Eculizumab and thrombotic microangiopathy after hematopoietic stem cell transplantation: A report on its efficacy and safety in two pediatric patients.

BACKGROUND: Thrombotic microangiopathy (TMA) is a severe complication after hematopoietic stem cell transplantation (HSCT), with the reported mortality rate in such cases usually reaching 90%. CASES: We report on two pediatric cases of patients successfully treated by eculizumab for severe HSCT-TMA, occurring in two girls (8.4 and 3.6 years). The first patient developed TMA with hematologic abnormalities and renal/pulmonary lesions after allogeneic HSCT for Philadelphia-positive acute lymphoblastic leukemia; she received eculizumab 7 months after HSCT, with a dramatic improvement of renal function. The second patient developed severe TMA (cardiac tamponade, renal failure requiring dialysis, gastritis) after autologous HSCT for metastatic neuroblastoma. She received eculizumab for 7 months, with a dramatic improvement of renal function. No side effects were observed. CONCLUSION: The use of eculizumab as first-line therapy in pediatric patients with severe HSCT-TMA with multisystemic lesions appears promising. Larger international studies are required to confirm its benefit and safety for this specific indication.

Identification of a promoter sequence from the BETL1 gene cluster able to confer transfer-cell-specific expression in transgenic maize

The maize (Zea mays L.) betl1 locus, encoding a basal endosperm transfer layer-specific protein, has been mapped and molecularly cloned in its entirety. The locus is shown to consist of three gene copies in the maize inbred line A69Y. To distinguish the three transcription units from the locus name, we have termed them BETL1a, BETL1b, and BETL1c. Two of the copies are expressed, whereas one is inactive and contains retrotransposon-like insertions in both promoter and intron regions. Based on this information, and a restriction site map covering 17 kb around the BETL1 locus, a DNA fragment putatively containing an active promoter sequence was identified. This fragment was tested for its ability to confer transfer-cell-specific expression in transient and stably transformed maize tissues. The transgenic maize plants obtained showed the predicted cell-type specificity of expression restricted to the basal endosperm transfer cells, although there were minor deviations in promoter strength and timing and accumulation of the transgene product from the corresponding BETL-1 endogene expression pattern.

Isolation of a gene encoding a glycosylated cytokinin oxidase from maize.

The major cytokinin oxidase in immature maize kernels was purified to homogeneity. Selected tryptic peptides were used to design degenerate oligonucleotide primers for PCR isolation of a fragment of the oxidase gene. Hybridization of the PCR fragment to a maize genomic library allowed isolation of a full-length cytokinin oxidase gene (ckx1). The gene encodes a protein of approximately 57 kDa that possesses a signal peptide, eight consensus N-glycosylation sequences and a consensus FAD binding sequence. Expression of ckx1 in Pichia caused secretion of active glycosylated cytokinin oxidase that contains a substrate-reducible FAD. The gene displays sequence homology with a putative oxidoreductase from Arabidopsis thaliana and with the fas5 gene from Rhodococcus fascians.

Disruption of Maize Kernel Growth and Development by Heat Stress (Role of Cytokinin/Abscisic Acid Balance).

Temperature stress during kernel development affects maize (Zea mays L.) grain growth and yield stability. Maize kernels (hybrid A619 x W64A) were cultured in vitro at 3 d after pollination and either maintained at 25[deg]C or transferred to 35[deg]C for 4 or 8 d, then returned to 25[deg]C until physiological maturity. Kernel fresh and dry matter accumulation was severely disrupted by the long-term heat stress (8 d at 35[deg]C) and did not recover when transferred back to 25[deg]C, resulting in abortion of 97% of the kernels. Kernels exposed to 35[deg]C for 4 d (short-term heat stress) exhibited a recovery in kernel growth and water content at about 18 d after pollination and kernel abortion was reduced to about 23%. During the cell division phase, abscisic acid (ABA) levels showed a steady decline in the control but maintained a moderate level in the heat-stressed kernels. However, later in development heat-stressed kernels had significantly higher levels of ABA than the control. Cytokinin analysis confirmed a peak in zeatin riboside and zeatin levels in control kernels at 10 to 12d after pollination. In contrast, kernels subjected to 4 d of heat stress had no detectable levels of zeatin and the zeatin riboside peak was reduced by 70% and delayed until 18 d after pollination. The long-term heat-stressed kernels showed low to nondetectable levels of either zeatin riboside or zeatin. Regression analysis of ABA level against cytokinin level during the endosperm cell division phase revealed a highly significant negative correlation in nonstressed kernels but no correlation in kernels exposed to short-term or long-term heat stress. Application of benzyladenine to heat-stressed, growth-chamber-grown plants increased thermotolerance in part by reducing kernel abortion at the tip and middle positions on the ear. These results confirm that shift in hormone balance of kernels is one mechanism by which heat stress disrupts maize kernel development. The maintenance of high levels of cytokinins in the kernels during heat stress appears to be important in increasing thermotolerance and providing yield stability of maize.

Regulation of sucrose phosphate synthase by gibberellins in soybean and spinach plants.

Exogenous applications of gibberellins (GAs) increased the extractable activity of leaf sucrose phosphate synthase (SPS) in soybean (Glycine max [L.]) and spinach (Spinacia oleracea [L.]). The response to GA applications was detectable within 2 h postapplication and was still observed 6 h, 24 h, and 7 d after treatment. When paclobutrazol, a GA biosynthesis inhibitor, was applied to intact soybean and spinach plants, decreased extractable SPS activity resulted within 24 h following the treatment. Different methods of GA application (spray, injection, capillary wick, and excised leaf systems) produced similar effects on SPS activity of soybean leaves. Protein synthesis in soybean leaves appeared to be necessary for GA-promoted SPS activity because gibberellic acid only partially reversed the inhibitory effect of pretreatment with cycloheximide. Levels of SPS protein from crude extracts of spinach plants were measured by a dot blot technique using monoclonal antibodies against SPS. Application of gibberellic acid to spinach leaves increased levels of SPS protein 2 h, 24 h, and 7 d after treatment. The results suggest that, in both soybean and spinach, GA is one of the endogenous hormonal factors that regulate the steady-state level of SPS protein and, hence, its activity.

Regulation of key enzymes of sucrose biosynthesis in soybean leaves : effect of dark and light conditions and role of gibberellins and abscisic Acid.

An important part in the understanding of the regulation of carbon partitioning within the leaf is to investigate the endogenous variations of parameters related to carbon metabolism. This study of diurnal changes in the activities of sucrose-synthesizing enzymes and levels of nonstructural carbohydrates in intact leaves of field-grown soybean plants (Glycine max [L.]) showed pronounced diurnal fluctuations in sucrose phosphate synthase (SPS) activity. However, there was no distinct diurnal change in the activity of fructose-1,6-bisphosphatase (F1,6BPase). SPS activity in leaves from plants grown in controlled environments presented two peaks during the light period. In contrast to field-grown plants, F1,6BPase activity in leaves from growth chamber-grown plants manifested one peak during the first half of the light period. In plants grown under both conditions, sucrose and starch accumulation rates were highest during early hours of the light period. By the end of the dark period, most of the starch was depleted. A pattern of diurnal fluctuations of abscisic acid (ABA) levels in leaves was also observed under all growing conditions. Either imposition of water stress or exogenous applications of ABA inhibited F1,6BPase activity. However, SPS-extractable activity increased following water deficit but did not change in response to ABA treatment. Gibberellin application to intact soybean leaves increased levels of both starch and sucrose. Both gibberellic acid (10(-6)m) and gibberellins 4 and 7 (10(-5)m) increased the activity of SPS but had an inconsistent effect on F1,6BPase. Correlation studies between the activities of SPS and F1,6BPase suggest that these two enzymes are coordinated in their function, but the factors that regulate them may be distinct because they respond differently to certain environmental and physiological changes.