Extracellular vesicles as potential biomarkers of acute graft-vs-host disease.

Acute graft-vs-host disease (GVHD) is a serious complication after allografting. We carried out an exploratory study to investigate a potential correlation of surface antigens on extracellular vesicles (EVs) and acute GVHD. EVs were extracted from serum samples from 41 multiple myeloma patients who underwent allografting. EVs were characterized by flow cytometry using a panel of 13 antibodies against specific membrane proteins that were reported to be predictive of acute GVHD. We observed a correlation between three potential biomarkers expressed on EV surface and acute GVHD onset by both logistic regression analysis and Cox proportional hazard model. In our study, CD146 (MCAM-1) was correlated with an increased risk-by almost 60%-of developing GVHD, whereas CD31 and CD140-α (PECAM-1 and PDGFR-α) with a decreased risk-by almost 40 and 60%, respectively. These biomarkers also showed a significant change in signal level from baseline to the onset of acute GVHD. Our novel study encourages future investigations into the potential correlation between EVs and acute GVHD. Larger prospective multicenter studies are currently in progress.

Cathepsin activities and membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling.

This study investigated enzymatic activity of cathepsins and the membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling. Stage III oocytes (>0.5mm), obtained through dissection of anaesthetised female fish and desegregation of ovarian cumulus, were exposed to 2M methanol or 2M DMSO (both prepared in Hank's medium) for 30min at 22 degrees C before being loaded into 0.5ml plastic straws and placed into a programmable cooler. After controlled slow freezing, samples were plunged into liquid nitrogen (LN) and held for at least 10min, and thawed by immersing straws into a 27 degrees C water bath for 10s. Thawed oocytes were washed twice in Hank's medium. Cathepsin activity and membrane integrity of oocytes were assessed both after cryoprotectant treatment at 22 degrees C and after freezing in LN. Cathepsin B and L colorimetric analyses were performed using substrates Z-Arg-ArgNNap and Z-Phe-Arg-4MbetaNA-HCl, respectively, and 2-naphthylamine and 4-methoxy-2-naphthylamine were used as standards. Cathepsin D activity was performed by analysing the level of hydrolytic action on haemoglobin. Oocytes membrane integrity was assessed using 0.2% Trypan blue staining for 5min. Analysis of cathepsin activities showed that whilst the activity of cathepsin B and D was not affected by 2M DMSO treatment, their activity was lowered when treated with 2M methanol. Following freezing to -196 degrees C, the activity of all cathepsins (B, D and L) was significantly decreased in both 2M DMSO and 2M methanol. Trypan blue staining showed that 63.0+/-11.3% and 72.7+/-5.2% oocytes membrane stayed intact after DMSO and methanol treatment for 30min at 22 degrees C, respectively, whilst 14.9+/-2.6% and 1.4+/-0.8% stayed intact after freezing in DMSO and methanol to -196 degrees C. The results indicate that cryoprotectant treatment and freezing modified the activities of lysosomal enzymes involved in oocyte maturation and yolk mobilisation.

Changes in cathepsin gene expression and relative enzymatic activity during gilthead sea bream oogenesis.

The aim of this study was to provide evidence on the modulation of lysosomal enzymes in terms of both gene expression and enzymatic activity during follicle maturation. For this purpose three lysosomal enzymes, cathepsins B, D, and L, were studied in relation to yolk formation and degradation, during the main phases of ovarian follicle growth in the pelagophil species, the sea bream Sparus aurata. Specific attention was focused on the gene expression quantification method, on the assay of enzymatic activities, and on the relationship between the proteolytic cleavage of yolk proteins (YPs), cathepsin gene expression and cathepsin activities. For the gene expression study, the cathepsins B-like and L-like mRNAs were isolated and partially or fully characterized, respectively; the sequences were used as design specific primers for the quantification of cathepsin gene expression by real-time PCR, in follicles at different stages of maturation. The enzymatic assays for cathepsins B, D, and L were optimized in terms of specificity, sensitivity and reliability, using specific substrates and inhibitors. In ovulated eggs, the lipovitellin I (LV I) was degraded and the changes in electrophoretic pattern were preceded by an increase in the activity of a cysteine proteinase, cathepsin L, and its mRNA. Cathepsin B did not appear to be involved in YP changes during the final maturation stage.

Role of cathepsins in ovarian follicle growth and maturation.

Several complex processes are involved in the production of viable eggs. The aim of this review is to provide an overview on the role played by lysosomal enzymes, especially cathepsins B, D, and L, during ovarian follicle growth and maturation. Specific attention is focused on the relationship between the second proteolytic cleavage of yolk proteins (YP) and the resumption of the meiosis during germinal vesicle break down (GVBD). Maturation represents the final stage of oocytes development prior to ovulation. Oocytes in this phase appear translucent. In many teleosts GVBD is accompanied by water uptake and among marine teleosts with pelagic eggs, most of the final volume is reached by this process. The last phase of maturation in benthonic eggs also occurs concomitant to a second proteolytic cleavage and is related with a slight hydration process. In vitro maturation by 17alpha,20beta-dihydroxy-4-pregnen-3one in class III Danio rerio oocytes, induced 80% of GVBD. The maturation of these oocytes is known to be associated with proteolysis of their major yolk components. In the present study, we show that inhibition of specific enzymes (cathepsins) involved in the second YP processing, did not affect the occurrence of GVBD as the oocytes become translucent and display a slight increase in size. More specifically, in vitro incubation of the maturing oocytes with a cathepsin B inhibitor suppressed both cathepsin B and L activities and the proteolysis of YP. On the contrary, the addition of cathepsin L inhibitor, only affected cathepsin L activity, indicating that cathepsin B is probably involved in Cathepsin L activation, and this enzyme is probably responsible for the second YP processing. These results, together with previous studies, indicate that the GVBD process is independent of the occurrence of the second proteolytic process. It supports the hypothesis that the maturation process is under K+ ion flux control, while yolk proteolysis is related to the temporal and specific activation of cathepsins by acidification of yolk spheres.

Body mass index in children and adolescents according to age and pubertal stage.

OBJECTIVE: To evaluate the dependence of body mass index (BMI) values on pubertal stage in subjects similar in age. DESIGN, SUBJECTS AND MEASUREMENTS: Height and weight were recorded cross-sectionally in school subjects from three provinces in central Italy. The subjects were subdivided into three groups: (1) 4271 school subjects (2125 males and 2146 females; 8.5-15.5 y old), in whom the pubertal development was also recorded, were selected to subdivide BMI values according to pubertal stage and age; (2) 6345 females (10.5-14.5 y old), who were asked whether or not they had had their first menstrual period, were selected to subdivide BMI values according to age in pre-menarche and post-menarche girls, separately; and (3) 1919 females (10.5-14.5 y old), who had presented their menarche within the previous 6 months, were selected to subdivide short-term post-menarche BMI values according to age. RESULTS: The medians and interquartile ranges of BMI varied according to age and pubertal stage. Kruskall-Wallis test performed in subjects similar in age demonstrated that significant differences existed among the medians of BMI values of subjects at different pubertal stages in 12-14-y-old males (P<0.05), and in 11-14-y- old females (P<0.001). The difference also proved to be significant between stage I and stage II (P<0.05) in 10-y-old females, but not in 10-11-y-old males. The Kruskal-Wallis test performed in subjects similar in pubertal stage demonstrated that significant differences among the medians of BMI at different ages existed only in females at stages II and III. A significant positive trend was observed in both genders according to pubertal stage for BMI values of subjects similar in age (z-test for trend, P<0.01). On the contrary, a negative age trend proved to be significant in females at stages I (P<0.01), II (P<0.01) and III (P<0.001), but not in males when the subdivision of BMI was made according to age in subjects similar in pubertal stage. BMI values were significantly higher in post-menarche girls as compared to pre-menarche girls similar in age (P<0.001). However, at partial regression analysis BMI values were influenced by pubertal stage and, to a lesser extent, by age, but not by menarcheal status. An inverse association between short-term post-menarche BMI and age was observed, with the highest values in girls presenting menarche at 11 y of age (P<0.05). The negative trend was demonstrated at the z-test for trend (P<0.001). CONCLUSIONS: BMI values depend on pubertal degree of maturation, especially in girls. This influence should be taken into account when BMI is evaluated in adolescents. SPONSOR: University of Perugia, Region of Umbria.