Exploring the damage limitation possibilities of mineral fibres for future integrated solutions: an in vitro study.

Owing to their possible carcinogenic effect, asbestos and other silica derivatives have been identified as priority substances for risk reduction and prevention of pollution. Neutralisation procedures have thus become a topical research subject in many European and American countries. In the present study, silica derivatives (asbestos-containing and asbestos substitutes like slag wool, rock wool, cement asbestos) were fully impregnated with an epoxy resin according to the procedure used for the in situ impregnation with viscous polymeric media, which penetrate and cement the fibres in place and reduce the risk of their dispersion in air. Untreated and treated samples were used to investigate their in vitro interaction with a human continuous epithelial cell line (NCTC 2544 keratinocytes) and test the resin's efficiency in passivating the surface activity of the fibrous particulate. SEM and morpho-quantitative data evidenced that impregnation with the epoxy resin modifies the mineral fibres' bioactivity (reduction of cell adhesion and decreased spread/round cell ratio) and demonstrated the value of in vitro cell testing after passivation as a risk-assessment procedure. These tests could be used for the rapid determination of the level of passivation of new synthetic mineral fibrous materials subjected to resin impregnation.

Effect of mono-dose intraperitoneal cecropins in experimental septic shock.

OBJECTIVE: To investigate the efficacy of three cecropins, cecropin A, cecropin B, and cecropin P1, in preventing lethality in a rat model of septic shock. DESIGN: Prospective, randomized, controlled animal study. SETTING: Research laboratory in a university hospital. SUBJECTS: Adult male Wistar rats. INTERVENTIONS: Rats were given an intraperitoneal injection of 2 x 10(10) colony forming units of Escherichia coli, with the exception of the uninfected control group (C0). Animals were randomized to receive, immediately after bacterial challenge, intraperitoneally isotonic sodium chloride solution (untreated control group C1), 1 mg/kg cecropin A (group 2), 1 mg/kg cecropin B (group 3), 1 mg/kg cecropin P1 (group 4), 20 mg/kg imipenem (group 5), or 60 mg/kg piperacillin (group 6). Each group included 15 animals. MEASUREMENTS AND MAIN RESULTS: We measured bacterial growth (quantitative agar culture) in abdominal exudate and plasma, endotoxin and tumor necrosis factor-alpha concentration in plasma, and mortality. Results were evaluated at 48 hrs after inoculation. Cecropins, piperacillin, and imipenem significantly reduced the lethality and the number of E. coli in abdominal fluid compared with saline treatment. In addition, cecropin B significantly decreased the lethality compared with piperacillin treatment. Finally, only cecropins significantly reduced plasma endotoxin concentration. CONCLUSIONS: Mono-dose cecropin treatment prevents bacterial growth, endotoxemia, and mortality in rats with septic shock. Cecropin B was the most effective compound in reducing all variables measured.

Mupirocin prophylaxis against methicillin-susceptible, methicillin-resistant, or vancomycin-intermediate Staphylococcus epidermidis vascular-graft infection.

A rat model was used to investigate the efficacy of mupirocin in the prevention of vascular prosthetic graft infection due to Staphylococcus epidermidis strains with different susceptibility patterns (methicillin susceptible, methicillin resistant, and with intermediate resistance to vancomycin). The effect of mupirocin-soaked Dacron was compared to that of perioperative intraperitoneal prophylaxis with vancomycin. Graft infections were established in the back subcutaneous tissue of adult male Wistar rats by implantation of Dacron prostheses (1 cm(2)) followed by topical inoculation with 5 x 10(7) CFU of one staphylococcal strain. The study included a control group (no graft contamination), three contaminated groups that did not receive any antibiotic prophylaxis, three contaminated groups that received mupirocin-soaked grafts, three contaminated groups in which perioperative intraperitoneal vancomycin prophylaxis (10 mg/kg of body weight) was administered, and three contaminated groups that received mupirocin-soaked grafts and perioperative intraperitoneal vancomycin prophylaxis (10 mg/kg). The grafts were sterilely removed 7 days after implantation, and the infection was evaluated by using sonication and quantitative agar culture. Data analysis showed the efficacy of mupirocin against all three strains, with growth of the strains in treated rats significantly different than that in the untreated control. In addition, mupirocin was more effective than vancomycin against the strain with intermediate susceptibility to the glycopeptide. Finally, the combination of mupirocin and vancomycin produced complete suppression of the growth of all of the strains.

Do men and women follow different trajectories to reach extreme longevity? Italian Multicenter Study on Centenarians (IMUSCE).

Gender accounts for important differences in the incidence and prevalence of a variety of age-related diseases. Considering people of far advanced age, demographic data document a clear-cut prevalence of females compared to males, suggesting that sex-specific mortality rates follow different trajectories during aging. In the present investigation, we report data from a nationwide study on Italian centenarians (a total of 1162 subjects), and from two studies on centenarians living in two distinct zones of Italy, i.e., the island of Sardinia (a total of 222 subjects) and the Mantova province (Northern Italy) (a total of 43 subjects). The female/male ratio was about 2:1 in Sardinia, 4:1 in the whole of Italy, and about 7:1 in the Mantova province. Thus, a complex interaction of environmental, historical and genetic factors, differently characterizing the various parts of Italy, likely plays an important role in determining the gender-specific probability of achieving longevity. Gender differences in the health status of centenarians are also reported, and an innovative score method to classify long-lived people in different health categories, according to clinical and functional parameters, is proposed. Our data indicate that not only is this selected group of people, as a whole, highly heterogeneous, but also that a marked gender difference exists, since male centenarians are less heterogeneous and more healthy than female centenarians. Immunological factors regarding the age-related increase in pro-inflammatory status, and the frequency of HLA ancestral haplotypes also show gender differences that likely contribute to the different strategies that men and women seem to follow to achieve longevity. Concerning the different impact of genetic factors on the probability of reaching the extreme limits of the human life-span, emerging evidence (regarding mtDNA haplogroups, Thyrosine Hydroxilase, and IL-6 genes) suggests that female longevity is less dependent on genetics than male longevity, and that female centenarians likely exploited a healthier life-style and more favorable environmental conditions, owing to gender-specific cultural and anthropological characteristics of the Italian society in the last 100 years.

Modulation of mouse brain cortex adrenoceptor in old mice by supplementation of zinc and thymomodulin.

BACKGROUND: Previous experiments have shown that the age-related decrease of mouse brain cortex adrenoceptor density can be recovered by grafting a neonatal thymus into old recipients. The question arises whether similar results can also be obtained in the presence of a single thymic factor such as thymomodulin (TMD). It is worth noting that the activity of some thymic factors is strictly zinc (Zn) dependent and that their age-related decreased production is recovered in old mice supplemented with Zn. OBJECTIVE: The above-mentioned evidences prompted us to verify whether Zn and TMD, either alone or combined, are able to induce some corrective effects on age-dependent alterations of adrenoceptor characteristics of the mouse brain cortex. METHODS: Thus, we performed experiments on four groups of Balb/c mice treated with saline, Zn, TMD, or both Zn and TMD. Treatments started when animals were 18 months old and ran for 6 months. The alpha(1)- and beta-Adrenoceptor characteristics were assayed by steady state binding analysis using labelled prazosine and iodocyanopindolol, respectively. Data were analyzed using one-way analysis of variance, followed, when appropriate, by multiple-comparison analysis. RESULTS: Results show an increase of beta-adrenoceptor density and a decrease of alpha(1)-adrenoceptor density in both Zn- and Zn+TMD-treated animals when compared to saline-treated controls, while receptor affinities did not change significantly. CONCLUSIONS: The lack of action of TMD suggests that this type of treatment cannot mimic the effects of grafting the whole gland; it cannot be excluded that different time-dose schedule could be more effective. Zn treatment, on the other hand, does modulate adrenoceptors; however, it shows a corrective action on the age-related decreased density of beta-adrenoceptors, but further decreases that of alpha(1)-adrenoceptors. This differential action could be due to their differential physiological role.

Contribution of zinc to reduce CD4+ risk factor for 'severe' infection relapse in aging: parallelism with HIV.

Aging and HIV have parallelism in immunodeficiency status because of the appearance of infections or relapse leading to death in both conditions. HIV-RNA is predictor for HIV progression correlated with CD4+ depletion. CD4+ and plasma zinc levels (zincaemia) may be predictors for infections relapse in aging because of zinc relevance for normal immune efficiency against infections and for CD4+ growth. Moreover, zincaemia decreases in aging and infection. A total of 67 elderly subjects affected by infections resistant to antibiotic therapy were recruited. A total of 28 HIV+ subjects with HAART therapy were also used. CD4+ depletion (507 mm3) and zincaemia deficiency (76 microg/dl), as compared to CD4+ (700-1100 mm3) and zincaemia (85-100 microg/dl; age 40-75 years) normal ranges, are possible limits (Cox hazard regression) for severe infections relapse, such as chronic obstructive bronchitis and bronchopneumonia by bacteria or Candida complication, in aging. CD4+ and zincaemia values are within the lower limits of normal range in urinary tract infections. Zincaemia and HIV-RNA or CD4+ are inversely correlated (r = 0.57 and r = 0.72, respectively) in HIV+ HAART treated subjects. Consequently there is no appearance of opportunistic infections. Parallelism between aging and HIV may exist because of the resemblance in marked zinc deficiency and CD4+ depletion with high scores in relative risks for severe infections relapse. Supplementing zinc (12 mg Zn++/day) for one month in infected elderly subjects and HAART therapy in HIV+ subjects reduces risk scores in CD4+ and zincaemia deficiencies for infections relapse, suggesting that the zinc beneficial effect may be independent either by HIV-virus or pathogen agents involved. While HAART may reduce the occurrence of opportunistic infections in HIV by means of also major zinc bioavailability, supplementing zinc can be recommended in elderly people as resistance to infections. Since zinc deficiency is correlated with CD4+ depletion, this latter may also be good diagnostic marker to detect 'clear immunodeficiency' in aging, as in HIV condition.

Decrease of rotenone inhibition is a sensitive parameter of complex I damage in brain non-synaptic mitochondria of aged rats.

We investigated NADH oxidation in non-synaptic and synaptic mitochondria from brain cortex of 4- and 24-month-old rats. The NADH oxidase activity was significantly lower in non-synaptic mitochondria from aged rats; we also found a significant decrease of sensitivity of NADH oxidation to the specific Complex I inhibitor, rotenone. Since the rotenone-binding site encompasses Complex I subunits encoded by mtDNA, these results are in accordance with the mitochondrial theory of aging, whereby somatic mtDNA mutations are at the basis of cellular senescence. Accordingly, a 5 kb deletion was detected only in the cortex of the aged animals.

Thyroid-thymus interaction on beta-adrenoceptor regulation during development and ageing.

Up-regulation of brain cortex beta-adrenoceptors (beta ARs) can be induced by very acute stimulation with a single injection of T4 or T3 in young Balb/c-nu mice. We have also shown that this very rapid receptor increase can also be demonstrated in ageing animals when stimulated with T3 but not T4 injection. The aim of the present paper was to verify the capability of the thymus to reverse these impairments which we often observed in other experiments on old mice and young athymic nudes. The up-regulation induced by T4 and T3 was studied in normal and athymic nude young, normal old, and normal old and athymic nude young mice grafted with neonatal thymus 1 month earlier. In addition, since brain cortex bears both beta AR subpopulations, the eventual differential behavior of beta 1AR and beta 2AR subtypes was also investigated. Animals were injected intraperitoneally with saline, or saline containing 0.5 microgram T3 or 6.4 micrograms T4 per g body weight and killed 15 or 60 min after injection. Results show that thymus can recover the modifications of basal levels as well as T3-induced up-regulation of beta ARs in nude and old mice. On the contrary, impaired response to T4 stimulation is corrected in nude but not in old mice. The peripheral conversion of T4 into T3 can explain their differential influence since a correct conversion only occurs in presence of an efficient beta-adrenergic function. Thus, a vicious circle may occur with a decreasing number of beta-adrenoceptors causing in old age altered T4 to T3 conversion, in turn responsible for altered beta-adrenergic responsiveness to T4.

Presence of a DNA-4236 bp deletion and 8-hydroxy-deoxyguanosine in mouse cardiac mitochondrial DNA during aging.

A particular mitochondrial DNA (mtDNA) deletion, the so-called "common deletion", accumulates progressively with age in human post-mitotic cells. We investigated the presence of age-related mtDNA deletions in mouse heart and, according to the free-radical theory of aging, the potential involvement of reactive oxygen species (ROS). Hearts from young, adult and old male Balb/c mice were homogenized and centrifuged in order to discard nuclear DNA. The supernatant was then utilized to prepare mtDNA by SDS-proteinase K digestion. The presence of a mtDNA4236 deletion was estimated by PCR analysis, by separating the amplificated segment on agarose gel. The incidence of the mtDNA4236 deletion was 16%, 28% and 78% in young, adult and old mice, respectively. 8-hydroxy-2'-deoxyguanosine (8-OH-dG), a marker of DNA oxidation, was also determined by HPLC-electrochemical analysis. 8-OH-dG was not detectable in young mice, while its concentrations (moles 8-OH-dG/10(6) moles dG; mean +/- SD) were 59.0 +/- 1.41 and 31.0 +/- 4.24 (p < 0.02) in adult and old mice, respectively. These data indicate that a mtDNA4236 deletion is progressively associated with aging in mouse hearts, and that oxidative damage to mtDNA is greater in middle-aged than senescent animals.

Assay of total protein kinase activity in mouse brain cortex.

In previous studies we demonstrated that the adrenergic system is impaired in old animals and that the main alterations were observed at the level of receptor density and adenylate-cyclase activity. The decreased ability to produce cAMP could influence the activity of the cAMP dependent protein kinase (PKA), one of the enzymes responsible for the phosphorylation of protein substrates. Since protein phosphorylation is one of the most common and important mechanisms through which a cell regulates its activity, the characteristics of the phosphorus incorporation reaction were studied. Kinase activity was measured in homogenate of young mouse brain cortex prepared avoiding gross manipulations in order to maintain conditions as close to those present in the living animal as possible. Results show that phosphate incorporation is proportional to protein content and strictly dependent on ATP availability. Increasing the ATP concentration from 10 to 500 mumol/l, the length of incorporation phase increases, suggesting that the limiting point of the reaction is better represented by energy availability than by enzyme or protein substrate concentrations.

Age-related regulation of mouse brain cortex adrenoceptors following camphor vapor exposition.

The paper deals with the ability of adrenergic receptors (AR) of mouse brain cortex to be differentially regulated in response to single or multiple expositions to camphor vapor. The regulation of alpha- and beta-adrenoceptors has been studied in young and old Balb/c-nu mice. Results confirm the decrease of total beta-adrenoceptor density previously observed in untreated mice with advancing age; in addition, receptor density decreases in both young and old mice after a single exposition to camphor vapor, followed by an adaptation after multiple stimuli. The beta1, subtype is mainly responsible for density decrease in young animals, while both beta1, and beta2 subtypes contribute to the decrease in old mice. On the contrary, beta2 subpopulation gives the major contribution to the adaptive recovery in both young and old mice. alpha-Adrenoceptors also show an age-related decrease in the control group; after a single exposition they show an increased density with the exception of alpha1-subset in the young group. Repeated expositions lead to a rather general adaptive response towards pre-stimuli conditions. The differential behaviour of receptor subtypes in response to camphor vapor exposition can be related to the differential alterations of receptor characteristics observed during aging and also suggests a possible mechanism through which these alterations may occur.

Immune enhancement by conditioning of senescent mice. Comparison of old and young mice in learning ability and in ability to increase natural killer cell activity and other host defense reactions in response to a conditioned stimulus.

It has been clearly demonstrated that immune responses may be conditioned in a manner similar to that of the classical Pavlovian experiments. Evidence of impaired immune function in aging has raised the question of whether psychological conditioning of an immune response can also be effective in old age. The knowledge that aged mice have decreased spleen cell natural killer (NK) activity and that NK cytotoxicity, at least in young mice, can be psychologically conditioned led us to explore in old mice the possibility of conditioning the response of NK cell activity using the odor of camphor as the conditioned stimulus (CS) and the injection of Poly I:C as the unconditioned stimulus (US). Young and old male mice were divided into five and six groups, respectively. They received the CS and/or the US in association (conditioning) trials (sessions 1-9). Mice were exposed to the camphor odor alone at 72 hours after the final association trial to observe the conditioning phenomenon (session 10). The group conditioned with Poly I:C and camphor and receiving the CS at session 10 showed statistically significant increases in spleen cell NK activity over those of the control groups that did not receive the CS treatment at session 10 (2.6- and 4.0-fold increase in young and old, respectively). Treatment with camphor odor alone had no effect on boosting NK cell activity. These findings demonstrate the possibility of conditioning immune responses in old age, offering a valuable tool for attenuating age-related immune deterioration in various species, including the human. In addition, these results again confirm highly significant immune enhancement by classical conditioning and extend previous findings from female mice to males as well.

Assay of estrogen and progesterone receptors in small samples of breast cancer: I. Mathematical analysis of total binding data.

The quantitative determination of Estrogen and Progesterone receptors in breast cancer is performed using about 500 mg of tissue. Such an amount of tissue sample is available with ever increasing difficulty. In the present paper a mathematical method of analysis of the total (specific+nonspecific) binding data is given. In this way about 50% of tissue can be saved as only one set of test tubes is required; in fact, the method does not need the second set of tubes used to determine nonspecific binding in the conventional assay. Additional advantages from the statistical point of view are also presented.

Assay of estrogen and progesterone receptors in small samples of breast cancer: II. The conventional procedure compared to the mathematical one.

Parallel assays of ER and PR receptors have been performed in the same tissue samples using both the conventional method employing two sets of test tubes and a mathematical method which does not need the second set of tubes containing excess cold ligand concentrations. Determinations have been performed in calf uterus and human breast cancer samples. Findings show that the two methods give quite similar results. In particular, they are very close in human BC tissues where receptor density values show a very wide range. The mathematical method seems quite useful when the samples are poor of receptors, a case where the precision of the method may influence both prognosis and therapy.

Parallel decrease of adenylyl cyclase activity and beta1-adrenoceptor density in brain cortex of aging mice.

Beta-adrenergic receptors (betaARs) and adenylyl cyclase (AC) activity have been found to undergo progressive alterations in the brain cortex of aging mice. In particular, betaAR changes are in charge of the beta1 subpopulation (beta1AR), beta2-adrenoceptors (beta2ARs) showing no age-related impairments. On these bases, the question arises whether AC alterations can be accounted for by beta1AR changes or, alternatively, whether there are also receptor independent AC modifications. Experiments have thus been performed on the brain cortex from young and old mice assaying beta1AR and beta2AR characteristics and isoproterenol (IPR) and forskolin stimulated AC activity in the same membrane preparations. Other membranes were previously treated with the reducing agent dithiothreitol (DTT) which is known to specifically destroy the binding capacity of beta1ARs. No statistically significant differences in basal AC activity have been found among the groups studied. Data on IPR-stimulated AC activity in old animals confirm previous findings demonstrating impaired responsiveness when compared with that of young mice. DTT treated membrane preparations from both young and old mice show decreased AC activities. The entity of the decrease is lower in aged animals due to the lower initial level of beta1ARs. Forskolin stimulation, which is assumed to directly activate AC, has been found impaired in aged mice when compared with young animals, suggesting that also post-receptor alterations occur with advancing age.

Age-related alterations of isoproterenol-stimulated adenylyl-cyclase activity are partially corrected by thymic graft.

Previous experimental results have demonstrated progressive impairments in beta-adrenergic responsiveness with advancing age. Beta-adrenoceptors are involved in the alterations as their density progressively decreases during aging. Alterations in both in vivo responsiveness and receptor density are corrected by neonatal thymic grafts. In the present paper adenylyl-cyclase (AC) activity has been studied in the same animal models used before. Results show that no statistically significant changes can be observed when AC is assayed in absence of beta-adrenergic stimulation. On the contrary, when assayed after Isoproterenol stimulation, AC activity shows a shift of the peak and a decrease of its height in aged animals. A neonatal thymus grafted into old recipients one month before the experiment was performed, is capable of correcting the altered height of the peak but not the peak concentration.