In vitro selection of RNA aptamers against a conserved region of the <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> erythrocyte membrane protein 1

The var-gene encoding Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is known to play a major role in the pathogenicity of the P. falciparum parasite. The protein enables the parasite to adhere to the endothelial linings of small blood vessels (cytoadherence) as well as to non-infected erythrocytes (rosetting), thus preventing clearance from the bloodstream. The development and spread of resistance towards most anti-malarial drugs used for treatment and prevention of the most severe form of malaria truly emphasise the importance of a continuous research and development of new drugs. In this study we use Systematic Evolution of Ligands by EXponential enrichment (SELEX) methodology to isolate high-affinity ligands (aptamers). To validate the results from the SELEX in vitro selection, different aptamers have been selected against PfEMP1 in a live cell assay of P. falciparum strain FCR3S1.2, a highly rosetting strain. We have been able to show the rosette disrupting capacity of these SELEX-aptamers at concentrations of 33 nM and with 100% disruption at 387 nM. The described results show that RNA aptamers are promising candidates for adjunct therapy in severe malaria.     

Validity of two recently-proposed prognostic grading indices for lung, gastro-intestinal, breast and renal cell cancer patients with radiosurgically-treated brain metastases

We tested the validity of two prognostic indices for stereotactic radiosurgically (SRS)-treated patients with brain metastases (BMs) from five major original cancer categories. The two indices are Diagnosis-Specific Graded Prognostic Assessment (DS-GPA) and our Modified Recursive Partitioning Analysis (RPA). Forty-six hundred and eight BM patients underwent gamma knife SRS during the 1998–2011 period. Primary cancer categories were non-small cell lung cancer (NSCLC, 2827 patients), small cell lung cancer (SCLC, 460), gastro-intestinal cancer (GIC, 582), breast cancer (BC, 547) and renal cell cancer (RCC, 192). There were statistically significant survival differences among patients stratified into four groups based on the DS-GPA systems (p < 0.001) in all five original cancer categories. In the NSCLC category, there were statistically significant mean survival time (MST) differences (p < 0.001) among the four groups without overlapping of 95 % confidence intervals (CIs) between any two pairs of groups with the DS-GPA system. However, among the SCLC, GIC, BC and RCC categories, MST differences between some pairs of groups failed to reach statistical significance with this system. There were, however, statistically significant MST differences (p < 0.001) among the three groups without overlapping of 95 % CIs between any two pairs of groups with the Modified RPA system in all five categories. The DS-GPA system is applicable to our set of patients with NSCLC only. However, the Modified RPA system was shown to be applicable to patients with five primary cancer categories. This index should be considered when designing future clinical trials involving BM patients.     

Depletion of white cells from platelet concentrates with a new adsorption filter

Removal of white cells (WBCs) from platelets may reduce alloimmunization to WBC antigens, prevent febrile reactions, and improve platelet increments in multiply transfused patients receiving HLA-matched platelets. A new surface-modified fibrous polyester filter was evaluated; it requires no special processing of pooled platelet concentrates and can be used at the patient's bedside. The studies were designed to measure WBC removal, platelet function, in vitro platelet recovery, and in vivo platelet survival. WBC mean removal was 99.8 percent +/- 0.56 (n = 37) when a pool similar in volume to 6 platelet concentrates was tested. The mean number of residual WBCs after filtration was 5.6 x 10(5). In vitro mean platelet recovery was 86.9 percent for a pool size of 6 units (n = 37). Clot retraction and platelet aggregation were unaffected by filtration. Survival studies of 111Indium-labeled platelets done with filtered autologous platelets showed no reduction in the normally expected survival. These studies indicated that the filter efficiently removes WBCs without substantially decreasing platelet number, survival, or function. This device offers the potential of considerably improving platelet transfusion therapy.     

Th activation of maternal and cord blood

Th activation of red cells is characterized by agglutination with the peanut lectin from Arachis hypogaea and is diminished by treatment with proteolytic enzymes. The first cases of Th activation were associated with bacterial infections. More recently, a high incidence of Th activation in congenital hypoplastic anemia has been reported, along with the finding that 13.5 percent of cord bloods are Th activated. The incidence of Th reactivity in newborn infants was confirmed by studying 200 paired samples of maternal and cord blood. Twenty-two (11%) of the cord samples and 13 (6.5%) of the maternal samples were Th activated. In 6 paired samples (6/22), both the mother and child had Th activation, a finding that demonstrates a high degree of concordance. Additionally, 3 (6%) of 50 pregnant women were Th positive. These findings indicate that Th activation is another of the red cell antigen alterations related to pregnancy.     

Subunit composition of plasma von Willebrand factor in patients with the myeloproliferative syndrome

In order to evaluate the role of proteolysis in acquired von Willebrand's disease (vWD) associated with the myeloproliferative syndrome, we have determined the relative quantity of von Willebrand factor (vWF) fragments as compared with the intact 225 kDa subunit in four patients. The plasma vWF of each individual lacked large multimers; each had a prolonged bleeding time; and both platelet and leukocyte counts were elevated. Plasma was obtained from blood drawn into 1 mmol/L leupeptin, 6 mmol/L N-ethylmaleimide, and 5 mmol/L EDTA to prevent in vitro proteolysis. vWF was isolated from plasma by immunoadsorbent chromatography, reduced, subjected to SDS-5% polyacrylamide gel electrophoresis, and immunoblotted with a mixture of 55 anti-vWF monoclonal antibodies. In three patients with essential thrombocytosis (ET) the 176 and 140 kDa fragments were increased in proportion to the intact 225 kDa subunit indicating increased proteolysis. Treatment of one ET patient with CCNU (Lomustine) decreased the platelet count and, to a lesser extent, the white blood cell count. This was associated with a correction of the bleeding time, a partial correction of the multimeric abnormality, and a lessening of vWF cleavage. In a patient with polycythemia rubra vera (PRV) the proportion of the 176 kDa fragment was increased to the upper limit of normal but there was no definite evidence of increased proteolysis. These studies provide evidence that proteolysis plays a role in the acquired von Willebrand's disease associated with the myeloproliferative syndrome. However, other mechanisms must also be considered.     

Hematogones: a multiparameter analysis of bone marrow precursor cells

Morphologically distinct lymphoid cells with homogeneous, condensed chromatin and scant cytoplasm can be observed in large numbers in the bone marrow of children with a variety of hematologic and nonhematologic disorders. In some patients, these cells may account for greater than 50% of the bone marrow cells, creating a picture that can be confused with acute lymphoblastic leukemia (ALL) or metastatic tumor. Although originally called hematogones (HGs), a variety of other names have been proposed for these unique cells. The clinical significance of expanded HGs has not been resolved, and the biologic features of these cells are incompletely described. In this study, we correlate the clinical, morphologic, cytochemical, flow cytometric, molecular, and cytogenetic properties of bone marrow samples from 12 children with substantial numbers of HGs (range 8% to 55% of bone marrow cells). Diagnoses in these patients included anemia, four; neutropenia, one; anemia and neutropenia, one; idiopathic thrombocytopenic purpura, two; retinoblastoma, two; Ewing's sarcoma, one; and germ cell tumor, one. Flow cytometric analyses of bone marrow cells demonstrated a spectrum extending from early B-cell precursors (CD10+, CD19+, TdT+, HLA-Dr+) to mature surface immunoglobulin-bearing B cells in these patients, corroborating our morphologic impression of HGs, intermediate forms, and mature lymphocytes. DNA content was normal, and no clonal abnormality was identified by either cytogenetic or immunoglobulin and T-cell receptor (TCR) gene rearrangement studies. Follow-up ranged from 3 months to 3 years. None of the patients has developed acute leukemia or bone marrow involvement by solid tumor. The possible role of HGs in immune recovery and hematopoiesis is presented.     

Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V

Purified human factor VIII procoagulant protein (VIII:C) was treated with purified human activated protein C (APC) and the loss of VIII:C activity correlated with proteolysis of the VIII:C polypeptides. APC proteolyzed all VIII:C polypeptides with mol wt = 92,000 or greater, but not the doublet at mol wt = 79-80,000. These results and our previous thrombin activation studies of purified VIII:C, are analogous with similar studies of factor V and form the basis for the following hypothesis: activated VIII:C consists of heavy and light chain polypeptides [mol wt = 92,000 and mol wt = 79-80,000 (or 71-72,000), respectively] which are similar in Mr to the heavy and light chains of activated factor V. Thrombin activates VIII:C and V by generating these polypeptide chains from larger precursors and APC inactivates both molecules by cleavage at a site located in the heavy chain region of activated VIII:C and V.