzene to t,t-MA may introduce some limitations in the application of this metabolite as an exposure index of low benzene exposure. Nevertheless, the t,t-MA/UBz ratio may be an important index of susceptibility to benzene toxicity.

Lead, cadmium, and selenium in the blood of patients with sporadic amyotrophic lateral sclerosis.

We determined whole blood lead and cadmium levels, and serum selenium levels in patients with sporadic amyotrophic lateral sclerosis and age- and sex-matched controls. Disability due to the disease directly correlated with lead levels, and there was a strong inverse correlation with selenium concentrations. Lead and selenium concentrations tended to be similar in the cases and controls, both in the study population as a whole and after the removal from the analysis of the patients with the highest degree of disability. In the patients with limited disability, cadmium concentrations were higher than in the controls. Our findings lend limited support to a possible involvement of cadmium, but not lead, in the etiology of sporadic amyotrophic lateral sclerosis, and strongly suggest that short-term indicators of exposure are inadequate to investigate the relationship between selenium and the disease.

Effect of PDGF and TGF-beta on the release of biogenic amines from invertebrate immunocytes and their possible role in the stress response.

PDGF-AB and TGF-beta 1 intervene in molluscan stress response, the former inhibiting and the latter inducing the release of norepinephrine and epinephrine from hemocytes. These amines are down-regulated even when TGF-beta 1 is added to hemolymph pre-incubated with PDGF-AB. The opposite behaviour is observed if the growth factors are reversed. The dopamine response is not affected in either case, even after the addition of CRH or ACTH. After pre-incubation with PDGF-AB or TGF-beta 1 in the presence of CRH or ACTH, norepinephrine and epinephrine release falls. These findings suggest that when the interaction is between growth factors, the order of combination is crucial, while in cases where the interaction is between growth factors and other peptides, such as CRH and ACTH, the order is of no importance.

Neutral endopeptidase-24.11 (NEP)-like activity in molluscan hemocytes.

Using a spectrofluorimetric procedure, we found that the plasma membrane from hemocytes of two freshwater snails, Planorbarius corneus and Viviparus ater, shows neutral endopeptidase-24.11 (NEP)-like activity. Moreover, the addition of ACTH(1-24) to the hemolymph provokes an increase in NEP-like activity. This increased NEP-like activity is blocked by phosphoramidon, a potent inhibitor of mammalian NEP. These findings suggest that this peptidase has been well conserved in the course of evolution and plays an important role in immune-neuroendocrine mechanisms.

Observations on the use of plasma, hair and tissue to evaluate trace element status in cancer.

Selenium, zinc and copper were measured in plasma, hair and tissue of patients affected by either breast or lung cancer and their controls. A decrease in plasma Se and Zn in women affected by breast cancer was observed, whereas plasma Cu was increased in lung cancer. No significant modification was found in hair trace element levels adjusted for the main confounders, in particular for hair treatment which altered Se content. The examined elements were highly concentrated in cancerous vs normal tissue, but results changed according to the unit used to express results. The usefulness and significance of these biomarkers of trace element status are discussed in the light of the most recent literature data.

Zinc and copper status and blood pressure.

In order to elucidate the relationships between Zn and Cu and blood pressure, the present case-control study was carried out. Zn and Cu status was evaluated in 60 subjects, pharmacologically untreated, affected by mild stable hypertension and in 60 normotensives matched for sex, age and smoking habits. Different markers of Zn and Cu status, including serum, erythrocyte and urine levels of the two trace elements and activities of some Zn- or Cu-dependent enzymes (alkaline phosphatase, lactic dehydrogenase, superoxide dismutase and lysyl oxidase) were evaluated. No significant difference between hypertensives and normotensives was observed in the mean levels of Zn and Cu as well as in Zn- or Cu-dependent enzymes, though higher levels of serum copper were associated with increased risk of hypertension. Interesting relationships between the biological parameters investigated were observed in the hypertensive subjects. Inverse correlations between blood pressures and serum Zn were observed. Furthermore, blood pressure was inversely related to lysyl oxidase activity. These findings give further support to the hypothesis that an imbalance of Zn and Cu bioavailability may be associated to hypertensive condition.

Zinc, copper, and zinc- or copper-dependent enzymes in human hypertension.

Imbalance of zinc and copper status has been hypothesized in human hypertension. A case-control study was carried out to elucidate the possible relationship between zinc and copper status and essential hypertension. Thirty-one subjects affected by mild stable hypertension, pharmacologically untreated, were investigated together with 31 normotensive controls individually matched for sex, age, and smoking habits. Zinc and copper in serum and urine wee measured, and serum activities of alkaline phosphatase (AP), lactic dehydrogenase (LDH), copper-zinc superoxide dismutase (Cu-Zn SOD), lysyl oxidase (LOX), and monoamine oxidase (MAO) were evaluated. No significant difference in serum and urine zinc and copper content as far as in serum activity of zinc (AP and LDH) or copper (Cu-Zn SOD, LOX, and MAO)-dependent enzymes was found between hypertensives and normotensives. Positive relationships were found in normotensives between serum and urine levels of zinc (r = 0.577; p = 0.001) and copper (r = 0.394; p = 0.028), and between serum copper and Cu-Zn SOD (r = 0.534; p = 0.002). In normotensives, diastolic blood pressure and serum zinc were positively related (r = 0.370; p = 0.041). In hypertensives, inverse correlations were observed between diastolic blood pressure and AP (r = -0.498; p = 0.004) and Cu-Zn SOD (r = 0.452; p = 0.011), and between systolic blood pressure and LOX (r = -0.385; p = 0.033). Diastolic blood pressure was related to LDH inversely in hypertensives (r = -0.357; p = 0.049) and positively in normotensives (r = 0.457; p = 0.010). In normotensives, diastolic blood pressure was inversely related with MAO (r = -0.360; p = 0.046). These findings support the hypothesis that an imbalance of zinc and copper status might be involved in human hypertension.

Cytokines and evolution: in vitro effects of IL-1 alpha, IL-1 beta, TNF-alpha and TNF-beta on an ancestral type of stress response.

Invertebrate hemocytes are immune-neuroendocrine cells which contain a variety of cytokines [Ottaviani et al. (1993) Biochem. Biophys. Res. Commun. 195, 984-988] and release biogenic amines when added to corticotropin-releasing factor (CRF), a phenomenon we have described as an evolutionary proto-type stress response [Ottaviani et al. (1991) Proc. R. Soc. Lond. B 245, 215-218]. Here we show in two molluscs, Planorbarius corneus and Viviparus ater, that this response is significantly reduced when hemocytes are pre-incubated with IL-1 alpha, IL-1 beta, TNF-alpha and TNF-beta before the addition of CRF. These results confirm and extend the hypothesis that a deep evolutionary relationship exists between cytokines and stress response. Moreover, these data offer an evolutionary basis for understanding the promiscuity of cytokine receptors.

Relationship between corticotropin-releasing factor and interleukin-2: evolutionary evidence.

The addition of corticotropin-releasing factor (CRF) to molluscan hemocytes induces the release of biogenic amines (norepinephrine, epinephrine, dopamine), a phenomenon we have considered as an ancestral type of stress response [(1992) Gen. Comp. Endocrinol. 87, 354-360]. A similar but less significant response was observed following the addition of interleukin-2 (IL-2). Pre-incubation of hemocytes with IL-2 or anti-IL-2 monoclonal antibody significantly reduced or completely eliminated the CRF-induced release of biogenic amines. Further direct evidence of competition between CRF and IL-2 was revealed by immunocytochemical and cytofluorimetric analysis. The data are compatible with the presence of a unique (ancestral?) receptor on molluscan hemocytes, capable of binding both CRF and IL-2, two key molecules of the neuroendocrine and immune system, respectively.

CRF provokes the release of norepinephrine by hemocytes of Viviparus ater (Gastropoda, Prosobranchia): further evidence in favour of the evolutionary hypothesis of the mobile immune-brain.

The concentration of biogenic amines (norepinephrine, epinephrine and dopamine) was determined by HPLC in serum and hemocytes of the mollusc Viviparus ater following in vitro incubation of the hemolymph with corticotropin-releasing factor (CRF) for different periods of time. CRF provoked the release of norepinephrine from hemocytes into the serum, the maximum level being observed after 15 min. incubation. Moreover, immunoreactive tyrosine hydroxylase and dopamine beta-hydroxylase, i.e., the enzymes involved in biogenic amine biosynthesis, have been demonstrated in hemocytes. These findings show that in invertebrates an ancestral type of stress response, similar to that performed by mammalian hypothalamic-pituitary-adrenal axis, occurs in phagocytic hemocytes, a cell type capable of both fundamental immune and neuroendocrine responses.

Stress response in the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata): interaction between CRF, ACTH, and biogenic amines.

Previous studies reported that ACTH molecules influence chemotactic and phagocytic activities of hemocytes in the freshwater snail, Planorbarius corneus. The present study reveals that ACTH and CRF affect the release of biogenic amines. Hemocytes from P. corneus hemolymph incubated in vitro with ACTH for 15, 30, and 45 min released epinephrine, norepinephrine, and dopamine. The greatest release occurred after 15 min, while after 45 min the values were similar to those of the controls. Similar incubations with CRF also provoked a release of biogenic amines, this being mainly mediated by the release of endogenous ACTH. These data suggest that (i) ACTH and CRF provoke the release of biogenic amines; (ii) there is a direct relationship between CRF, ACTH, and biogenic amines, with the hemocytes as the target; (iii) exogenous ACTH can mimic an ancestral type of stress response; (iv) the major pathway of the stress response in P. corneus is mediated by a CRF-ACTH-biogenic amine axis. These data should help to unravel part of the complex molecular signaling mechanisms involved in the physiological/endocrinological reaction of invertebrate organisms to stress, and suggest that a stress response unexpectedly similar to that present in mammalian cells is detectable in invertebrates.

Evolution, immune responses and stress: studies on molluscan cells.

Using a variety of techniques we found the presence of ACTH and opioid-like molecules in the phagocytic spreading hemocytes (SH) and the serum of the mollusc Planorbarius corneus. In vitro experiments have shown that ACTH and beta-endorphin exert chemotactic activity on SH, while ACTH, but not beta-endorphin increases the phagocytic activity of SH. Moreover, ACTH and CRF provoked the release of biogenic amines from the hemocytes, mimicking a proto-stress type of response. Thus, the same mobile cell is capable of immune and proto-stress responses by using as mediators neuropeptides which remained fundamentally similar throughout evolution.

Cadmium in blood, urine and hair related to human hypertension.

A case-control study was performed to assess whether cadmium is related to hypertension in a non-occupationally exposed population. 63 male subjects affected by mild stable hypertension, pharmacologically untreated, were investigated together with 63 male normotensive controls individually matched for sex, age, body mass index, smoking habits and work activities. Cadmium in blood, zinc and copper in serum, the three elements in urine and hair, together with some biological parameters involved in pathogenesis of hypertension, were investigated. The mean Cd blood value in hypertensives (H) was 0.58 micrograms/L vs 0.44 micrograms/L in normotensives (N) (t = 2.03; p less than 0.05) with a greater difference in non-smokers (0.41 micrograms/L vs 0.25 micrograms/L) (t = 2.69, p less than 0.01). Furthermore, both systolic and diastolic blood pressure were significantly related to cadmium blood levels (r = 0.20 and 0.19 respectively, p less than 0.05). Smoking habit affected cadmium levels only in the blood, not in the other biological matrices examined. No significant difference of cadmium content in urine and hair was found between normotensives and hypertensives but Cd/Cu ratio in urine was significantly lowered in the second group.

Effect of DL-penicillamine on the aorta of growing chickens. Ultrastructural and biochemical studies.

The effect of DL-penicillamine on the architecture of the aortic wall of growing chickens was studied, with particular attention to elastin and collagen. Penicillamine was added to the diet (0.2% and 0.4%, in the presence or not of 10 mg/kg CuSO4 and 100 mg/kg vitamin B6) from hatching, for periods from 7 days up to 2 months. The same regions of the thoracic aortas were examined and compared in all the different experimental conditions. The results showed that penicillamine induced relevant modifications in the process of elastin fibrogenesis. The alterations consisted of an increase of elastin in the extracellular space, associated with an increase in the number of elastin fibers per unit area, and a decrease of the mean profile area of the fibers. Interestingly, penicillamine induced the formation of numerous bundles of microfibrils associated or not with elastin fibers. After prolonged treatment, elastin tended to diminish and the fibers tended to fuse into polymorphic syncytia. Collagen fibrils were larger, showed more heterogeneous cross diameters, were less numerous, and were more spread out within the tissue. All the other components of the aortic wall appeared not to be altered by the chemical. Penicillamine did not modify the copper content of chick aortas, whereas it induced a 40-50% reduction of the activity of both salt and 4 M urea-soluble peptidyl lysyl oxidases in the same tissue. These data may help in understanding some of the pathologic manifestations in human beings during D-penicillamine treatment.