The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action

Blood coagulation is initiated when plasma factor VII(a) binds to its essential cofactor tissue factor (TF) and proteolytically activates factors X and IX. Progressive inhibition of TF activity occurs upon its addition to plasma. This process is reversible and requires the presence of VII(a), catalytically active Xa, Ca2+, and another component that appears to be associated with the lipoproteins in plasma, a lipoprotein-associated coagulation inhibitor (LACI). A protein, LACI(HG2), possessing the same inhibitory properties as LACI, has recently been isolated from the conditioned media of cultured human liver cells (HepG2). Rabbit antisera raised against a synthetic peptide based on the N-terminal sequence of LACI(HG2) and purified IgG from a rabbit immunized with intact LACI(HG2) inhibit the LACI activity in human serum. In a reaction mixture containing VIIa, Xa, Ca2+, and purified LACI(HG2), the apparent half-life (t1/2) for TF activity was 20 seconds. The presence of heparin accelerated the initial rate of inhibition threefold. Antithrombin III alpha alone had no effect, but antithrombin III alpha with heparin abrogated the TF inhibition. LACI(HG2) also inhibited Xa with an apparent t1/2 of 50 seconds. Heparin enhanced the rate of Xa inhibition 2.5-fold, whereas phospholipids and Ca2+ slowed the reaction 2.5-fold. Xa inhibition was demonstrable with both chromogenic substrate (S-2222) and bioassays, but no complex between Xa and LACI(HG2) could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nondenaturing PAGE, however, showed that LACI(HG2) bound to Xa but not to X or Xa inactivated by diisopropyl fluorophosphate. Thus, LACI(HG2) appears to bind to Xa at or near its active site. Bovine factor Xa lacking its gamma-carboxyglutamic acid-containing domain, BXa(-GD), through treatment with alpha-chymotrypsin, was used to further investigate the Xa requirement for VIIa/TF inhibition by LACI(HG2). LACI(HG2) bound to BXa(-GD) and inhibited its catalytic activity against a small molecular substrate (Spectrozyme Xa), though at a rate approximately sevenfold slower than native BXa. Preincubation of LACI(HG2) with saturating concentrations of BXa(-GD) markedly retarded the subsequent inhibition of BXa. The VII(a)/TF complex was not inhibited by LACI(HG2) in the presence of BXa(-GD), and further, preincubation of LACI(HG2) with BXa(-GD) slowed the inhibition of VIIa/TF after the addition of native Xa. The results are consistent with the hypothesis that inhibition of VII(a)/TF involves the formation of a VIIa-TF-XA-LACI complex that requires the GD of XA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of clonal rearrangements of the Ig heavy chain locus in acute leukemia

Clonal rearrangements of the Ig heavy chain (IGH) locus occur in nearly all cases of B-cell precursor acute leukemia (BCP-ALL). Some of these rearrangements may be detected by polymerase chain reaction (PCR) using VH gene framework III (FRIII) and JH consensus primers. However, about 20% of BCP-ALLs fail to amplify with this technique. To determine the causes of these PCR failures and to investigate any possible association with specific subgroups of disease, we analyzed 72 acute leukemias of defined immunophenotype and cytogenetics, comparing FRIII with VH-family leader-specific PCR methods and Southern blotting. Of 37 BCP-ALL cases, 6 (16.2%) failed totally to amplify with FRIII and JH primers. None of these cases amplified with VH leader primers. Additionally, all cases retained germline VH6 genes and 5 of 11 rearranged alleles amplified with a consensus DH primer, indicating that these rearrangements represented biallelic DH-JH recombinations. Among the 6 FRIII and VH leader PCR-negative BCP-ALL cases, there was no common immunophenotype or consistent cytogenetic abnormality, although all showed structural chromosomal abnormalities and 3 of 5 successfully karyotyped had abnormalities of chromosome 12p. 13 cases with t(9;22)(q34;q11) Philadelphia chromosome-positive [Ph+]) and IGH rearrangements (9 BCP-ALL and 4 biphenotypic cases) were also analyzed. Of 23 rearranged IGH alleles, 19 (82%) were positive by FRIII PCR, and all 4 remaining alleles were amplified by VH leader primers. Use of the leader primers in these Ph+ cases also detected 3 additional clonal rearrangements that were not anticipated from Southern blotting; such unexpected bands were not observed in 21 other Ph- cases. The additional bands represented "new" and unrelated VH rearrangements rather than VH-VH replacement events. We conclude that biallelic DHJH rearrangements occur in a subgroup of BCP-ALL; in these cases, the activation of the full VHDHJH recombination mechanism had not occurred. Therefore, these cases of BCP-ALL were arrested at an early stage of B- cell differentiation. In contrast, all Ph+ BCP-ALLs and biphenotypic acute leukemias, which may represent the transformation of multipotent hemopoietic stem cells, had undergone VHDHJH recombination. Of 9 Ph+ BCP-ALL cases, 3 also showed ongoing VHDHJH rearrangement, reflecting the persistent expression of the VHDHJH recombinase. Finally, sequence analysis of 33 rearranged VHDHJH genes showed that only 3 including 2 Ph+ BCP-ALL maintained an intact open-reading frame. Loss of the open- reading frame occurred not only because of out-of-frame VHDH and DHJH joining, but also because of VH gene mutation and deletion. These data show that most BCP-ALLs may represent the neoplastic transformation of BCPs destined to die in the bone marrow.     

Commentary: revision lumbar surgery and revisiting the role of preoperative depression screening

COMMENTARY ON: Adogwa O, Parker SL, Shau DN, et?al. Preoperative Zung Depression Scale predicts outcome after revision lumbar surgery for adjacent segment disease, recurrent stenosis, and pseudarthrosis. Spine J 2012;12:179-85 (in this issue).     

Taking refuge from modernity: 21st century hermits

Idiopathic environmental intolerances, such as ‘multiple chemical sensitivity’ and ‘electrosensitivity,’ can drastically affect the quality of life of those affected. A proportion of severely affected patients remove themselves from modern society, to live in isolation away from the purported causal agent of their ill health. This is not a new phenomenon; reports of hermits extend back to the 3^rd century AD. We conducted a literature review of case reports relating to ancient hermits and modern day reclusion resulting from idiopathic environmental intolerance, in order to explore whether there are similarities between these two groups and whether the symptoms of these ‘illnesses of modernity’ are simply a present-day way of reaching the end-point of reclusion. Whilst there were some differences between the cases, recurring themes in ancient and modern cases included: dissatisfaction with society, a compulsion to flee, reports of a constant struggle and a feeling of fighting against the establishment. The similarities which exist between the modern-day cases and the historical hermits may provide some insight into the extreme behaviours exhibited by this population. The desire to retreat from society in order to escape from harm has existed for many centuries, but in different guises.     

自动BACTEC960系统检测结核分枝杆菌药物敏感性的多中心评估分析

地点：巴西东南部3个分枝杆菌参比实验室。目的：评估自动化分枝杆菌生长指示管（MGIT）检测结核分枝杆菌药物敏感性的能力。设计：用自动BECTECMGIT960（M960）对95份结核分枝杆菌临床分离株进行链霉素（SM），异烟肼（INH）、利福平（RMP）、乙胺丁醇（EMB）的敏感性检测，并与BACTEC460（B460）、比例法（PM）、抗性比率法（RRM）的检测结果进行比较，其中88株以BACTEC460、比例法、抗性比率法中的至少两种方法获得一致结果作为最终结果，并与M960的结果相比较。结果：M960与B460的符合率为95．2％，与PM为96．6%,与RRM为93．4％；其中与RRM在SM上符合率最低，与B460在EMB上符合率最低。M960结果与最终结果的符合率为97．9％，INH与RMP的符合率为99．2%,SM与EMB的符合率分别为96．2%和96．9％，M960报告结果的平均时间为6．9d。结论：与比例法和抗性比率法相比，M960显示出了极大的优势，并且有助于患者的治疗。     

A mutation of the common receptor subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and IL-5 that leads to ligand independence and tumorigenicity

The cytokines interleukin-3, interleukin-5, and granulocyte-macrophage colony-stimulating factor bind with high affinity to a receptor complex that contains a ligand-specific alpha-chain and a common beta-chain, h beta c. We report here the isolation of a mutant form of h beta c, from growth factor-independent cells, that arose spontaneously after infection of a murine factor-dependent hematopoietic cell line (FDC-P1) with a retroviral h beta c expression construct. Analysis of this h beta c mutation shows that a small (37 amino acid) duplication of extracellular sequence that includes two conserved sequence motifs is sufficient to confer ligand-independent growth on these cells and lead to tumourigenicity. Because this is a conserved region in the cytokine receptor superfamily, our results suggest that the large family of cytokine receptors has the capacity to become oncogenically active.