Improving the efficiency of PBPC collection by pre-apheresis peripheral blood and mid-apheresis product measurements of CD34 cells

BACKGROUND: The adequacy of HPC collection for BMT is typically assessed by the number of CD34 cells. However, during a series of leukapheresis procedures (LP) the CD34 value on the final HPC product may not be available for testing until late evening, sometimes resulting in additional, retrospectively unnecessary, LP in order to ensure an adequate HPC collection (>5x10(6) CD34/kg). We hypothesized that an estimate of the CD34 content of HPC products prior to 16:00 h on the day of LP would permit improved HPC collection planning. We therefore assessed the effectiveness of predicting the total amount of CD34 cells that would be collected in a given LP by either (a) the concentration of CD34 cells/microL in peripheral blood prior to LP (pre-CD34) or (b) the predicted total amount of CD34 cells to be collected based on sampling the LP product at the mid-point of each LP. We also compared the number of LP per patient and total HPC collected for the study group with data from the previous calendar year. METHODS: Allogeneic and autologous BMT donors who completed a 20-L HPC collection between September 2002 and February 2003 were eligible. CD34 cells were measured on blood drawn prior to LP and from the HPC product at the mid-point (10 L) of LP. The CD34 content of the final LP was predicted by doubling the value of total CD34 cells at the mid-run (MRp-CD34). The MRp-CD34/kg and the cumulative CD34/kg collected were made available before 16:00 h and used to determine the need for additional LP. The true CD34 content of each HPC collection was also measured from the final product the next day (CD34-FP). RESULTS: A 20-L LP was completed and data were available from 31 patients and nine allogeneic donors who underwent a total of 85 LP for diagnoses, including 11 myeloma, 10 lymphoma, seven HD, three acute leukemia and five others. The mean (range) and correlation (R2) vs. the CD34-FP were, for pre-CD34, 54 CD34/microL (0.3-232), R2=0.66 (P<0.01), and for MRp-CD34, 3.2x10(6) CD34/kg (0.04-22.48), R2=0.90 (P<0.01). The mean number of CD34/kg collected per LP in the patients/donors was 3.4x10(6) CD34/kg (0.05-18.94). The median number of CD34 cells employed for transplant in the study group vs. controls (5.7 vs. 5.6x10(6)/kg) and the time to engraftment of neutrophils (12 vs. 11 days) and platelets (12 vs. 12 days) was similar to historical controls. However, the study group had a significantly lower median number of LP (three vs. two; P<0.02) to obtain the required collection of 5x10(6) CD34 cells/kg. DISCUSSION: Both the pre-CD34 and the MRp-CD34 were significantly correlated with CD34-FP. However, the CD34-FP was more reliably predicted by MRp-CD34. Early availability of mid-run CD34 values was associated with a significant reduction in the number of LP required to collect 5x10(6) CD34 cells/kg, without reduction in the number of CD34 cells for transplant or prolongation of days to neutrophil or platelet engraftment.     

Neurological Involvement in Primary Sj?gren Syndrome: A Focus on Central Nervous System

Objectives

Sjögren syndrome is an autoimmune disease involving mainly salivary and lacrimal glands. Beyond widely described PNS involvement, high variable prevalence of CNS manifestations ranging from 2.5 and 60% of all pSS patients has been reported, without specific syndrome definition. The aim of this cohort study was to evaluate the prevalence of CNS signs and symptoms in pSS patients and to identify possible biomarkers of CNS damage.

Methods

120 patients with pSS diagnosis according to the 2002 American-European Consensus Group criteria were enrolled after exclusion of secondary causes. All patients underwent to a wide neurological, neuropsychological, psychiatric, neuroradiological and ultrasonographic evaluation.

Results

Central and peripheral nervous system involvement was observed in 81 patients with a prevalence of 67.5%. The prevalence of CNS involvement was significantly higher than PNS disease (p 0.001). 68 patients (84%) shown non-focal CNS symptoms and 64 (79%) focal CNS deficits with headache as the most common feature (46.9%), followed by cognitive (44.4%) and mood disorders (38.3%). Particularly, we observed a high prevalence of migraine without aura, subcortical frontal executive functions and verbal memory impairment and apathy/alexythimia. MR spectroscopy revealed a reduction of NAA levels or NAA/Cr ratio decrease in subcortical frontal and basal ganglia white matter, while ultrasonography showed an impairment of microvasculature response. At multivariate analysis, headache, cognitive disorders and psychiatric symptoms was significantly associated to serological markers (anti-SSA), MRS and ultrasonographic features.

Conclusions

The higher prevalence of MWO-mimic headache, cognitive dys-esecutive syndrome and mood disorders observed in this series confirmed previous evidences of a higher diffused CNS compromission rather than focal involvement such as SM-like clinical course or NMO-like syndrome. The association with immunological biomarkers, metabolic cerebral dysfunction and microvascular damage suggests a possible endothelial dysfunction of the cerebral microcirculation or a potential inflammation-mediated shift of the neurovascular coupling.     

Linkage disequilibrium and sequence diversity in a 500-kbp region around the adh1 locus in elite maize germplasm

Linkage disequilibrium (LD) at the adh1 locus was examined in two sets of maize inbreds. A set of 32 was chosen to represent most of the genetic diversity in the cultivated North American elite maize breeding pool. A second set of 192 inbreds was chosen to sample more deeply the two major heterotic groups in elite maize germplasm. Analysis of several loci in the vicinity of the adh1 gene shows that LD as measured by D and r² extends greater than 500 kbp in this germplasm. The presence of this exceptionally long segment of high LD may be suggestive of selection acting on one of the genes in the vicinity of adh1 or of a locally reduced rate of recombination.Electronic Supplementary Material Supplementary material is available for this article at .     

Analysis of autoreactive CD4 T cells in experimental autoimmune encephalomyelitis after primary and secondary challenge using MHC class II tetramers

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is primarily mediated by CD4 T cells specific for Ags in the CNS. Using MHC class II tetramers, we assessed expansion and phenotypic differentiation of polyclonal self-reactive CD4 T cells during EAE after primary and secondary challenge with the specific Ag. After EAE induction in SJL mice with proteolipid protein 139-151, CNS-specific T cells up-regulated activation markers and expanded in the draining lymph nodes and in the spleen. Less than 20% of total autoreactive T cells entered the CNS simultaneously with Th cells of other specificities. Almost all tetramer-positive cells in the CNS were activated and phenotypically distinct from the large peripheral pool. When EAE was induced in Ag-experienced mice, disease symptoms developed earlier and persisted longer; autoreactive T cells were more rapidly activated and invaded the CNS earlier. In striking contrast to specific CTLs that respond after secondary viral challenge, the absolute numbers of autoreactive CD4 T cells were not increased, indicating that the accelerated autoreactivity in Ag-experienced mice is not related to higher frequencies of autoreactive CD4 T cells.     

A transposable element insertion within ZmGE2 gene is associated with increase in embryo to endosperm ratio in maize

Most of the maize kernel oil is located in the embryo while the majority of starch is located in the endosperm. Maize kernel composition and value are affected significantly by the ratio of the embryo size to the endosperm size; however, the genetic regulation of embryo to endosperm ratio (EER) in maize is unknown. Here we identified ZmGE2 gene, which encodes a cytochrome p450 protein, as a gene associated with EER variation in maize. We first expressed rice Giant Embryo (GE) gene driven by oleosin promoter in maize and detected a 23.2?% reduction in EER in transgenic seeds, demonstrating the existence of evolutionarily conserved mechanisms for EER determination in rice and maize. We next identified maize GE2, a homolog of rice GE sharing 70?% identity in amino sequence, as a candidate based on the similar expression pattern and co-localization with a previously detected QTL for EER. Followed by linkage and association mapping, a 247-bp transposable element (TE) insertion in 3′-untranslated region of ZmGE2 gene was identified to be associated with increase in EER and kernel oil content. Expression level of the favorable ZmGE2 allele containing the 247-bp TE insertion was strongly reduced. In addition, the 247-bp TE insertion site was a selection target during the artificial long-term selection for the high EER trait in a high oil population. This is the first report that demonstrates an association of ZmGE2 with EER variation in maize and identifies ZmGE2 gene as a promising target for manipulation of EER and grain composition by either transgenic approach or molecular breeding in maize.     

Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes

Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet beta-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 microM) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action.     

Active anthocyanin degradation in <Emphasis Type="Italic">Brunfelsia calycina</Emphasis> (yesterday–today–tomorrow) flowers

Anthocyanins are the largest group of plant pigments responsible for colors ranging from red to violet and blue. The biosynthesis of anthocyanins, as part of the larger phenylpropanoid pathway, has been characterized in great detail. In contrast to the detailed molecular knowledge available on anthocyanin synthesis, very little is known about the stability and catabolism of anthocyanins in plants. In this study we present a preliminary characterization of active in planta degradation of anthocyanins, requiring novel mRNA and protein synthesis, in Brunfelsia calycina flowers. Brunfelsia is a unique system for this study, since the decrease in pigment concentration in its flowers (from dark purple to white) is extreme and rapid, and occurs at a specific and well-defined stage of flower development. Treatment of detached flowers with protein and mRNA synthesis inhibitors, at specific stages of flower development, prevented degradation. In addition, treatment of detached flowers with cytokinins delayed senescence without changing the rate of anthocyanin degradation, suggesting that degradation of anthocyanins is not part of the general senescence process of the flowers but rather a distinctive and specific pathway. Based on studies on anthocyanin degradation in wine and juices, peroxidases are reasonable candidates for the in vivo degradation. A significant increase in peroxidase activity was shown to correlate in time with the rate of anthocyanin degradation. An additional indication that oxidative enzymes are involved in the process is the fact that treatment of flowers with reducing agents, such as DTT and glutathione, caused inhibition of degradation. This study represents the first step in the elucidation of the molecular mechanism behind in vivo anthocyanin degradation in plants.     

Relationship between radiation induced adaptive response in human fibroblasts and changes in chromatin conformation

Chromatin conformation changes in the normal human fibroblasts VH-10 were studied by the method of anomalous viscosity time dependence (AVTD). Gamma-irradiation of cells in a dose range of 0.1–3 Gy caused an increase in maximal viscosity of cell lysates. Conversely, irradiation of cells with low doses of 0.5 or 2 cGy resulted in a decrease in the AVTD peaks with a maximum effect approximately 40 min after irradiation. The same exposure conditions were used to study a possible adaptive effect of low doses, measured by changes in cell survival. A primary dose of 2 cGy caused significant modification of cell response to a challenge dose. Approximately 20% protection to challenge doses of 0.5 Gy (p < 0.003), 2 Gy (p < 0.02) and 2.5 Gy (p < 0.002) was observed. However, the direction of this effect (adaptation or synergism) was found to be dependent on a challenge dose. The combined effect of 2 cGy and 1 Gy was significantly synergistic, while no modification was observed for 1.5 Gy and 3 Gy. A partial correlation was found between the AVTD changes and cell survival when the combined effect of a primary dose of 2 cGy and challenge dose was examined. The dose of 2 cGy alone increased survival by 16% (p < 0.0003). These results suggest that the low-dose induced effects on survival may be related to chromatin reorganization.     

RNA approaches the B‐form in stacked single strand dinucleotide contexts

Duplex RNA adopts an A‐form structure, while duplex DNA interconverts between the A‐ and B‐forms depending on the environment. The C2′‐endo sugar pucker seen in B‐form DNA can occur infrequently in ribose sugars as well, but RNA is not understood to assume B‐form conformations. Through analysis of over 45,000 stacked single strand dinucleotide (SSD) crystal structure conformations, this study demonstrates that RNA is capable of adopting a wide conformational range between the canonical A‐ and B‐forms at the localized SSD level, including many B‐form‐like conformations. It does so through C2′‐endo ribose conformations in one or both nucleotides, and B‐form‐like neighboring base stacking patterns. As chemical reactions on nucleic acids involve localized changes in chemical bonds, the understanding of how enzymes distinguish between DNA and RNA nucleotides is altered by the energetic accessibility of these rare B‐form‐like RNA SSD conformations. The existence of these conformations also has direct implications in parametrization of molecular mechanics energy functions used extensively to model nucleic acid behavior., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 65–82, 2016     

Plant N-glycan profiling of minute amounts of material

Development of convenient strategies for identification of plant N-glycan profiles has been driven by the emergence of plants as an expression system for therapeutic proteins. In this article, we reinvestigated qualitative and quantitative aspects of plant N-glycan profiling. The extraction of plant proteins through a phenol/ammonium acetate procedure followed by deglycosylation with peptide N-glycosidase A (PNGase A) and coupling to 2-aminobenzamide provides an oligosaccharide preparation containing reduced amounts of contaminants from plant cell wall polysaccharides. Such a preparation was also suitable for accurate qualitative and quantitative evaluation of the N-glycan content by mass spectrometry. Combining these approaches allows the profiling to be carried out from as low as 500 mg of fresh leaf material. We also demonstrated that collision-induced dissociation (CID) mass spectrometry in negative mode of N-glycans harboring α(1,3)- or α(1,6)-fucose residue on the proximal GlcNAc leads to specific fragmentation patterns, thereby allowing the discrimination of plant N-glycans from those arising from mammalian contamination.