[Energy expenditure in construction industry]. / Il dispendio energetico in edilizia.

The aim of this study is to show the results obtained from measuring energy expenditure (EE) during work, through portable devices, in a group of male construction workers. After defining cardio-respiratory parameters in laboratory, authors applied to all subjects an heart rate monitor for measuring the heart rate (HR) and, at the same time, a calorimeter for measuring energy expenditure (EE). To analyse data obtained, authors calculate the Relative Aerobic Strain (RAS), both for the measurements of EE and for HR detected. Results confirm that in many of the typical activities of construction industry, in particular in those characterised by an higher component of manual engagement compared to foreman, workloads are exceeding limits of the probable threshold fatigue (33% of RAS), both for energy expenditure than for HR measured.

Muscle myostatin signalling is enhanced in experimental cancer cachexia.

BACKGROUND/AIMS: Myostatin belongs to the transforming growth factor-beta superfamily and negatively regulates skeletal muscle mass. Its deletion induces muscle overgrowth, while, on the contrary, its overexpression or systemic administration cause muscle atrophy. The present study was aimed at investigating whether muscle depletion as occurring in an experimental model of cancer cachexia, the rat bearing the Yoshida AH-130 hepatoma, is associated with modulations of myostatin signalling and whether the cytokine tumour necrosis factor-alpha may be relevant in this regard. MATERIALS AND METHODS: Protein levels of myostatin, follistatin (myostatin endogenous inhibitor) and the activin receptor type IIB have been evaluated in the gastrocnemius of tumour-bearing rats by Western blotting. Circulating myostatin and follistatin in tumour hosts were evaluated by immunoprecipitation, while the DNA-binding activity of the SMAD transcription factors was determined by electrophoretic-mobility shift assay. RESULTS: In day 4 tumour hosts muscle myostatin levels were comparable to controls, yet follistatin was reduced, and SMAD DNA-binding activity was enhanced. At day 7, both myostatin and follistatin increased in tumour bearers, while SMAD DNA-binding activity was unchanged. To investigate whether tumour necrosis factor-alpha contributed to induce such changes, rats were administered pentoxifylline, an inhibitor of tumour necrosis factor-alpha synthesis that partially corrects muscle depletion in tumour-bearing rats. The drug reduced both myostatin expression and SMAD DNA-binding activity in day 4 tumour hosts and up-regulated follistatin at day 7. CONCLUSIONS: These observations suggest that myostatin pathway should be regarded as a potential therapeutic target in cancer cachexia.

Evaluation of the antifungal effect of EDTA, a metal chelator agent, on Candida albicans biofilm.

OBJECTIVE: Candida albicans biofilm is frequently found on artificial surfaces and the infections related to biofilm are difficult to eliminate, as they require the removal of artificial devices and treatment with antifungal drugs. Nowadays, fungal growth in biofilms is difficult to eradicate with conventional antifungal drugs such as fluconazole. Among chelating agents, disodium salt-Ethylene Diamine Tetraacetic Acid (EDTA) is known to have antifungal activity. In this study, we examined the in vitro activity of the EDTA and the antifungal drug fluconazole against C. albicans mature biofilm. MATERIALS AND METHODS: C. albicans ATCC 20191, fluconazole-susceptible strain, was grown at an inoculum starter of 1 x 106 cells/ml for 72 h in 24-well microtiter plates and was further treated for 24 h with EDTA and/or fluconazole. Antifungal activities in biofilms were expressed as reduction in optical density (OD) determined by a 2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay and compared to untreated biofilms. RESULTS: Colorimetric readings revealed that EDTA alone (at 25 and 2.5 mM) significantly reduced fungal metabolic activity in preformed biofilms. Also, EDTA combined with fluconazole significantly reduced the growth of biofilm when compared to biofilm treated with fluconazole alone (at 25 and 2.5 µg/ml). CONCLUSIONS: Our data suggest that the employment of EDTA or other chemicals destabilizers of the biofilm matrix, in combination with antifungal drugs, could lead to the development of new strategies for the management of infections associated to Candida biofilm. Another relevant result of our study suggests that the initial cell concentration, probably through mechanisms of quorum sensing, affects the cellular viability during the process of biofilm formation.

Potassium and calcium currents activated by foetal calf serum in Balb-c 3T3 fibroblasts.

In quiescent Balb-c mouse 3T3 fibroblasts, the application of whole or dialyzed 10% foetal calf serum elicits a biphasic electrical response, consisting of a transient outward current, flowing through Ca(2+)-activated K+ channels, followed by an inward one, lasting up to 15 min. On the basis of experiments with ion substitutions and blockers, the inward current can be attributed to the opening of cationic channels permeable to Na+ and Ca2+ ions. This current could mediate the calcium influx involved in the sustained elevation of [Ca2+]i that has been observed in many preparations in response to mitogen stimulation and that is involved in triggering cell proliferation.

Two currents activated by epidermal growth factor in EGFR-T17 fibroblasts.

Application of 10 nM Epidermal Growth Factor (EGF) to single EGFR-T17 fibroblasts induced a marked hyperpolarization that could last for tens of minutes; in many cases the first transient was followed by a series of oscillations of the membrane potential. The outward current responsible for the hyperpolarizing response could be recorded simultaneously to an increase in the intracellular calcium concentration, as measured with the fluorescent indicator fura-2. The conductance was nearly linear in the voltage range from -100 to +50 mV. While the EGF-induced current had many characteristics of a K+ current and was strongly reduced by 50 nM charybdotoxin (ChTx), its reversal potential was apparently more negative than the potassium equilibrium potential (VK). The application of 2 microM ouabain prior to EGF stimulation produced responses that were similar to those obtained without ouabain; however, under these conditions the EGF-induced current showed a reversal potential of -96.6 +/- 3.2 mV, very close to VK. Simultaneous application of both 2 microM ouabain and 50 nM ChTx completely abolished the response. It can be concluded that the response to EGF stimulation in EGFR-T17 cells consists of two components: the first is a current carried through Ca(2+)-activated K+ channels; the second is due to the acceleration of the operation of the Na+/K(+)-ATPase.

Mice lacking TNFalpha receptors 1 and 2 are resistant to death and fulminant liver injury induced by agonistic anti-Fas antibody.

The liver is particularly susceptible to Fas-mediated cytotoxicity. Mice given an adequate parenteral dose of agonistic anti-Fas antibody (aFas) or of FasL are known to develop a devastating liver injury and to die in a few hours. The present work shows that mice lacking TNFR1 and TNFR2 (R(-)) both survive a single dose of aFas, otherwise rapidly lethal, and develop a mild form of hepatic damage, compared to the much more severe liver injury that in a few hours strikes wild-type mice (R(+)), eventually involving increased activity of proteases of different families (caspase 3-, 8-, and 9-like, calpains, cathepsin B). Neither the overall tissue levels of Fas and FasL nor Fas expression at the hepatocyte surface are altered in the liver of R(-) animals. The DNA-binding activity of the NF-kappaB transcription factor is enhanced after aFas treatment, but much more markedly in R(-) than in R(+) mice. Bcl2, while unchanged in untreated animals, is markedly upregulated in R(-) but not in R(+) mice challenged with aFas. The requirement of a normal TNFR1/TNFR2 phenotype for full deployment of the general and liver-specific aFas toxicity in mice most likely implies that treatment with aFas in some way results in activation of the TNFalpha-TNFRs system and that this activation synergizes with Fas-mediated signals in causing the fulminant liver injury and the animal death. The precise cellular and molecular details underlying this interplay between Fas- and TNFRs-mediated signaling systems in the general and liver-specific aFas toxicity largely remain to be clarified.

Rapid and extensive lethal action of clofibrate on hepatoma cells in vitro.

Clofibrate, for a long time in use as a hypolipidemic drug, is a well known peroxisomal proliferator (PP) and hepatocarcinogen in rodents. We show here that in vitro 1 mM clofibrate induces a rapid and massive death of rat AH-130 hepatoma cells. Cell death was prominent already after 4 h of treatment, with a characteristic ;apoptotic' pattern by conventional microscopy. This was further supported by the pronounced chromatin condensation detectable on 4',6-diamine-2'-phenylindole dihydrochloride (DAPI) staining, the clearcut internucleosomal DNA fragmentation on agarose-gel electrophoresis (ladder pattern), and the accumulation of markedly hypochromic cells observed in flow cytometric DNA histograms. Consistently with the apoptotic features of the process, some parameters commonly used to detect cell death, such as plasma membrane permeabilization to trypan blue or propidium iodide, lack of mitochondrial retention of rhodamine 123, or extracellular release of lactate dehydrogenase, were all virtually negative. However, these same parameters became markedly positive after 24 h of treatment, which was suggestive for the occurrence of ;secondary' necrosis among AH-130 cells. By a combination of flow cytometric parameters, after 4 h on 1 mM clofibrate only 41% of the AH-130 cells could still be categorized as viable (i.e., non-apoptotic and non-necrotic), while 46% of cells appeared apoptotic and 13% necrotic. At 24 h, 67% of cells were necrotic, 20% apoptotic and only 13% non-apoptotic and non-necrotic. Apoptosis was also extensive in AH-130 cells treated with another PP such as nafenopin at 1 mM concentration and in human hepatoma HepG2 cells treated with clofibrate. By contrast, clofibrate did not cause apoptosis on primary rat hepatocyte cultures. These observations indicate that: (i) apart from their well-known cell growth-promoting action, PPs such as clofibrate or nafenopin may exert a substantial cytotoxic action on targets such as the AH-130 or HepG2 hepatoma cells; (ii) this cell death evolves from an initial 'apoptotic' to an eventual ;necrotic' pattern; (iii) detection of cell death requires the adoption of a full panel of tests, adequate to cover the whole evolving death pattern, while such tests may even be substantially misleading whenever applied individually; (iv) the cytotoxicity of clofibrate and similar agents on normal and, particularly, tumoural cells may deserve careful reevaluation.

Skeletal muscle wasting in tumor-bearing rats is associated with MyoD down-regulation.

Cachexia is a syndrome characterized by profound skeletal muscle wasting that frequently complicates malignancies. A number of studies indicate that protein hypercatabolism, largely mediated by classical hormones and cytokines, is the major component of muscle depletion. Impaired regeneration has been suggested to contribute to the reduction of muscle size. In particular, it has been shown that the expression of MyoD, a muscle-specific transcription factor, is down-regulated by cytokines such as TNFalpha and IFNgamma in a NF-kappaB-dependent posttranscriptional manner. The present study investigated whether modulations of the transcription factor MyoD are associated with the onset of muscle wasting in a well established model of cancer cachexia. Rats bearing the Yoshida AH-130 hepatoma develop a condition of muscle protein hypercatabolism, largely dependent on TNFalpha bioactivity. In the gastrocnemius of these animals the expression of MyoD was markedly reduced, paralleling the decrease of muscle weight. This pattern is associated with increased nuclear translocation of AP-1, while DNA-binding assays did not detect any change in NF-kappaB activity. This is the first observation demonstrating that muscle depletion in tumor-bearing rats is associated with a down-regulation of MyoD levels. Although the underlying mechanisms remain to be clarified, this change is compatible with the hypothesis that a reduced expression of molecules involved in the regulation of the regenerative response may concur to muscle wasting in cancer cachexia.

In vitro and in vivo conditional sensitization of hepatocellular carcinoma cells to TNF-induced apoptosis by taxol.

High mortality among hepatocellular carcinoma (HCC) patients reflects both late diagnosis and low curability, due to pharmacoresistance. Taxol (TAX) is toxic for many human HCC-derived cell lines, yet its clinical efficacy on HCCs is poor. Combining TAX with other drugs appears a promising possibility to overcome such refractoriness. We analyzed whether combining tumor necrosis factor (TNF) with TAX would improve their toxicity. Human HCC-derived cell lines were treated with TAX or TNF, alone or combined. Apoptosis was assessed by morphology and flow-cytometry. Several pro- and anti-apoptotic molecules were evaluated by western blotting and/or enzymatic assay. After a 24 hour treatment, TNF was ineffective and TAX modestly cytotoxic, whereas HCC cells were conditionally sensitized to TNF by TAX. Indeed some relevant parameters were shifted to a prodeath setting: TNF-receptor 1 was increased, SOCS3, c-FLIP and pSTAT3 were markedly downregulated. These observations provide a significant clue to critically improve the drug susceptibility of HCC cells by combining 2 agents, TAX and TNF. The sequential application of TAX at a low dosage followed by TNF for only a short time triggered a strong apoptotic response. Of interest, prior TAX administration could also sensitize to TNF-induced apoptosis in the Yoshida AH-130 hepatoma transplanted in mice. Therefore, scrutinizing the possibility to develop similar combination drug regimens in suitable preclinical models seems highly advisable.

Role of mitogen-induced calcium influx in the control of the cell cycle in Balb-c 3T3 fibroblasts.

The role of mitogen-activated calcium influx from the extracellular medium in the control of cell proliferation was studied in Balb-c 3T3 fibroblasts. Stimulation of serum-deprived, quiescent cells with 10% foetal calf serum (FCS) induced a long-lasting (up to 70 min) elevation of intracellular free calcium concentration ([Ca2+]i). Both the sustained [Ca2+]i increase and the related inward current, described in a previous paper [Lovisolo D. Munaron L. Baccino FM. Bonelli G. (1992) Potassium and calcium currents activated by foetal calf serum in Balb-c 3T3 fibroblasts. Biochim. Biophys. Acta, 1104, 73-82], could be abolished either by chelation of extracellular calcium with EGTA or by SK&F 96365, an imidazole derivative that can block receptor-activated calcium channels. The effect of the abolition of these ionic signals on FCS-induced proliferation was investigated by adding either EGTA or SK&F 96365 to the culture medium during the first hours of stimulation of quiescent cells with 10% FCS. As measured after 24 h, a 22% inhibition of growth was observed when SK&F 96365 was added for the first hour, and stronger inhibitions, up to 56%, were obtained by adding the blocker for the first 2 or 4 h. Similar effects were observed with addition of 3 mM EGTA, though the inhibition was less marked for the 4 h treatment. By contrast, incubation with either substance in the next 4 h of serum stimulation did not influence cell growth, except for a slight inhibition observed when SK&F 96365 was applied from the 4th to the 8th hour. The reduction in growth resulting from the abolition of the early calcium influx was paralleled by an accumulation of cells in the G2/M phase. Both growth inhibition and G2/M accumulation were reversible, since after further 24 h in 10% FCS cells had fully recovered the exponential growth. These data indicate that the early calcium influx seen in response to mitogen stimulation develops on a timescale long enough to play a significant role in cell cycle progression, and that its block in the early G1 phase can lead to a reduction of proliferation by arresting cells in later stages of the cycle.

Effect of 4-Hydroxynonenal on cell cycle progression and expression of differentiation-associated antigens in HL-60 cells.

4-Hydroxynonenal (HNE) is a highly reactive aldehyde produced by lipid peroxidation of cellular membranes that inhibits growth and induces differentiation in HL-60 cells. Its mechanisms of action were investigated by analyzing the cell cycle distribution and the appearance of differentiated phenotypes in HL-60 cells. Data obtained by exposing cells to DMSO for 7.5 h (same time as for HNE treatment) or for the whole length of the experiments (5 d) were used for comparison. HNE induced a marked increase in the proportion of G0/G1 cells after 1 and 2 d. The brief DMSO treatment did not affect the distribution, whereas continuous exposure led to a progressive accumulation of cells in G0/G1 (maximal at day 5). The proportion of phagocytic cells gradually increased in HNE-treated and DMSO long-exposed cultures from day 2 and peaked at day 5 (35 and 63%, respectively), whereas the effect of the brief DMSO treatment was negligible. The expression of CD11b and CD67 increased in cells treated with HNE or continuously exposed to DMSO, whereas CD36 was expressed at low levels on both treatments. These results indicate that the pathway of the granulocytic differentiation induced by HNE in HL-60 cells differs from that of DMSO: with HNE, growth inhibition precedes the onset of differentiation, whereas in DMSO-treated cells the two processes are chronologically associated.

Decreased expression of the high-mobility group protein T160 by antisense RNA impairs the growth of mouse fibroblasts.

The T160 protein belongs to the HMG-1 box protein family and preferentially binds to non-B-DNA conformations with no sequence specificity. Its exact role has yet to be defined, though it seems to participate in processes involving DNA, such as replication, transcription and recombination. We have used an antisense RNA strategy to investigate its role in cell growth and proliferation. T160 expression is strongly suppressed by stable introduction of an antisense construct into NIH3T3 cells, and this decrease is accompanied by substantial changes in the growth properties of the stable transfectants. Impaired growth of T160- cells was mainly related to two mechanisms: i) decreased rates of cell proliferation at normal serum concentration; and ii) occurrence of cell death by apoptosis at low serum concentration, as demonstrated by both flow cytometry and microscopy. The finding that decreased T160 availability affects cell proliferation, provides further evidence of its involvement in a basic cell function, such as DNA replication.

Italian multicenter study of reversible cerebral ischemic attacks. Part 5. Risk factors and cerebral atherosclerosis.

As part of a prospective study, the influence of several premorbid and environmental factors on the presence, extent and severity of cerebral vessel atherosclerosis was studied in 462 patients with clinical diagnosis of RIA who underwent cerebral angiography. The extent and severity of atherosclerosis of the cerebral vessels was quantified using extracranial and intracranial cerebrovascular scores (ECS, ICS) based on the number and severity of the lesions in 11 extracranial and 21 intracranial arterial segments. Results of univariate and multivariate analyses indicate that the presence of atherosclerotic changes of cerebral vessels, as shown by angiography, was strongly related with age in both sexes. The lesions were more frequent in males, in particular under age 55. Elevated cholesterol was associated with a higher incidence of atherosclerotic lesions. Smoking was associated with a higher incidence of extracranial lesions. Age, smoking and history of hypertension were the best predictors of the extent and severity of cerebral vessel atherosclerosis.

Cryosurgery plus adjuvant systemic alpha2-interferon for HPV-associated lesions.

OBJECTIVE: The authors report their experience in patients with adjuvant systemic 2-interferon with the aim of defining the effectiveness, side-effects, indications and limitations of this treatment. MATERIALS AND METHODS: From January 1989 to December 1996, 123 patients with genital, anorectal and perineal HPV lesions were treated with cryosurgery; adjuvant systemic a2-interferon was administered to 38 of them. There were 76 female and 47 male patients (median age of 29 years, range; 15-56 years). Clinical examinations included: digital rectal examination, head and neck examination, urethral meatus inspection and, in female patients, gynaecological examination; they underwent colposcopylurethroscopy, proctosigmoidoscopy, cystoscopy (in advanced disease); scraping for cytology and PCR analysis, and biopsy for histology. Twenty-three percent of patients had more than one site involved; upper digestive tract involvement was observed in 6.6% and 47% had lesions larger than 6 sqcm. Twenty-five females with genital lesions had esocervical lesions only; ten of them had SIL1, while seven a SIL3. RESULTS: Ninety-eight out of 123 patients (79.7%) were recurrence-free after a median follow-up of 32 months. A recurrence was observed in 25 patients: in univariate analysis, recurrence of disease occurred more frequently in females (p = 0.04), in patients with longer duration of symptoms (p = 0.0002),with wider lesions (p = 0.00015), with head and neck involvement (p < 0.01), and in HIV-positive patients (p = 0.03). In multivariate analysis, duration of symptoms (p = 0.005), head and neck involvement (p = 0.01), and width of lesion > 3 sq cm (p = 0.025) were associated with increased risk CONCLUSION: Our findings confirm the value of cryosurgery in the treatment HPV lesions; it is less traumatic, and gives good aesthetic and functional results; moreover, large lesions may be treated and the depth of cryonecrosis is more suitably adapted. Patients amenable to adjuvant treatment with a2-interferon should have multiorgan involvement, HPV type 16 or 18, lesions >3 sqcm, long lasting symptoms (>6 months) and presence of SIL.

Inhibition of colchicine binding to rat liver tubulin by aldehydes and by linoleic acid hydroperoxide.

The onset of fat accumulation within CCl4 poisoned hepatocytes, occurring as early as 1 h after treatment, is known to be provoked by a block in lipoprotein secretion. Lipoprotein secretion involves the function of the microtubular system. Several data indicate that this early block in lipoprotein secretion is not primarily the consequence of impaired protein synthesis. Therefore effects of some derivatives of lipid peroxidation, i.e. aldehydes and linoleic acid hydroperoxide were investigated. The results described in this paper shown that the above mentioned lipid peroxidation derivatives inhibit, with different activities, [3H] colchicine binding to liver high-speed supernates. Percentage binding inhibition is directly related to concentrations of aldehydes of LAHPO. LAHPO is more effective than aldehydes. Among the aldehydes tested, 4-hydroxypentenal, produced during lipid peroxidation of biological materials, was the most active. The presence of thiols, added to the incubation medium, partially protects against the inhibition of [3H] colchicine binding by aldehydes. This suggests that aldehydes act by reacting with -SH groups of tubulin. The possibility that interaction between lipoperoxidation derivatives and tubulin in vivo may contribute to the onset of fat infiltration in CCl4 poisoning is discussed.

Aliphatic aldehydes inhibit the proliferative response of human peripheral blood lymphocytes to phytohemagglutinin and alloantigens.

The effect of methyl glyoxal (MG) and various 4-hydroxyalkenals on the response of human peripheral blood lymphocytes (PBL) to phytohemagglutinin (PHA) or allogeneic cells has been investigated. Pretreatment of PBL with aldehydes significantly reduced the percentage of blast-transformed cells and [3H]thymidine incorporation into DNA in both PHA- and alloantigen-stimulated cultures, hydroxyalkenals being more effective than MG. Further experiments showed that these aldehydes also affected the proliferation of pre-activated lymphocytes. The percentage of blasts as well as [3H]thymidine incorporation into DNA were significantly decreased when the aldehydes were added until 72 h after application of the mitogenic stimulus.

Effect of two aliphatic aldehydes, methylglyoxal and 4-hydroxypentenal, on the growth of Yoshida ascites hepatoma AH-130.

The influence of a ketoaldehyde, methylglyoxal (MG), and a hydroxyalkenal, 4-hydroxypentenal (HPE), on the growth of a highly-deviated tumour has been investigated. MG and HPE, administered intraperitoneally, strongly depressed in rats the proliferative activity of the Yoshida ascites hepatoma AH-130, reducing its mitotic and labelling indices as well as the proportion of cycling cells (growth fraction). Monitoring the effects on the cell cycle by the labelled mitoses method showed that the percentage of labelled mitoses was markedly lowered after either aldehyde, which is indicative for a blocking effect in the S phase. In addition, the mean cell cycle time was slightly prolonged by MG, probably due to accumulation of cells in G1, whereas HPE delayed the first mitotic peak and increased the mean DNA synthetic period without modifying the overall cycle time. The effects of HPE on the cell cycle were prevented by pretreatment with polyamines. Repeated doses of MG significantly increased the fraction of tumour-bearing rats surviving at 90 days ('indefinite' survivors) as well as the survival time of those which succumbed, implying that the carcinostatic effect of MG persisted over several cell cycles. By contrast, HPE did not significantly modify the survival of AH-130-bearing rats, suggesting that its influence on tumour growth was rapidly reversible.

Cell death induced in L-cells by treatment with thymidine: staging of the process and relationship to apoptosis.

The purpose of this study was to characterize the stages in the development of thymidine-induced cell death. L-cells were characterized by both morphologic and quantitative techniques and evaluated at 24, 48, and 72 h of treatment. Cells first enlarged (stage I); about 50% of these enlarged cells then decreased in size with blebbing and compacting (stage II). This residual cell body transformed into a smooth eosinophilic hyaline body (stage III) by 72 h, many of which could be identified within the vacuolar system of viable cells. These changes were reflected in morphologic counts and Coulter sizing. Cell death (loss of labeled DNA) began in stage II and was most prominent in stage III. No cleavage of DNA into oligonucleosomal fragments was detected by agarose gel electrophoresis at any stage. The similarity of these changes to the complete spectrum of apoptosis in vivo is discussed.

[Various personality traits of patients with psoriatic arthropathy]. / Su alcuni aspetti della personalità in pazienti affetti da artropatia psoriasica.

It is well known that patients affected by rheumatic diseases may present specific pathological trends in personality structure, as has been extensively reported in literature. Our study was aimed at investigating several aspects of the personality traits of 20 patients with psoriatic arthropathy, compared with a group of 20 patients with rheumatoid arthritis. All patients were evaluated with appropriate rating scales assigned in auto and hetero-administration. The study results points to a personality trait disturbance in psoriatic arthritis patients, which can be clearly differentiated from the anxious habitus and/or reactive-depressive state observed in patients with rheumatoid arthritis.