Retinoic acid-resistant HL-60 cells exclusively contain mutant retinoic acid receptor-alpha

Sequence analysis of the retinoic acid receptor-alpha (RAR alpha) gene from a subline of HL-60 cells (RA-res) stably resistant to all-trans retinoic acid (RA) disclosed a single-base change in codon number 411, the same C to T transition previously reported in an independently selected HL-60 RA resistant clone by Robertson et al (Blood 80:1885, 1992). This mutation eliminates a FokI restriction endonuclease site. Using primers framing this mutation in exon 9 of the RAR alpha gene, we showed that polymerase chain reaction products amplified from either mRNA or genomic DNA templates from the RA-res subline were completely resistant to FokI digestion whereas those from wild-type (wt) HL-60 cells could be digested to completion. The lack of a normal allele in the RA-res cells was confirmed by mixing experiments and hybridization analyses. Southern blot analysis of DNA from the RA-res and wt cells versus control placental DNA indicated that the RAR alpha gene is not haploid. The independent isolation of the same RAR alpha mutation in different laboratories suggests either that the mutation exits in a small subpopulation in the wt line or that this is a mutational "hot spot." Furthermore, the results indicate that if a dominant negative mode of resistance is involved in the RA-res subline, this must involve interference with the function of heterologous receptor proteins such as the retinoid X receptors. The lack of any normal RAR alpha in this subline may facilitate studies of the mode of action of retinoids.     

Molecular evidence for a single clonal origin in biphenotypic concomitant chronic lymphocytic leukemia and multiple myeloma

To establish the clonal origin of a case of concomitant B-cell chronic lymphocytic leukemia (IgM kappa) and multiple myeloma (IGA lambda), we analyzed the immunoglobulin (Ig) gene rearrangements in the patient's blood and bone marrow. Despite the different isotypes, pretreatment investigation of the heavy chain gene (JH) revealed a germline fragment and two identical rearrangements in the blood and marrow. Both kappa and lambda light-chain genes were rearranged in the blood, suggesting peripheral blood lymphocyte involvement in the myeloma. Analysis of the Ig genes after chemotherapy demonstrated no change in the JH or CK rearrangements; however, the lambda genes were now in a germline configuration. Our results suggest that in this patient both chronic lymphocytic leukemia and myeloma originated from the same B-cell progenitor.     

Plastic-adherent progenitor cells in mobilized peripheral blood progenitor cell collections

The use of peripheral blood progenitor cells (PBPC) to reconstitute hematopoiesis after high-dose chemoradiotherapy is now commonplace in the treatment of malignancies. Attempts to characterize these cells have concentrated primarily on their phenotype and their content of clonogenic colony-forming cells (CFC). We have used a plastic-adherent delta (P delta) assay system to evaluate the quantity and quality of more primitive cells in addition to the conventional measurements of CFC and CD34-positive cells. The leukapheresis products from 20 patients mobilized using cyclophosphamide (Cy) and granulocyte colony- stimulating factor (G-CSF) were examined for progenitor cell content. The mean number of mononuclear cells (MNC), colony-forming units- granulocyte/macrophage (CFU-GM), and CD34-positive cells from two leukaphereses per patients were 7.9 x 10(8)/kg, 47.3 x 10(4)/kg, and 10.5 x 10(6)/kg, respectively. The mean number of P delta progenitors was 9.3 x 10(4)/kg. Limiting dilution analyses showed the frequency of P delta progenitors in PBPC to be between 1 and 5.3 per 10(5) MNC and that each P delta progenitor has the proliferative capability to generate an overall mean of 4.5 CFU-GM. Of the 20 patients, 16 underwent autografting with PBPC alone. Fifteen patients engrafted neutrophils and platelets within 16 days. One patient had delayed engraftment associated with inadequate etoposide clearance. Statistical analysis showed a strong correlation between numbers of CFU-GM and CD34 positivity. The numbers of plastic-adherent P delta progenitor cells did not correlate with CFU-GM or CD34-positive cells. We conclude that the plastic-adherent P delta progenitor cell assay is capable of measuring primitive hematopoietic cells and that it may be useful for the investigation of primitive progenitors in PBPC harvests.     

Stable multilineage hematopoietic chimerism in alpha-thalassemic mice induced by a bone marrow subpopulation that excludes the majority of day-12 spleen colony-forming units

We have investigated the contribution of highly purified day-12 spleen colony-forming units (CFU-S-12) as well as more primitive cells to sustained blood cell production using in vivo and in vitro assays that allow frequency analysis. Normal or day-6 post-5-fluorouracil light- density bone marrow (BM) was sorted on the basis of differences in rhodamine-123 (Rh123) retention or wheat germ agglutinin (WGA) affinity and tested in vivo using a recently developed alpha-thalassemic chimeric mouse model. In addition, short-term and long-term clonal activity was assessed in vitro using a limiting dilution-type long-term BM culture, the cobblestone area forming cell assay. When sublethally irradiated alpha-thalassemic mice were transplanted with as many as 281 purified WGAbright CFU-S-12, derived from a fraction containing 95% of all CFU-S-12 from day-6 post-5-fluorouracil light-density BM of wild- type mice, detectable chimerism was not observed at 6 months posttransplantation. In contrast, only three CFU-S-12 were included in the Rh123dull and WGAdim subpopulations that induced 29% to 58% and 21% to 31% stable multilineage donor-type chimerism of erythrocytes and leukocytes, respectively. The Rh123dull and WGAdim cells were up to 240- fold enriched for long-term repopulating ability (LTRA) as compared with unseparated BM. A comparable level of chimerism was found in the different hematopoietic organs and at the level of BM CFU-S-12. The frequency of the LTRA unit capable of inducing a 10% sustained level of donor-type erythrocytes was calculated to be 1 to 2 per 10(5) BM cells. Several reports have suggested that LTRA and spleen colony formation could be capacities of the same stem cell subset. However, the present results show that the majority of CFU-S-12 have only short-term repopulating ability and are physically separable from more primitive stem cells with long-term multilineage reconstituting capacities.     

Blood cell dynamics in P-selectin-deficient mice

P-selectin is expressed on the surfaces of activated platelets and endothelium where it mediates binding to leukocytes. P-selectin- deficient mice were shown to exhibit peripheral neutrophilia (Mayadas et al: Cell 74:541, 1993). We now show that this is not caused by changes in bone marrow precursors nor by a lack of neutrophil margination. Both P-selectin-positive and -negative animals displayed similar increases in peripheral blood neutrophil numbers after injection of epinephrine. However, clearance of 51Chromium-labeled neutrophils is delayed in mice deficient for P-selectin, indicating that the neutrophilia is at least in part the result of delayed removal. We detected no obvious alterations in lymphocyte differentiation, distribution, or adhesion to high endothelial venules in peripheral lymph nodes. Through intravital microscopy, we examined the impact of P-selectin deficiency on leukocyte/endothelial interaction beyond the initial stages of inflammation. Four hours after the administration of an inflammatory irritant, leukocyte rolling was observed even in the absence of P-selectin. There were significantly fewer rolling cells relative to wild-type mice, and their velocity was reduced. Moreover, in the peritonitis model, the number of peritoneal macrophages in wild-type mice increased threefold at 48 hours, whereas the macrophages in the mutant mice remained near baseline levels. Thus, whereas P-selectin is known to be involved in early stages of an inflammatory response, our results indicate that it is additionally responsible for leukocyte rolling and macrophage recruitment in more prolonged tissue injury.     

Recycling with nicotine patches in smoking cessation

The aim was to evaluate if recycling of failures from a smoking cessation study may be of value. The study comprised 126 smokers (50%) of 252 failures, from a double-blind smoking cessation trial with nicotine patch, who accepted recycling after 1 year. Subjects were allocated nicotine patches delivering 15, 20 or 25 mg of nicotine (over 16 hours) according to their base-line saliva cotinine concentrations in an open trial. The treatment period was 12 weeks followed by tapering over 6 weeks. The percentage of quitters after 3, 12, 26, and 52 weeks was 44, 20, 7 and 6%, respectively. After 26 weeks, all subjects had relapsed in the group previously treated with active nicotine patch compared with 12% abstainers in the previous placebo subjects. The sustained abstinence rate without slips after one year was 2%. Recycling does not seem to be of long-term clinical relevance in our set-up for subjects initially treated with nicotine, but of some value in subjects quitting without nicotine therapy initially.     

Immunologic characterization of canine factor VIII

Canine factor VIII (FVIII) preparations isolated from cryoprecipitates by gel chromatography were pooled to provide one batch of antigen for simultaneous immunization of two rabbits and a goat. The goat and rabbit antisera had similar FVIII-neutralizing titers, but the latter had seven to ten times more precipitating titer for FVIII-related antigen (FVIII-RA). Absorption with material low in FVIII had little effect on the precipitating titer of the rabbit antibody, but it abolished the precipitating capacity of the goat antibody and caused a 20% reduction in the neutralizing titer of both antisera. Results obtained in the Laurell assay with the two different antisera were similar. This finding was true whether the FVIII-RA levels were reduced, normal, or elevated, as well as for heat-treated and frozen- thawed plasmas. Both antisera were neutralized by the same canine plasma to a similar extent. Analysis of FVIII concentrates by crossed immunoelectrophoresis suggested that canine FVIII-RA was heterogeneous, with slow-and fast-migrating components. The presence of more than one antigenic site on the FVIII complex was also supported by the disparity between the FVIII-neutralizing and -precipitating titers of goat antiserum and by the demonstration that FVIII-RA, FVIII-neutralizing antigen, and procoagulant activity varied independently.     

呼吸衰竭患者入急诊抢救室时机与疾病预后的相关性研究

目的：探讨呼吸衰竭患者进入急诊抢救室的时机与患者预后的相关性。方法：选取我院2011年6月至2014年3月收治的97例呼吸衰竭患者,根据入院时间分为观察组（58例）和对照组（39例）,观察组患者为法定工作时间进入急诊室抢救的患者,对照组为非法定工作时间进入抢救室的患者,比较两组患者的入院生理指标及抢救成功率差异;并根据入院时的急性生理与慢性健康评分（A-PACHEⅡ）进行分及患者转归进行分层分析。结果：观察组和对照组患者的年龄、性别、血PH值、动脉血氧分压（PaO2）、动脉血二氧化碳分压（PaCO2）、血液中乳酸含量、APACHEⅡ评分、D－二聚体、纤维蛋白原（ Fib）、死亡率差异均不具有统计学意义（ P＞0．05）。97例患者存活77例,抢救无效死亡20例,存活组和死亡组的血PH值、PaO2、PaCO2、血液中乳酸含量、APACHEⅡ评分、D－二聚体、Fib差异均具有显著性,差异具有统计学意义（ P＜0．05）。 APACHEⅡ评分＜20分的观察组和对照组患者的死亡率差异不显著（P＞0．05）;APACHEⅡ评分≥20分的对照组患者死亡率46．67％高于对观察组的14．81％,差异具有统计学意义（ P＜0．05）。结论：APACHEⅡ评分＜20分的呼吸衰竭患者入急救室时机对患者的转归无影响,但APACHEⅡ评分≥20分的患者,入院时机会对患者结局产生影响,应该采取相应措施降低因入院时机造成的患者不良结局。     

Randomized controlled comparative study on effect of training to improve lower limb motor paralysis in convalescent patients with post-stroke hemiplegia

[Purpose] The motor paralysis-improving effect on the hemiplegic lower limb was compared among mirror therapy, integrated volitional-control electrical stimulation, therapeutic electrical stimulation, repetitive facilitative exercises, and the standard training method in post-stroke hemiplegia patients. [Subjects and Methods] Eighty one stroke patients admitted to a convalescent rehabilitation ward were randomly allocated to the above 5 treatment groups. Each patient performed functional training of the paralytic lower limb for 20 minutes a day for 4 weeks, and changes in the lower limb function were investigated using the Stroke Impairment Assessment Set. [Results] The hip and knee joint functions did not significantly improve in the standard training control group, but significant improvements were observed after 4 weeks in the other intervention groups. Significant improvement was noted in the ankle joint function in all groups. [Conclusion] Although the results were influenced by spontaneous recovery and the standard training in the control group, the hip and knee joints were more markedly improved by the interventions in the other 4 groups of patients with moderate paralysis, compared to the control group.Key words: Stroke, Motor paralysis, Intervention