Comparison of bone mass and quality determinants in adolescents and young adults with juvenile systemic lupus erythematosus (JSLE) and juvenile idiopathic arthritis (JIA).

BACKGROUND: Few prospective data have been published on the comparison of bone density and quality in homogeneous groups of patients with juvenile systemic lupus erythematosus (JSLE) and juvenile idiopathic arthritis (JIA). OBJECTIVE AND HYPOTHESIS: The objective of this study is to perform a longitudinal evaluation of the prevalence and the characteristics of bone mass and quality and to evaluate the differences on the bone parameters, using DXA, pQCT and QUS. POPULATION AND/OR METHODS: Forty-three JSLE patients (35 females, 8 males, median age 18.8, range 14.0-34.1 years) have been studied with DXA, pQCT and QUS scans and compared with 138 JIA patients (112 females, 26 males, median age 18.9, range 13.4-33.2 years), and 79 controls (59 females, 20 males; median age 19.3, range 13.5-36.5 years). Of these, 39 patients (32 females and 7 males, median age 20.3, range 16.6-36.8 years) with JSLE were followed longitudinally and compared with 131 patients (108 females, 23 males median age 20.7, range 15.8-37.1 years) with JIA and 63 controls (48 females, 15 males; median age 21.9, range 15.5-38.3 years). RESULTS: JSLE patients have a higher bone cortical density (CrtBMD) than controls and JIA patients (p < 0.005). However, JSLE and JIA patients have a significantly reduced bone trabecular density (TrbBMD) compared to controls (p < 0.0001), with no differences between JSLE and JIA. In addition, JIA patients show a significantly reduced muscle area (MuscleCSA) compared to JSLE and controls (p < 0.001). Conversely, fat area (FatCSA) is significantly increased both in JIA and JSLE patients when compared to controls (p < 0.001), with no differences between the JSLE and JIA groups. Analogous results are observed in the polar resistance to stress (SSIp). On longitudinal evaluation, contrary to CrtBMD, the difference between BMAD SDS, TrbBMD, MuscleCSA and FatCSA remains unchanged; in JSLE patients, SSIp is stable in comparison to JIA and controls without any difference between the two groups. CONCLUSIONS: The evaluation of bone density and structure parameters in JSLE patients highlights significant differences compared with JIA patients and controls. These data might indicate a different pathogenesis of bone damage in the two entities, and suggest a different diagnostic and therapeutic approach to improve the peak bone mass.

A new Rayleigh-like wave in guided propagation of antiplane waves in couple stress materials.

Motivated by the unexpected appearance of shear horizontal Rayleigh surface waves, we investigate the mechanics of antiplane wave reflection and propagation in couple stress (CS) elastic materials. Surface waves arise by mode conversion at a free surface, whereby bulk travelling waves trigger inhomogeneous modes. Indeed, Rayleigh waves are perturbations of the travelling mode and stem from its reflection at grazing incidence. As is well known, they correspond to the real zeros of the Rayleigh function. Interestingly, we show that the same generating mechanism sustains a new inhomogeneous wave, corresponding to a purely imaginary zero of the Rayleigh function. This wave emerges from 'reflection' of a bulk standing mode: This produces a new type of Rayleigh-like wave that travels away from, as opposed to along, the free surface, with a speed lower than that of bulk shear waves. Besides, a third complex zero of the Rayleigh function may exist, which represents waves attenuating/exploding both along and away from the surface. Since none of these zeros correspond to leaky waves, a new classification of the Rayleigh zeros is proposed. Furthermore, we extend to CS elasticity Mindlin's boundary conditions, by which partial waves are identified, whose interference lends Rayleigh-Lamb guided waves. Finally, asymptotic analysis in the thin-plate limit provides equivalent one-dimensional models.

Iron released from an erythrocyte lysate by oxidative stress is diffusible and in redox active form.

The incubation of a ghost-free erythrocyte lysate with the oxidizing agent phenylhydrazine resulted in both methemoglobin formation and release of iron in a desferrioxamine (DFO)-chelatable form. The released iron was diffusible, as shown by a dialysis carried out simultaneously with the incubation. When the dialysate was added to erythrocyte ghosts or to microsomes from liver or brain, lipid peroxidation developed in the membranes, indicating that the diffusible iron was in a redox active form. The addition of ATP to the lysate markedly increased both iron diffusion and lipid peroxidation in the membranes subsequently added to the dialysate. The possible implication of these data in some well known pathologies is discussed.

Iron release, membrane protein oxidation and erythrocyte ageing.

The aerobic incubation of erythrocytes in phosphate buffer for 24-60 h (a model of rapid in vitro ageing) induced progressive iron release and methemoglobin formation. Membrane proteins showed electrophoretic alterations and increase in carbonyl groups (as documented by IR spectroscopy). None of these phenomena were seen when the erythrocytes were incubated under anaerobic conditions. The membranes from aerobically incubated cells bound a much higher amount of autologous IgG than those from anaerobically incubated ones, suggesting that the aerobic incubation gives rise to the senescent antigen. The addition of ferrozine during the aerobic incubation prevented both the IgG binding and the protein alterations seen in the IR spectra, suggesting an intracellular chelation of the released iron by ferrozine.

Protection against oxidative damage of erythrocyte membrane by the flavonoid quercetin and its relation to iron chelating activity.

Incubation of glutathione (GSH) depleted mouse erythrocytes with the oxidants phenylhydrazine, acrolein, divicine and isouramil resulted in the release of free iron and in lipid peroxidation and hemolysis. The addition of the flavonoid quercetin, which chelates iron and penetrates erythrocytes, resulted in remarkable protection against lipid peroxidation and hemolysis. The protection seems to be due to intracellular chelation of iron, since a semi-stoichiometric ratio between released iron and the amount of quercetin necessary to prevent lipid peroxidation and hemolysis was found. Incubation of GSH depleted human erythrocytes with divicine and isouramil did not induce lipid peroxidation and hemolysis in spite of a substantial release of iron. However, divicine and isouramil produced alterations of membrane proteins, such as spectrin and band 3, as well as formation of senescent cell antigen. The addition of quercetin prevented these alterations.

Iron release and erythrocyte damage in allyl alcohol intoxication in mice.

Allyl alcohol administration in a toxic dose (1.5 mmol/kg) to starved mice causes the development of hemolysis in nearly 50% of the animals. Malonic dialdehyde (MDA) appears in plasma of the animals showing hemolysis. The treatment of mice with desferrioxamine after allyl alcohol intoxication completely prevents lipid peroxidation and hemolysis, suggesting the involvement of iron in the allyl alcohol-induced erythrocyte damage. Erythrocytes obtained from intoxicated mice before the development of hemolysis show, upon incubation, release of iron, lipid peroxidation and lysis. Studies carried out with reconstituted systems of erythrocyte lysates, containing ghosts and different fractions of erythrocyte cytosol and incubated in the presence of acrolein (the major metabolite of allyl alcohol), strongly suggest that iron is released from hemoglobin. This iron appears to promote lipid peroxidation which is accompanied by erythrocyte lysis. Thus, the allyl alcohol-induced hemolysis appears to be a model for iron delocalization from iron stores.

Release of free, redox-active iron in the liver and DNA oxidative damage following phenylhydrazine intoxication.

Following the subchronic intoxication of rats with phenylhydrazine, resulting in marked anemia, reticulocytosis, methemoglobinemia and increased hemocatheresis, the hepatic content of total iron was increased, as was hepatic ferritin and its saturation by iron. A striking increase (approximately 7-fold) was also observed in free iron which appeared to be redox-active. The increase in liver free iron involved the hepatocellular component of the liver. Since DNA is one of the cellular targets of redox active iron, liver DNA from phenylhydrazine-treated rats was analyzed by electrophoresis and found to be markedly fragmented. Experiments with isolated hepatocytes in culture or in suspension challenged with phenylhydrazine or Fe-nitrilotriacetate strongly suggested that the DNA damage was due to reactive iron rather than to the hepatic metabolism of phenylhydrazine. The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a specific marker of oxidative DNA damage, were significantly higher in phenylhydrazine-treated rats as compared to untreated controls. The prolongation of phenylhydrazine treatment over a period of 6 weeks resulted in a persistent damage to DNA and in phenotypic changes such as an increase in hepatocyte gamma-glutamyl transpeptidase (gamma-GT, EC 2.3.2.2) activity. Possible relationships between iron overload, iron release, DNA damage and tumor initiation are discussed.

Protection of erythrocytes against oxidative damage and autologous immunoglobulin G (IgG) binding by iron chelator fluor-benzoil-pyridoxal hydrazone.

Iron is released in a free desferrioxamine-chelatable form when erythrocytes are challenged by an oxidative stress. The release of iron is believed to play an important role in inducing destructive damage (lipid peroxidation and hemolysis) or in producing membrane protein oxidation and generation of senescent cell antigens (SCA). In this report, we further tested the hypothesis that intracellular chelation of iron released under conditions of oxidative stress prevents erythrocyte damage or SCA formation. Fluor-benzoil-pyridoxal hydrazone (FBPH), an iron-chelating molecule of the family of aromatic hydrazones, was prepared by synthesis and used for the above purpose after the capacity of the product to enter cells had been ascertained. GSH-depleted mouse erythrocytes were incubated with the oxidant drug phenylhydrazine in order to produce iron release, lipid peroxidation, and hemolysis. FBPH at a concentration of 200 microM prevented lipid peroxidation and hemolysis in spite of equal values of iron release. FBPH was active even at a lower concentration (100 microM) when the erythrocytes were preincubated with it for 15 min. No preventive effect was seen when FBPH saturated with iron was used. Prolonged aerobic incubation (60 hr) of erythrocytes produced iron release and formation of SCA as determined by autologous immunoglobulin G (IgG) binding. The IgG binding was detected by using an anti-IgG antibody labeled with fluorescein and by examining the cells for fluorescence by confocal microscopy. FBPH prevented SCA formation in a dose-related manner. These results lend further support to the hypothesis that iron release is a key factor in erythrocyte ageing.

Iron release in erythrocytes from patients with beta-thalassemia.

Our previous studies have shown that iron is released in a free (desferrioxamine-chelatable) form when erythrocytes undergo oxidative stress (incubation with oxidizing agents or aerobic incubation in buffer for 24-60 h (a model of rapid in vitro ageing)). The release is accompanied by oxidative alterations of membrane proteins as well as by the appearance of senescent antigen, a signal for termination of old erythrocytes. In hemolytic anemias by hereditary hemoglobin alterations an accelerated removal of erythrocytes occurs. An increased susceptibility to oxidative damage has been reported in beta-thalassemic erythrocytes. Therefore we have investigated whether an increased iron level and an increased susceptibility to iron release could be observed in the erythrocytes from patients with beta-thalassemia. Erythrocytes from subjects with thalassemia intermedia showed an extremely higher content (0 time value) of free iron and methemoglobin as compared to controls. An increase, although non-statistically-significant, was seen in erythrocytes from subjects with thalassemia major. Upon aerobic incubation for 24 h the release of iron in beta-thalassemic erythrocytes was by far greater than in controls, with the exception of thalassemia minor. When the individual values for free iron content (0 time) seen in thalassemia major and intermedia were plotted against the corresponding values for HbF, a positive correlation (P < 0.001) was observed. Also, a positive correlation (P < 0.01) was seen between the values for free iron release (24 h incubation) and the values for HbF. These results suggest that the presence of HbF is a condition favourable to iron release. Since in beta-thalassemia the persistance of HbF is related to the lack or deficiency of beta chains and therefore to the excess of alpha chains, the observed correlation between free iron and HbF, is consistent with the hypothesis by others that excess of alpha chains represents a prooxidant factor.

Hemolytic drugs aniline and dapsone induce iron release in erythrocytes and increase the free iron pool in spleen and liver.

Incubation of rat erythrocytes with the hydroxylated metabolites of aniline and dapsone (4-4'-diaminodiphenylsulfone), phenylhydroxylamine and dapsone hydroxylamine, respectively, induced marked release of iron and methemoglobin formation. On the contrary, no release of iron nor methemoglobin formation was seen when the erythrocytes were incubated with the parent compounds (aniline and dapsone). The acute intoxication of rats with aniline or dapsone induced a marked increase in the erythrocyte content of free iron and methemoglobin, indicating that the xenobiotics are effective only after biotransformation to toxic metabolites in vivo. Prolonged administration of aniline or dapsone to rats produced continuous release of iron from erythrocytes. Marked iron overload was seen in the spleen and in the liver Kupffer cells, as detected histochemically. The spleen weight in these subchronically treated animals was significantly increased. The free iron pool was markedly increased in the spleen and to a lower extent in the liver. The possible relationships between iron release in erythrocytes, oxidative damage seen in senescent cells, hemolysis, overwhelmed capacity of spleen and liver to keep iron in storage forms and subsequent increase in low molecular weight, catalitically active iron is discussed.

Development of horse polyclonal antiserum inhibiting all in vitro biological functions of human IFN-gamma.

Following a standard immunization protocol with recombinant human interferon-gamma (IFN-gamma), a horse polyclonal antiserum was obtained and evaluated for its ability to interfere with multiple IFN-gamma activities in vitro. Data obtained show that polyclonal horse antiserum neutralizes the antiproliferative activity of IFN-gamma, inhibits the binding of IFN-gamma to cellular receptors, and can up-regulate HLA-DR antigen expression and interfere with its antiviral activity. The broad neutralizing capacity of horse polyclonal antiserum has been assessed on cell lines which differ as to origin and sensitivity to IFN-gamma. Moreover, we observed that this antiserum could inhibit the binding of radiolabeled IFN-gamma to its cellular receptor, its subsequent internalization into the target cell, and its antiviral activity. As it is able to inhibit all the biological activities of IFN-gamma, this antiserum might provide new therapeutic approaches to diseases with evidence of activated cell-mediated immunity.

Identification of Mycobacterium xenopi by gas chromatography.

For the purposes of the following study we cultured 32 strains of Mycobacterium xenopi isolated from clinical specimens and several strains of other slowly growing mycobacteria. The cultures were grown in liquid medium and then analysed--after saponification, methylation, extraction with organic solvent and washing of the organic phase--using a highly sensitive manual gas-liquid chromatographic assay for the determination of secondary alcohol 2-OH-docosanol. The percentage of this compound was compared with that previously measured in strains of Mycobacterium xenopi grown on solid medium. The presence of this specific alcohol was always apparent, even though its quantity was lower than that obtained by growing mycobacteria on solid medium. The absence of interference peaks around the compound was checked by analyzing strains of other slowly growing mycobacteria in the same conditions.

Gas chromatographic assay of cellular fatty acids and alcohols for the identification of Mycobacterium species.

Ten mycobacterial species obtained from 141 cultures isolated from clinical specimens were studied. The cultures were grown on solid medium and then analysed-after saponification, methylation, extraction with organic solvent and washing of the organic phase--by capillary gas-liquid chromatography for fatty acid and secondary alcohol composition. The absence of secondary alcohols was characteristic of M. genavense, M. tuberculosis and the following Mycobacterium species with specific branched-chain fatty acids allowing their direct identification: M. gordonae, M. kansasii and M. marinum. The presence of secondary alcohols was characteristic of M. avium, M. phlei, M. scrofulaceum, M. terrae and M. xenopi. In the case of M. xenopi direct identification was made possible by the presence of a specific alcohol.

The quality assurance system in clinical chemistry.

The quality assurance system in clinical chemistry allows for the identification of errors and control actions to correct them. It is well known that laboratory errors can be classified as: pre-analytical, analytical and post-analytical. While pre-analytical and post-analytical errors are very difficult to identify, the analytical variability (both imprecision and inaccuracy) can be monitored with internal quality control (IQC) programs and external quality assessment (EQA) schemes. The purpose of IQC is mainly to verify the stability of laboratory estimates with time and therefore it is essentially a control of imprecision. IQC programs are based on the use of control samples which are analyzed in each analytical series. The easiest method of representing IQC data is by the use of Shewhart's chart, although "cusum" chart and Youden plot are often useful. As for the criteria according to which an analytical series should be accepted or rejected, the use of practical control rules is widely spread in laboratories. Participation in EQA schemes allows the laboratory to have a retrospective estimate of its performance in terms of both imprecision and inaccuracy, if definitive or reference methods are available. In lack of definitive or reference methods, consensus mean or median can be derived from the data obtained by all the participants or, in some cases, by the participants using the same analytical method (e.g. for analytes not yet completely characterized and measured with immunoassays.

Assessment of qualitative tests.

For qualitative (2-class) tests, which provide binary (yes/no) information, the correctness of specimen classification remains the most important criterion for performance evaluation. However, a more informative picture emerges from the relationship between percentage of positive results and analyte concentration, which allows some inherent test characteristics to be derived (the positive/negative discrimination concentration and the "grey zone" around it). The information content of evaluation approaches is decidedly improved by the availability of numerical results (counts per minute, absorbance) in most situations of clinical interest. The concentration/response functions underlying the quality of individual tests may thus be derived and compared, and laboratory staff given a more objective criterion to judge individual performance. Examples are drawn from the authors' experience in running external quality assessment programs for tests for infectivity markers.

Italian external quality assessment scheme in immunoassay.

This paper deals with the organization, the data processing and some of the results obtained in Italian external quality assessment (EQA) schemes for hormones, tumor markers and hepatitis B markers. The EQA for hormones and tumor markers includes up to sixteen analytes together with the participation, in 1990, of about 250 laboratories. Laboratory results were used to prepare periodic and end-of-period reports. The former includes the results (with the related statistical parameters) obtained by all participants and by laboratories using the same method, as well as the histogram of the data. The end-of-period report contains estimates of imprecision and average bias for all laboratories, for each laboratory and for the more widely employed kits. From 1980 to 1988, laboratory variability improved significantly for TSH, progesterone, estradiol, testosterone, CEA and ferritin, slightly for cortisol, FSH, prolactin and AFP, while there was no improvement for both total T3 and T4. For LH we found an unusually high variability mainly due to systematic differences between kits based on different monoclonal antibodies. About 200 laboratories participated in the EQA for hepatitis B markers (HBsAg and anti-HBs) organized in 1990. For these analytes the periodic reports show the percentage of negative and positive results and the histogram of the responses (absorbance or counts) normalized with respect to the cut off.

External quality assessment of assays for hormones and tumor markers in Italian laboratories.

External quality assessment (EQA) programs run by CNR/Tecnostandard for immunoassays of hormones and tumor markers, started in 1980, presently include as many as 20 analytes; about 300 laboratories are involved in these programs. For all immunoassays submitted to the EQA, the inspection of cumulative results allows the current situation to be documented for total variability and its within-kit and between-kit components (the former accounting for the reproducibility and robustness of the kits and the latter for their systematic differences of estimation). For 13 assays subjected to EQA for longer, the variability trends over time are depicted, and single factors affecting the overall quality of particular assays are identified. Among these, experimental simplification of kit structure, alignment of calibrators with an acknowledged reference material, and adoption of monoclonal-antibody based two-sites assays can be mentioned. On the contrary, neither automation of the procedures nor (more expectedly) increasing use of nonisotopic techniques has proved effective in significantly improving the analytical quality.

Immunoassay external quality assessment in Italy: HBsAg and anti-HBs tests.

The mode of operation of the CNR/Tecnostandard external quality assessment scheme for HBsAg and anti-HBs assays is outlined, and the relevant results reported. Emphasis is given to the retrospective evaluation of data in an attempt to derive a picture of the state of the art in Italian laboratories. The approaches followed for this evaluation include analysis of the rate of correct results and inspection of the relationships between analyte concentration and either the percentage of positive classifications of samples or the numerical test responses. Information is given on the "average" performance of individual kits as actually used in participants' laboratories.

[Oxidative stress and Rett syndrome]. / La sindrome di Rett: ruolo dello stress ossidativo.

The oxidative stress (OS) hypothesis is able to explain several features of Rett syndrome (RTT), a pervasive development disorder almost exclusively affecting females mainly caused by a mutation in the X-linked methyl-CpG binding protein 2 (MeCP2) gene. In particular, the generation of an OS imbalance is related to MeCP2 gene mutation type, as well as natural history, clinical heterogeneity of the disease, and is compatible with the potential reversibility of the disease observed in the RTT animal models. In addition, our findings indicate the importance of blood as a suitable biological fluid for detecting markers of central nervous system oxidative damage in RTT and underline the key role of interaction between organic chemists, OS biochemists, and clinicians in revealing potential new markers of the disease and identifying potential new targets and interventional strategies aimed at improving the quality of life of these patients, affected by a so far incurable disease. Further efforts in the near future are needed in order to dissect the "black box" of the molecular events likely linking the MeCP2 gene mutation to OS derangement and subsequent disease expression.