Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow

The characterization of many cytokines involved in the control of hematopoiesis has led to intense investigation into their potential use in ex vivo culture to expand progenitor numbers. We have established the optimum ex vivo culture conditions that allow substantial amplification of transient engrafting murine stem cells and which, simultaneously, augment the ability to sustain serial bone marrow transplantation (BMT). Short-term incubation of unfractionated BM cells in liquid culture with stem cell factor (SCF) and interleukin-11 (IL- 11) produced a 50-fold amplification of clonogenic multipotential progenitors (CFU-A). Following such ex vivo expansion, substantially fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, BM cells expanded ex vivo resulted in significantly more rapid hematopoietic recovery. In a serial transplantation model, unmanipulated BM was only able to consistently sustain secondary BMT recipients, but BM expanded ex vivo has sustained quaternary BMT recipients that remain alive and well more than 140 days after 4th degree BMT. These results show augmentation of both short-term recovery posttransplant and the ability to serially transplant marrow by preincubation in culture with SCF and IL-11.     

Clonal evolution in B-lineage acute lymphoblastic leukemia by contemporaneous VH-VH gene replacements and VH-DJH gene rearrangements

B-cell acute lymphoblastic leukemia (B-ALL), more frequently than any other B-lineage neoplasm, exhibits oligoclonal Ig heavy chain (IgH) gene rearrangement in 15% to 43% of all cases studied. To study the molecular processes that promote multiple IgH rearrangements, a comprehensive sequence analysis of a B-ALL case was performed in which seven clonal IgH gene rearrangements were identified. The genetic profiles suggested that a single leukemic progenitor clone evolved into several subclones through dual processes of variable (VH) to preexisting diversity-joining (DJH) gene segment rearrangement and VH to VH gene replacement. Predominant IgH-V usage and the uniquely rearranged clonotype-specific VHDJH region gene sequences were identified using a novel DNA-based gene amplification strategy. Polymerase chain reaction (PCR) was directed by an IgH-J generic primer and a complement of family-specific IgH-V primers that defined the major B-cell IgH-V gene usage. Clonality of rearranged VHDJH bands was substantiated by high resolution denaturant gel electrophoretic analysis. Sequence patterns of the amplified VHDJH fragments segregated into two groups defined by common DJH sequences. Partial N region homology at the VHD junction as well as shared DJH sequences firmly established VH to VHDJH gene replacement as a mechanism generating clonal evolution in one group. In the second subset, oligoclonality was propagated by independent VH gene rearrangements to a common DJH precursor. The contributions of all clonal Ig-VHDJH repertoires for each group was approximately 50% and reflected a symmetric distribution of leukemic subclones generated by either process. Thus, oligoclonal rearrangements evolved by two independent, yet seemingly contemporaneous molecular genetic mechanisms. All seven clones displayed nonfunctional Ig-VHDJH recombinations. These observations may have relevance to the recombinatorial opportunities available during normal B-cell maturation.     

Gamma (immune) interferon production by leukocytes from a patient with a TG cell proliferative disease

We report a patient with a disease characterized by proliferation of T cells with Fc receptors for IgG (TG). However, unlike lymphoid cells from normal individuals or from patients with other lymphoid malignancies, the patient's lymphocytes spontaneously produced gamma interferon (IFN-gamma) in vitro. The peripheral lymphocytes consisted of 95% TG cells, which exhibited the morphological characteristics of T- cell chronic lymphocytic leukemia (CLL) and were normal on cytochemical and chromosome analysis. The majority of TG cells were OKT3+, OKT8+, and OKT4-, 3A1-. These cells failed to express suppressor cell activity and displayed depressed levels of natural killer activity, but mediated antibody-dependent cell-mediated cytotoxicity. The spontaneous production of IFN-gamma by human peripheral lymphoid cells as demonstrated in this study may serve as a probe for studying the relationship between IFN-gamma and the proliferation of human T-cell subsets.     

Thiamin-responsive maple-syrup-urine disease: decreased affinity of the mutant branched-chain alpha-keto acid dehydrogenase for alpha-ketoisovalerate and thiamin pyrophosphate.

The biochemical basis for the therapeutic effects of thiamin in thiamin-responsive maple-syrup-urine disease (MSUD) was investigated in intact and disrupted fibroblast cultures from normals and patients with various forms of MSUD. Decarboxylation of alpha-keto[1-14C]isovalerate (KIV) by intact cells from a thiamin-responsive MSUD patient was at 30-40% of the normal rate with or without thiamin in the incubation medium. Under similar conditions, intact classical MSUD fibroblasts failed to decarboxylate KIV. Branched-chain alpha-keto acid (BCKA) dehydrogenase activity measured in disrupted cells from the thiamin-responsive subject showed sigmoidal kinetics in the absence of thiamin pyrophosphate (TPP), with an increased concentration of substrate needed for half-maximal velocity (K0.5 for KIV = 7 mM vs. 0.05 mM in normal cells). When assayed with 0.2 mM TPP present, the mutant enzyme showed (i) a shift in kinetics to near Michaelis-Menten type as observed with the normal BCKA dehydrogenase and (ii) a lower K0.5 value of 4 mM for KIV, suggesting a TPP-mediated increase in the mutant enzyme's affinity for substrate. By contrast, TPP increased only the Vmax and was without effect on the apparent Km for KIV of the BCKA dehydrogenase from cells of normals and patients with classical MSUD and variant thiamin-responsive MSUD (grade 3). Measurement of the apparent Km for TPP of the BCKA dehydrogenase from thiamin-responsive mutant MSUd cells showed a 16-fold increase in the constant to 25 microM compared to enzymes from normal or classical MSUD cells. These findings demonstrate that the primary defect in the thiamin-responsive MSUD patient is a reduced affinity of the mutant BCKA dehydrogenase for TPP that results in impaired oxidative decarboxylation of BCKA.     

布鲁氏菌抗体胶体金免疫层析法快速检测技术的建立

目的建立一种快速检测布鲁氏菌抗体的新方法。方法利用胶体金免疫层析技术,以大肠埃希菌表达、纯化的OMP31与BP26重组蛋白作为检测抗原,以金黄葡萄球菌A蛋白(SPA)作为胶体金标记物,制备胶体金免疫层析试纸条,用于检测布鲁氏菌抗体,并分析方法的敏感性和特异性。结果以粒径为40nm胶体金制备的试纸条检测布鲁氏菌抗体的敏感性最高,胶体金最佳标记pH为6.2,SPA蛋白最适标记量为6μg/ml。交叉试验证明试纸条不与其他非相关疾病感染血清反应,特异性高。试纸条检测结果与琥红平板试验方法的符合率为92%。结论制备的布鲁氏菌抗体胶体金免疫层析试纸条具有敏感、特异、简便、快速的特点,可用于鉴别布鲁氏菌自然感染和人工免疫,并可区分牛、羊种布鲁氏菌感染,可在基层推广使用。     

药物洗脱球囊在冠状动脉特殊病变介入治疗中的应用分析

目的分析药物洗脱球囊（DEB）在再狭窄病变、支架内闭塞病变、分叉病变及小血管病变中的应用情况。方法根据冠状动脉造影结果选择不同型号的DEB,严格按照DEB使用要求进行操作。结果 DEB在支架内再狭窄病变中应用26例（27处病变）,使用28个DEB,其中左主干（LM）1个、左前降支（LAD）12个、右冠状动脉（RCA）12个、回旋支（LCX）1个、钝缘支（OM）2个,出现1例冠状动脉夹层,给予裸金属支架置入;DEB在分叉病变中应用27例（28处病变）,使用28个DEB,其中LM至LCX开口6个、LM至LAD开口1个、LAD与第一对角支（D1）开口17个、LCX至OM 2个、RCA至左心室后支（PL）2个;DEB在小血管病变中应用13例（13处病变）,使用13个DEB,其中LCX 6个、LAD 3个、D1 2个、OM 1个、PL 1个;DEB在支架内闭塞病变中应用10例（10处病变）,使用12个DEB,其中LAD 8个、LCX 2个、OM 1个、中间支1个。术中及术后未见并发症发生,随访至今未发生主要不良心血管事件（MACE）。结论 DEB在再狭窄病变、支架内闭塞病变、分叉病变及小血管病变介入治疗过程中是安全的。     

Regional Variation of Climatic Influences on West Nile Virus Outbreaks in the United States

The national resurgence of human West Nile virus (WNV) disease in 2012 raised questions about the factors responsible for WNV outbreaks. Interannual climatic variations may influence WNV amplification and transmission to humans through multiple pathways, including mosquito breeding habitats, gonotrophic cycles, extrinsic incubation, avian communities, and human behavior. We examined the influences of temperature and precipitation anomalies on interannual variation in human WNV cases in three regions of the United States. There were consistent positive influences of winter temperatures, weaker and more variable positive effects of spring and summer temperatures, and highly variable precipitation effects that ranged from positive to negative. The overwintering period may be a particularly important climatic constraint on the dynamics of WNV in cold-temperate regions of North America. Geographic differences in the seasonal timing and relative importance of climatic drivers of WNV risk likely reflect underlying variability in key ecological and social characteristics.     

Interleukin-2 induction of lymphokine-activated killer (LAK) activity in the peripheral blood and bone marrow of acute leukemia patients: II. Feasibility of LAK generation in children with active disease and in remission

Activation and expansion in culture with rIL-2 of peripheral blood (PB) and/or bone marrow (BM) specimens derived from children with ALL and ANLL, with active disease (AP) and in remission were studied (RP). Baseline NK cytolytic activity from AP was found to be depressed, whereas RP-derived cells had normal NK activity, as assayed against K562 targets. Culture in rIL-2 significantly enhanced the NK activity of both AP- and RP-derived cells and generated LAK activity, as assayed by 4-hour 51Cr release, against NK-resistant Raji cell line and against fresh, allogeneic, and autologous tumor cells. Lytic activity against fresh, cryopreserved leukemia blasts was of lower than that found against cell lines. In three patients higher lytic activity against autologous than against allogeneic blasts was demonstrated. Expansion in culture with rIL-2 varied from twofold to 120-fold. rIL-2 activation and expansion was better in RP than in AP. The predominant phenotype of activated cells, as determined by flow cytometry, was [mean % (SD)]: CD3- = 54 (12), CD8+ = 55 (17), and NKH1+ = 26 (7). The consistently high level of CD8+ cells was accompanied by very low levels of CD4+ cells: mean = 11% (14). Double-marker analysis showed mean of 33% (10) for CD3+/NKH1+ cells and mean = 32 (11) for CD8+/NKH1+ cells, implying that these populations were overlapping. Kinetics of expression of cell surface markers during 2 to 3 weeks in culture showed that CD8+ and NKH1+ enrichment occurred during the first week and lasted for up to 4 weeks, whereas CD4+ expression decreased after the second week. A significant decrease in the expression of IL-2 receptors (CD25) was observed from the second week of culture. This study shows the feasibility of in vitro generation of killer cells from PB and BM of pediatric leukemia patients.     

Effect of various surface protections on the margin microleakage of resin-modified glass ionomer cements

STATEMENT OF PROBLEM: Because conventional glass ionomer cements are moisture sensitive, a surface coating is recommended during the initial setting stage. It is unknown whether resin-modified glass ionomer cements also need surface protection. PURPOSE: This study investigated the effect of various surface protections on microleakage with Class V resin-modified glass ionomer restorations. MATERIAL AND METHODS: Forty extracted molars with buccal and lingual Class V cavity preparations were restored with a resin-modified glass ionomer (Fuji II LC). The occlusal margin of each restoration was on enamel and the cervical margin on dentin. After immediate finishing and polishing, the teeth were divided into 4 groups according to the following surface protection treatments: group I, unprotected; group II, Fuji varnish; group III, resin adhesive; and group IV, acid etching and resin adhesive. After these procedures, all teeth were stored in isotonic saline for 24 hours, thermocycled 1500 times at 5 degrees C to 60 degrees C, and soaked in dye solution for 24 hours. The teeth then were longitudinally sectioned and observed under a stereomicroscope. The degree of dye penetration was recorded and analyzed with the Kruskal-Wallis and Mann-Whitney tests (P<.05) RESULTS: None of the 4 groups demonstrated complete margin sealing at either the occlusal or cervical margins. Groups II and III displayed the least microleakage at cervical margins; a significant difference existed between groups I and III (P=.034). Compared with the other 3 groups, group IV showed significantly greater microleakage at the cervical margins. CONCLUSION: Although resin-modified glass ionomers can be finished immediately, they remain moisture sensitive. Within the limitations of this study, the results suggest that resin adhesive should be used as a surface protection to reduce margin microleakage of resin-modified glass ionomer restorations.