Inactivation of factor XIa in human plasma assessed by measuring factor XIa-protease inhibitor complexes: major role for C1-inhibitor

From experiments with purified proteins, it has been concluded that factor XIa (FXIa) is inhibited in plasma mainly by alpha 1-antitrypsin (a1AT), followed by antithrombin III (ATIII), C1-inhibitor (C1Inh), and alpha 2-antiplasmin (a2AP). However, the validity of this concept has never been studied in plasma. We established the relative contribution of different inhibitors to the inactivation of FXIa in human plasma, using enzyme-linked immunosorbent assays (ELISAs) for the quantification of complexes of FXIa with a1AT, C1Inh, a2AP, and ATIII. We found that 47% of FXIa added to plasma formed complexes with C1Inh, 24.5% with a2AP, 23.5% with a1AT, and 5% with ATIII. The distribution of FXIa between these inhibitors in plasma was independent of whether FXIa was added to plasma, or was activated endogenously by kaolin, celite, or glass. However, in the presence of heparin (1 or 50 U/mL), C1Inh appeared to be the major inhibitor of FXIa, followed by ATIII. Furthermore, at lower temperatures, less FXIa-C1Inh and FXIa-a1AT complexes but more FXIa-a2AP complexes were formed. These data demonstrate that the contribution of the different inhibitors to inactivation of FXIa in plasma may vary, but C1Inh is the principal inhibitor under most conditions.     

The value of flow cytometric analysis of platelet glycoprotein expression of CD34+ cells measured under conditions that prevent P- selectin-mediated binding of platelets

In the present study, we show by adhesion assays and ultrastructural studies that platelets can bind to CD34+ cells from human blood and bone marrow and that this interaction interferes with the accurate detection of endogenously expressed platelet glycoproteins (GPs). The interaction between these cells was found to be reversible, dependent on divalent cations, and mediated by P-selectin. Enzymatic characterization showed the involvement of sialic acid residues, protein(s). The demonstration of mRNA for the P-selectin glycoprotein ligand 1 (PSGL-1) in the CD34+ cells by polymerase chain reaction (PCR) analysis suggests that this molecule is present in these cells. Under conditions that prevent platelet adhesion, a small but distinct subpopulation of CD34+ cells diffusely expressed the platelet GPIIb/IIIa complex. These cells were visualized by immunochemical studies. Furthermore, synthesis of mRNA for GPIIb and GPIIIa by CD34+ cells was shown using PCR analysis. The semiquantitative PCR results show relatively higher amounts of GPIIb mRNA than of PF4 mRNA in CD34+CD41+ cells in comparison with this ratio in platelets. This finding is a strong indication that the PCR results are not caused by contaminating adhering platelets. MoAbs against GPIa GPIb alpha, GPV, P- selectin, and the alpha-chain of the vitronectin receptor did not react with CD34+ cells. The number of CD34+ cells expressing GPIIb/IIIa present in peripheral blood stem cell (PBSC) transplants was determined and was correlated with platelet recovery after intensive chemotherapy in 27 patients. The number of CD34+CD41+ cells correlated significantly better with the time of platelet recovery after PBSC transplantation (r = .83, P = .04) than did the total number of CD34+ cells (r = .55). Statistical analysis produced a threshold value for rapid platelet recovery of 0.34 x 10(6) CD34+CD41+ cells/kg. This study suggests that if performed in the presence of EDTA the flow cytometric measurement of GPIIb/IIIa on CD34+ cells provides the most accurate indication of the platelet reconstitutive capacity of the PBSC transplant.     

Monoclonal antibody F1 binds to the kringle domain of factor XII and induces enhanced susceptibility for cleavage by kallikrein

In a previous study we have shown that monoclonal antibody F1 (MoAb F1), directed against an epitope on the heavy chain of factor XII distinct from the binding site for anionic surfaces, is able to activate factor XII in plasma (Nuijens JH, et al: J Biol Chem 264; 12941, 1989). Here, we studied in detail the mechanism underlying the activation of factor XII by MoAb F1 using purified proteins. Formation of factor XIIa was assessed by measuring its amidolytic activity towards the chromogenic substrate H-D-Pro-Phe-Arg-pNA (S-2302) in the presence of soybean trypsin inhibitor and by assessing cleavage on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Upon incubation with MoAb F1 alone, factor XII was auto-activated in a time-dependent fashion, activation being maximal after 30 hours. Factor XII incubated in the absence of MoAb F1 was hardly activated by kallikrein, whereas in the presence of MoAb F1, but not in that of a control MoAb, the rate of factor XII activation by kallikrein was promoted at least 60-fold. Maximal activation of factor XII with kallikrein in the presence of MoAb F1 was reached within 1 hour. This effect of kallikrein on the cleavage of factor XII bound to MoAb F1 was specific because the fibrinolytic enzymes plasmin, urokinase, and tissue-type plasminogen activator could not substitute for kallikrein. Also, trypsin could easily activate factor XII, but in contrast to kallikrein, this activation was independent of MoAb F1. SDS-PAGE analysis showed that the appearance of amidolytic activity correlated well with cleavage of factor XII. MoAb F1-induced activation of factor XII in this purified system was not dependent on the presence of high- molecular-weight kininogen (HK), in contrast to the activation of the contact system in plasma by MoAb F1. Experiments with deletion mutants revealed that the epitopic region for MoAb F1 on factor XII is located on the kringle domain. Thus, this study shows that binding of ligands to the kringle domain, which does not contribute to the proposed binding site for negatively charged surfaces, may induce activation of factor XII. Therefore, these findings point to the existence of multiple mechanisms of activation of factor XII.     

Frequent ongoing T-cell receptor rearrangements in childhood B- precursor acute lymphoblastic leukemia: implications for monitoring minimal residual disease

Crosslineage T-cell receptor delta (TCR delta) rearrangements are widely used as tumor markers for the follow up of minimal residual disease in childhood B-precursor acute lymphoblastic leukemia (ALL) by polymerase chain reaction (PCR). The major drawback of this approach is the risk of false-negative results due to clonal evolution. We investigated the stability of V delta 2D delta 3 rearrangements in a group of 56 childhood B-precursor ALL patients by PCR and Southern blot analysis. At the PCR level, V delta 2D delta 3-to-J alpha rearranged subclones (one pathway for secondary TCR delta recombination) were demonstrated in 85.2% of V delta 2D delta 3-positive patients tested, which showed that small subclones are present in the large majority of patients despite apparently monoclonal TCR delta Southern blot patterns. Sequence analysis of V delta 2D delta 3J alpha rearrangements showed a biased J alpha gene usage, with HAPO5 and J alpha F in 26 of 32 and 6 of 32 clones, respectively. Comparison of V delta 2D delta 3 rearrangement status between diagnosis and first relapse showed differences in seven of eight patients studied. In contrast, from first relapse onward, no clonal changes were observed in six patients studied. To investigate the occurrence of crosslineage TCR delta rearrangements in normal B and T cells, fluorescence-activated cell sorter-sorted peripheral blood CD19+/CD3- and CD19-/CD3+ cell populations from three healthy donors were analyzed. V delta 2D delta 3 rearrangements were detected at low frequencies in both B and T cells, which suggests that V delta 2-to-D delta 3 joining also occurs during normal B-cell differentiation. A model for crosslineage TCR delta rearrangements in B-precursor ALL is deduced that explains the observed clonal changes between diagnosis and relapse and is compatible with multistep leukemogenesis of B-precursor ALL.     

Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation

We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady- state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages.     

Monoclonal antibodies to factor VIII: their application in immunoblotting for the visualization of factor VIII in therapeutic concentrates and plasma

Two monoclonal antibodies (McAb) termed 12A4 and 19C1 have been raised against human factor VIII. In immunoassays 12A4 bound to factor VIII antigen (VIII:Ag) in plasma but not serum whilst 19C1 bound to VIII:Ag in both plasma and serum. Both McAb were shown by immunoblotting to react with the carboxy (C) terminal polypeptide of factor VIII which appeared as a doublet with a molecular weight (Mr) of 77,000/75,000. The C terminal factor VIII polypeptide was detectable by immunoblotting in each of 12 therapeutic factor VIII concentrates, from six different manufacturers, although its level was variable. Factor VIII was visualized in plasma by immunoblotting following its immunoadsorption and elution from agarose-bound monoclonal antibodies. No Mr 77,000/75,000 bands were detectable in plasma obtained from 13 unrelated CRM- haemophiliacs whilst 11 CRM+ haemophilic plasmas from seven kindred were shown to have a C terminal factor VIII polypeptide of normal molecular size.     

Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease

Huntington's disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAG/polyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2/HSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently transfecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43-74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2/HSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2/HSDJ in protein folding.     

DNA microsatellite instability and mismatch repair protein loss in adenomas presenting in hereditary non-polyposis colorectal cancer

BACKGROUND AND AIM: Hereditary non-polyposis colorectal cancer (HNPCC), as its name implies, is associated with few adenomas, and the early evolution of colorectal neoplasia is poorly understood. In this study our aim was to clarify the genetic profiles of benign polyps in subjects with HNPCC using a combined molecular and immunohistochemical approach. METHODS: Thirty adenomas and 17 hyperplastic polyps were obtained from 24 affected HNPCC subjects. DNA was extracted from paraffin embedded tissue by microdissection and analysed for the presence of microsatellite instability (MSI) and mutations in five genes known to be targets in mismatch repair deficiency (TGFbetaRII, IGF2R, BAX, hMSH3, and hMSH6). Serial sections were stained by immunohistochemistry for hMLH1 and hMSH2. RESULTS: Twenty four (80%) of 30 adenomas showed MSI. Of MSI positive adenomas, 66.7% showed MSI at more than 40% of markers (high level of MSI (MSI-H)). Two of 17 hyperplastic polyps revealed MSI at one marker (low level of MSI (MSI-L)). A significant association was found between MSI-H and high grade dysplasia in adenomas (p=0.004). Eight of nine adenomas with mutations of coding sequences revealed high grade dysplasia and all nine were MSI-H. Four of the nine ranged in size from 2 to 5 mm. The presence of the hMSH6 mutation was significantly correlated with high levels of MSI (80% of markers) (p<0.02). Twenty four adenomas gave evaluable results with immunohistochemistry. One of six (17%) microsatellite stable, six of seven (86%) MSI-L, and 11 of 11 (100%) MSI-H adenomas showed loss of either hMLH1 or hMSH2. CONCLUSIONS: Most adenomas in subjects with a definite diagnosis of HNPCC show MSI (80%). The finding of MSI-L is usually associated with loss of expression of hMLH1 or hMSH2, unlike the situation in MSI-L sporadic colorectal cancer. The transition from MSI-L to MSI-H correlated with the finding of high grade dysplasia and mutation of coding sequences and may be driven by mutation of secondary mutators such as hMSH3 and hMSH6. Advanced genetic changes may be present in adenomas of minute size.     

Metformin therapy in patients with chronic kidney disease

Metformin therapy is limited in patients with chronic kidney disease (CKD) due to the potential risk of lactic acidosis. This open‐label observational study investigated metformin and lactate concentrations in patients with CKD (n = 22; creatinine clearances 15–40 ml/min) and in two dialysed patients. Patients were prescribed a range of metformin doses (250–2000 mg daily) and metformin concentrations were compared with data from healthy subjects (scaled to 1500 mg twice daily). A subset of patients (n = 7) was controlled on low doses of metformin (250 or 500 mg daily). No correlation between metformin and lactate concentrations was observed. Three patients had high lactate concentrations (>2.7 mmol/l) and two had high metformin concentrations (3–5 mg/l), but none had any symptoms of lactic acidosis. Reducing metformin dosage and monitoring metformin concentrations will allow the safe use of metformin in CKD, provided that renal function is stable.