Clonal Analysis of Agnogenic Myeloid Metaplasia

Agnogenic myeloid metaplasia (AMM) is a chronic myeloproliferative disorder that leads to a sustained proliferation of megakaryocytes and an increase of reticulin fibers within the bone marrow. Blood and bone marrow samples from patients with advanced AMM with fully developed myelofibrosis as well as cases in the cellular phase of the disease were investigated for clonality. Clonality was studied by X-linked restriction length polymorphism in conjunction with DNA methylation patterns. Granulocytes and total bone marrow cells proved to be monoclonal in origin whereas at least a minor portion of the peripheral lymphocytes were not clonally derived. Our findings indicate that the cellular phase of AMM as well as the fully developed disease progressed to myelofibrosis represent a monoclonal proliferation of pluripotent hematopoietic stem cells.     

Haemophilus aphrophilus bleb infection after a mitomycin trabeculectomy

Background: Haemophilus aphrophilus is a rare cause of ocular infection. It has been reported once as a cause of late-onset endophthalmitis in a patient with an inadvertent bleb after cataract surgery. We present a case of Haemophilus aphrophilus bleb infection after a mitomycin trabeculectomy.
Methods: A 56-year-old woman presented with a bleb infection 10 weeks after a mitomycin C augmented trabeculectomy at a University tertiary referral practice of one of the authors (GET). The causative organism was Haemophilus aphrophilus , identified by the Toronto Public Health Laboratory, Ontario, Canada.
Results: The bleb infection resolved following topical, subconjunctival and intravenous antibiotic therapy. A formal bleb revision was required to repair a persistent bleb leak.
Conclusion: Patients who have had trabeculectomies augmented with mitomycin C may be predisposed to bleb infection with unusual organisms. Prompt diagnosis and treatment is necessary to control the infection. Increased awareness and communication with laboratory personnel may increase the isolation of this fastidious organism.     

人亲环素A基因克隆及在大肠杆菌中表达

目的 :用基因重组技术表达人亲环素 A( Cy PA) ,以避免从人组织材料中提取纯化该蛋白的麻烦。　 方法 :应用反转录聚合酶链反应 ( RT- PCR)技术从人淋巴细胞系 ( MT4 )总 RNA中扩增得到 Cy PA基因片段 ,并用重组 DNA技术对该基因片段进行克隆 ,构建表达载体 ,转入大肠杆菌进行表达。　 结果 :DNA序列分析表明 ,得到的基因片段与设计编码 Cy PA的结构基因序列完全相同。所构建的表达载体 p ET11/Cy PA转入大肠杆菌获得表达 ,重组蛋白表达量占菌体可溶性蛋白的 4 1% ,经测定具有肽基脯氨酸顺 /反异构酶活性。　 结论 :利用基因重组技术使大肠杆菌高效表达出有生物活性的人 Cy PA。     

An empirical evaluation of the performance of antibody identification tasks

Four empirical studies were conducted for better understanding of the nature of problem-solving activities by medical technologists and medical technology students when performing antibody identification tasks. The results indicated the importance of strategies that ensure the collection of converging evidence, as these strategies protect against the fallibility of commonly used heuristics and against errors due to simple slips. The results also indicate that not only do students make significant numbers of errors, but so do practicing technologists. In one of the studies covering a 1-year period, for instance, a group of 16 technologists made a total of 41 errors in 1057 cases. On the basis of these findings, several alternatives are proposed to reduce errors.     

Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation

Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1-166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47- phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates assembly of the p47 and p67 oxidase components on the cytosolic face of the plasma membrane via cytoskeletal reorganization, whereas Rap1a functions downstream as the final activation switch involving direct physical interaction with the transmembrane flavocytochrome component of the NADPH oxidase.     

Adjuvant selenium supplementation in the form of sodium selenite in postoperative critically ill patients with severe sepsis

Introduction

Plasma selenium (Se) concentrations are reduced in critically ill surgical patients, and lower plasma Se concentrations are associated with worse outcomes. We investigated whether adjuvant Se supplementation in the form of sodium selenite could improve outcomes in surgical patients with sepsis.

Methods

In this retrospective study, all adult patients admitted to a 50-bed surgical ICU with severe sepsis between January 2004 and April 2010 were included and analysed according to whether they had received adjuvant Se supplementation, which was given at the discretion of the attending physician. When prescribed, Se was administered in the form of sodium selenite pentahydrate (Na₂SeO₃∙5H₂O), in which 100 μg of Se corresponds to 333 μg of sodium selenite. A bolus of sodium selenite corresponding to 1,000 μg of Se was injected intravenously through a central venous line for 30 minutes, followed by infusion of 1,000 μg/day for 24 hours for 14 days until ICU discharge or death. We performed logistic regression analysis to investigate the impact of adjuvant Se supplementation on hospital mortality.

Results

Adjuvant Se was administered to 413 (39.7%) of the 1,047 patients admitted with severe sepsis. Age and sex were similar between patients who received adjuvant Se and those who did not. Compared with patients who did not receive adjuvant Se supplementation, patients who did had higher scores on the Simplified Acute Physiology Score II, a greater prevalence of cancer upon admission to the ICU and were more commonly admitted after abdominal surgery. Compared with patients who did not receive adjuvant Se, patients who did had higher hospital mortality rates (46% versus 39.1%; P = 0.027), and longer median (interquartile range (IQR)) ICU stays (15 days (6 to 24) versus 11 days (4 to 24); P = 0.01) and hospital lengths of stay (33 days (21 to 52) versus 28 days (17 to 46); P = 0.001). In multivariable analysis, adjuvant Se supplementation was not independently associated with favourable outcome (odds ratio = 1.19, 95% confidence interval = 0.86 to 1.65; P = 0.288).

Conclusions

In this retrospective analysis of a large cohort of surgical ICU patients with severe sepsis, adjuvant Se supplementation in the form of sodium selenite had no impact on in-hospital death rates after adjustment for confounders.     

Chronic graft-versus-host disease in 52 patients: adverse natural course and successful treatment with combination immunosuppression

Fifty-two of 175 (30%) survivors of allogeneic marrow transplantation developed chronic graft-versus-hose diseases (GVHD). Five with limited chronic GVHD had an indolent clinical course with involvement of only the skin and liver. Forty-seven with extensive chronic GVHD had an unfavorable multiorgan disorder that resembled several autoimmune diseases. Thirteen patients with extensive disease (group I) were not treated and only 2 survive with Karnofsky scores >- 70%. Mortality resulted from infections and morbidity from sica syndrome, pulmonary and hepatic insufficiency, scleroderma-like skin disease, and contractures. Another 13 (group II) received a median of 8 mo prednisone and/or a brief course of antithymocyte globulin, and 3 survive without disability. The other 21 (group III) were treated with a combination of prednisone (1.0 mg/kg/q.o.d.) and either cyclophosphamide, procarbazine, or azathioprine (all 1.5 mg/kg/day) for a median of 13 mo. Combination therapy was well tolerated with only modest myelotoxicity. Fifteen in group III had a good and 4 a fair response to treatment while 2 with no response died. Azathioprine and prednisone was the most effective regimen. All therapy has been discontinued in 12 group III patients: GVHD returned in 5 (including 2 who died in spite of retreatment) while 7 remain free of GVHD for a median of 11 (range 6-30) mo observation. Only I group III survivor is disabled and 16 of the original 21 are alive 2-4 yr after transplant with Karnofsky scores of 70%-100%. Thus, combination immmunosuppression appears to favorably affect and, in some cases, premanently arrest the adverse natural course of extensive chronic GVHD.     

Protective, restorative, and therapeutic properties of recombinant colony-stimulating factors

Pretreatment of mice with recombinant murine (rM) colony-stimulating factor-granulocyte-macrophage (CSF-gm) or recombinant human (rH) CSF-g provides partial protection from the lethal effects of ionizing radiation or the alkylating agent cyclophosphamide (CTX). In addition, these agents can significantly prolong survival if administered following lethal doses of irradiation or CTX. To induce protective activity, cytokines were injected 20 hours before lethal irradiation or CTX administration. To accelerate recovery from lethal irradiation, the cytokines must be administered shortly following irradiation, and the induction of maximal levels of activity is dependent on chronic administration. In contrast, because of their longer half-lives, accelerated recovery from alkylating agents requires a delay of at least 24 to 48 hours to allow complete clearance of CTX before administration of a CSF. Studies quantitating peripheral blood leukocytes and bone marrow cellularity as well as colony-forming units per culture (CFU-C) frequency and CFU-C per femur revealed a significant correlation between these parameters and the ability to survive lethal irradiation. This is a US government work. There are no restrictions on its use.