骨髓干细胞移植对心肌梗死大鼠血流动力学指标和心脏功能的影响

目的:心肌梗死所致的细胞缺失和瘢痕形成是心力衰竭乃至死亡的病理基础,目前药物治疗、介入治疗和外科手术均不能替代坏死心肌和彻底改善心脏功能。观察骨髓干细胞移植对心肌梗死大鼠血流动力学指标和心功能的影响。方法:实验于2005-03/2006-10在哈尔滨医科大学附属第一医院细胞移植中心完成。①实验动物:SD雄性大鼠60只作为细胞移植的受体,随机数字表法分成假手术组、心肌梗死组、细胞移植组,20只/组。另取SD雄性幼鼠10只作为骨髓干细胞的供体。实验过程中对动物的处置符合动物伦理学标准。②实验方法:取幼鼠股骨骨髓,Percoll分离后收取细胞层,加入含体积分数为0.1的胎牛血清、100IU/mL青霉素、100g/mL链霉素的DMEM营养液,差速贴壁法分离骨髓干细胞,达80%～90%融合时采用胰酶 乙二胺四乙酸消化传代。向含有第3代骨髓干细胞的培养液中加入5-氮杂胞嘧啶核苷进行诱导,3周后行BrdU标记,离心后配制成1×1012L-1的细胞悬液用于移植。心肌梗死组、细胞移植组大鼠建立心肌梗死模型,假手术组未结扎冠状动脉。细胞移植组吸取0.2mL骨髓干细胞悬液注射到瘢痕组织中,心肌梗死组注入等量干细胞培养液基质,假手术组不予任何移植处理。③实验评估:术后4周,利用导管和心动超声技术检测各组大鼠左室舒张末期内压、左室收缩末期内压、左室压力最大变化值、左室压力最小变化值、等容时间常数和心率。结果:术后4周,与假手术组比较,心肌梗死组左室收缩末期内压、左室压力最大变化值、左室压力最小变化值均明显降低(P<0.01),左室舒张末期内压、等容时间常数均明显增高(P<0.01);与心肌梗死组比较,细胞移植组以上各项指标均明显好转(P<0.01)。结论:骨髓干细胞移植到瘢痕心肌组织中,能改善心肌梗死后大鼠的血流动力学参数和心脏功能。     

Factors affecting Yersinia enterocolitica (serotype O:8) viability in deliberately inoculated blood

Interpretation of in vitro experiments using Yersinia enterocolitica in blood components requires information on factors affecting the organism's survival. Several factors were found to influence the survival of Y. enterocolitica (serotype O:8) in blood components. A 20- minute room-temperature incubation with plasma-containing components resulted in approximately 2 log10 inactivation. Inactivation could be prevented by preincubation treatment of the plasma at 55 degrees C for 1 hour, which suggests the involvement of heat-labile plasma factors. No antibacterial activity was observed in washed red cells during the 20-minute room-temperature incubation. However, Y. enterocolitica colony-forming units declined by up to 2 log10 in washed red cells during the first days of 4 degrees C storage. Use of a white cell- reduction filter on freshly inoculated samples removed approximately 1 log10 of the organism regardless of whether bacteria were suspended in saline or washed red cells. Thus, bacterial levels may be affected by plasma, cellular components, and white cell-reduction filters. However, caution should be exercised in interpreting in vitro spiking studies designed to investigate the potential benefits of white cell reduction to eliminate the growth of Y. enterocolitica because of potential differences between naturally infected and experimentally inoculated blood.     

Memory B cells at successive stages of differentiation. Affinity maturation and the role of IgD receptors

The following evidence, mainly presented here, suggests that IgD receptors play a crucial role in determining the potential for affinity maturation in memory B cell populations. IgD receptors are present on the first memory B cells to appear after priming. These memory cells give rise to more-mature memory cells that have lost their IgD receptors. The proportions of early (IgD(+)) and mature (IgD(-)) memory cells found in individual donors vary with time, priming conditions, and the availability of T cell help, and both populations frequently coexist for long periods of time. IgD(+) and IgD(-) memory cells carry IgG receptors and give rise to IgG responses with identical isotype representation in adoptive recipients. IgD(+) memory cells, however, always give rise to predominantly low-affinity antibody responses, whereas IgD(-) memory cells consistently generate responses of substantially higher average affinity. This affinity differential is maintained between early and mature memory populations in the same donor and does not appear to be a result of selective differentiation of higher-affinity IgD(+) memory cells into the IgD(-) memory pool. Thus, the selective forces responsible for affinity maturation appear to operate mainly in mature memory cell populations that have already lost IgD receptors; or, stated conversely, little or no selection towards high-affinity memory appears to occur among memory cells that retain IgD receptors. In discussing these findings, we suggest that the IgD receptors themselves are responsible for maintaining early memory populations at a lower average affinity than IgD(-) populations in the same animal. The IgD receptors, we argue, serve to increase the antigen-binding capacity of lower-affinity memory cells so that these cells can survive, expand, and differentiate (to IgD(-)) at antigen concentrations that select against expansion of low- affinity memory cells no longer carrying IgD receptors. Thus, when antigen is limiting, IgD(-) memory populations will be selectively expanded to higher average affinities, whereas coexisting IgD(+) populations will retain their initial affinity profile. This hypothesis suggests that mechanisms that regulate expression and loss of IgD receptors are central to the adaptability of the immune system in its response to invading pathogens. Two related roles can be envisioned for the IgD receptors in this regard. First, they extend the lower boundary of the affinity range of early memory cell populations induced by a given antigenic stimulus and therefore broaden the diversity of responses obtainable from these populations. Secondly, they support the persistence of low-affinity memory populations under conditions where antigen becomes limiting and eventually disappears. These persisting populations then serve as a diversely reactive reservoir from which mature memory populations can be drawn with higher affinities either for the original antigen or, more importantly, for related antigens that the animal may subsequently encounter. Thus the existence of IgD receptors on early memory cells maintains the full range of response diversity despite ongoing selective expansion of (mature) memory populations to produce antibodies with high combining affinities for individual antigens. The flexibility inherent in such an organizational system, we believe, could be expected to account for the evolutionary development of IgD receptors and the regulatory capabilities that support operation of the system.     

Augmentation of spontaneous macrophage-mediated cytolysis by eosinophil peroxidase

Eosinophil peroxidase (EPO), a cationic protein purified from horse blood, adhered to four different types of tumor cells, markedly potentiating their lysis by preformed or enzymatically generated H(2)0(2) (up to 76-fold, as assayed in serum-containing tissue culture medium without supplemental halide). Similarly, compared with uncoated tumor cells, EPO-coated tumor cells were up to 32 times more sensitive to lysis when incubated with macrophages or granulocytes whose respiratory burst was triggered by PMA. However, EPO-coated tumor cells were also readily lysed by bacillus Calmette- Guerin-activated macrophages in the absence of exogenous triggering agents. This spontaneous cytolysis was rapid (50 percent at 2 h) and potent (50 percent lysis at macrophage/tumor cell ratios of 1.5 to 4.6), and was observed with both a peroxide-sensitive tumor (TLX9) and a peroxide-resistant tumor (NK lymphoma). Under the conditions used, neither EPO alone nor macrophages alone were spontaneously cytolytic. Neither EPO nor EPO-coated tumor cells triggered a detectable increment in H(2)0(2) release from macrophages. Nonetheless, spontaneous macrophage-mediated cytolysis of EPO- coated tumor cells was completely inhibitable by catalase (50 percent inhibition, 23 U/ml), although not by heated catalase, indicating a requirement for H(2)0(2). Cytolysis was also completely inhibitable by azide (50 percent inhibition, 2.6 X 10 -5 M), indicating a requirement for enzymatic activity of EPO. Thus, a cytophilic peroxidase from eosinophils and H(2)0(2) spontaneously released from activated macrophages interacted synergistically in a physiologic medium to destroy tumor cells.     

Can the reading for serologic reactivity following 37 degrees C incubation be omitted?

The need to detect antibodies that agglutinate and/or hemolyze red cells (RBCs) directly at 37 degrees C, but do not react in subsequently performed indirect antiglobulin tests (IATs), is of concern relative to the streamlining and automation of antibody detection methods. To determine incidence and significance of such reactions, data from 87,480 tests, which used low-ionic-strength saline, 10-minute incubation at 37 degrees C, and anti-IgG, were analyzed for unexpected antibodies. There were 3590 positive tests, of which 475 showed reactions at 37 degrees C but not in subsequently performed IATs (37 + IAT-). Of these, 196 reactions were due to autoantibodies or other factors usually considered insignificant with respect to the survival of transfused incompatible RBCs, 176 were due to alloantibodies of questionable clinical significance (M, Lea, P1, etc.), and 103 were associated with alloantibodies of potential clinical significance (63 E, 27 K, 5 Jka, 4 D, 3 cE, and 1 C). This latter reaction was seen in 72 patients, with two 37 + IAT-antibodies occurring in each of 3 patients. Of the 75 potentially significant 37 + IAT-antibodies, 57 were seen in patients recently exposed to homologous RBCs, 13 in patients with a history of transfusion and/or pregnancy, and 5 in patients with no known exposure to homologous RBCs. IAT reactivity was observed in subsequent samples with 27 of these antibodies. The predictive value of a 37 + IAT-test was 21.7 percent for a potentially significant antibody. The incidence was 0.12 percent of all tests for unexpected antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment of a system to standardize acceptability criteria for alanine aminotransferase activity in donated blood

A multilaboratory study was conducted to develop a system for standardizing alanine aminotransferase (ALT) acceptability criteria ("cutoffs") for donated blood. Without standardized cutoffs, each laboratory must develop its own cutoff, and this may not make optimal use of ALT testing to reduce transmission of non-A, non-B hepatitis (NANB). Defining an ALT acceptability criterion in absolute terms is necessary because relative cutoffs based on local donor populations may be affected by the prevalence of NANB in each community. This study involved 16 laboratories using 23 different analytic systems. The ALT results of the analysis of a plasma reference sample could be used to translate mathematically a single, absolute cutoff to units applicable to each analytic system. The distribution of ALT results in 1.4 million donations from across the country was established; basing the cutoff on this sample avoids the problems inherent in using a local donor base to establish a cutoff. We propose the implementation of a system to standardize ALT acceptability criteria to an activity level defined by analysis of a nationwide donor sample.     

Complete nucleotide sequence of a begomovirus and associated betasatellite infecting croton (Croton bonplandianus) in Pakistan

The complete sequences of a begomovirus and an associated betasatellite isolated from Croton bonplandianus originating from Pakistan were determined. The sequence of the begomovirus showed the highest level of nucleotide sequence identity (88.9%) to an isolate of papaya leaf curl virus and thus represents a new species, for which we propose the name Croton yellow vein virus (CYVV). The sequence of the betasatellite showed the highest levels of sequence identity (82 to 98.4%) to six sequences in the databases that have yet to be reported, followed by isolates of tomato leaf curl Joydebpur betasatellite (48.7 to 52.5%). This indicates that the betasatellite identified here (and the six sequences in the databases) is an isolate of a newly identified species for which the name Croton yellow vein mosaic betasatellite (CroYVMB) is proposed. For the begomovirus, an analysis of the sequence indicates that it has a recombinant origin.     

Identification of a major pathogenicity determinant and suppressors of RNA silencing encoded by a South Pacific isolate of <Emphasis Type="Italic">Banana bunchy top virus</Emphasis> originating from Pakistan

Five genes encoded by Banana bunchy top virus (BBTV) originating from Pakistan were expressed in Nicotiana benthamiana using a Potato virus X (PVX) vector. Expression of the master replication-associated protein (mRep) and movement protein (MP) resulted in necrotic cell death of inoculated tissues, as well as leaf curling and necrosis along the veins in newly emerging leaves. The systemic necrosis induced by the expression of MP was discolored (dark) in comparison to that induced by mRep. Expression of the cell-cycle link protein (Clink), the coat protein (CP), and the nuclear shuttle protein from the PVX vector induced somewhat milder symptoms, consisting of mild leaf curling and mosaic, although expression of the CP caused a necrotic response in inoculated leaf. The accumulation of viral RNA was enhanced by MP, Clink, and CP. Of the five BBTV-encoded gene products two, the MP and Clink, stabilized GFP-specific mRNA and reduced GFP-specific small interfering RNA in N. benthamiana line 16c when expressed under the control of the 35S promoter and co-inoculated with a construct for the expression of GFP hairpin RNA construct. These results identified MP and Clink as suppressors of RNA silencing. Taken together the ability of MP to induce severe symptoms in plants and suppress RNA silencing implicates this product as a major pathogenicity determinant of BBTV.     

βC1 encoded by tomato yellow leaf curl China betasatellite forms multimeric complexes in vitro and in vivo

The βC1 protein encoded by betasatellites associated with begomoviruses is multi-functional. To investigate its properties, the βC1 protein encoded by tomato yellow leaf curl China betasatellite (TYLCCNB) was expressed in Escherichia coli and analyzed for its ability to self-interaction. The βC1 protein formed large soluble multimeric complexes in vitro and in vivo. Mutations that prevented formation of multimeric complexes in vitro, also prevented formation of granular bodies in vivo, suggesting that granular bodies resulted from βC1 oligomerization. Similarly, βC1 mutants unable to form complexes also did not induce typical symptoms in plants when expressed from a Potato virus X (PVX) vector, suggesting that βC1 self-interaction was required for symptom induction in planta. Deletion analysis revealed that amino acid sequences spanning two predicted α-helices at the C-terminal end of the protein were important in multimerization.