Anti-factor VIII antibodies of hemophiliac patients are frequently directed towards nonfunctional determinants and do not exhibit isotypic restriction

A significant proportion of hemophilia A patients receiving transfusions of factor VIII (FVIII) develop a specific antibody response towards FVIII. These antibodies are usually detected by assays in which they inhibit the function of the molecule, such as the Bethesda clotting test. We have prepared anti-FVIII antibodies by specific immunoadsorption from the plasma of four hemophiliacs with stable inhibitor levels. The isotypic distribution of such antibodies was determined and their capacity to bind to insolubilized FVIII was compared with their inhibitory activity in two functional assays, namely, the Bethesda assay and a chromogenic assay. In addition, the FVIII epitope specificity was determined by competition with monoclonal antibodies for the binding to insolubilized FVIII. We show here that (1) anti-FVIII antibodies are not isotypically restricted; thus, a significant proportion of specific IgG2 was found; (2) antibodies are frequently directed towards epitopes of FVIII that are not directly involved in the function of the molecule and therefore escape detection in the Bethesda method or chromogenic assay; and (3) each patient shows a unique pattern of FVIII epitope recognition. We conclude that evaluation of anti-FVIII antibodies by a functional method does not provide an accurate evaluation of the specific antibody response. These findings have important implications for the comparison of the immunogenicity of FVIII molecules produced by different technologies and for the development of methods to control anti-FVIII antibody production.     

Suppression of apoptosis in hematopoietic factor-dependent progenitor cell lines by expression of the FAC gene

Fanconi anemia (FA) is a genetically heterogeneous, inherited blood disorder characterized by bone marrow failure, congenital malformations, and a predisposition to leukemias. Because FA cells are hypersensitive to DNA cross-linking agents and have chromosomal instability, FA has been viewed as a disorder of DNA repair. However, the exact cellular defect in FA cells has not been identified. Sequence analysis of the gene defective in group C patients (FAC) has shown no significant homologies to other known genes. The FAC protein has been localized to the cytoplasm, indicating that FAC may either play an indirect role in DNA repair or is involved in a different cellular pathway. Recent evidence has indicated that FA cells may be predisposed to apoptosis, especially after treatment with DNA cross-linking agents. The demonstration that genes can suppress apoptosis has been accomplished by overexpression of such genes in growth factor-dependent cell lines that die by apoptosis after factor withdrawal. Using retroviral-mediated gene transfer, we present evidence that expression of FAC in the hematopoietic factor-dependent progenitor cell lines 32D and MO7e can suppress apoptosis induced by growth factor withdrawal. Flow cytometry and morphologic analysis of propidium iodide stained cells showed significantly lower levels of apoptosis in FAC-retroviral transduced cells after growth factor deprivation. Expression of FAC in both cell lines promoted increased viability rather than proliferation, which is consistent with other apoptosis-inhibiting genes such as Bcl- 2. These findings imply that FAC may act as a mediator of an apoptotic pathway initiated by growth factor withdrawal. Furthermore, the congenital malformations and hematologic abnormalities characterizing FA may be related to an increased predisposition of FA progenitor cells to undergo apoptosis, particularly in the absence of extracellular signals.     

Electrocardiographic imaging of cardiac resynchronization therapy in heart failure: observation of variable electrophysiologic responses.

BACKGROUND: Cardiac resynchronization therapy (CRT) for congestive heart failure patients with delayed left ventricular (LV) conduction is clinically beneficial in approximately 70% of patients. Unresolved issues include patient selection, lead placement, and efficacy of LV pacing alone. Being an electrical approach, detailed electrical information during CRT is critical to resolving these issues. However, electrical data from patients have been limited because of the requirement for invasive mapping. OBJECTIVES: The purpose of this study was to provide observations and insights on the variable electrophysiologic responses of the heart to CRT using electrocardiographic imaging (ECGI). METHODS: ECGI is a novel modality for noninvasive epicardial mapping. ECGI was conducted in eight patients undergoing CRT during native rhythm and various pacing modes. RESULTS: In native rhythm (six patients), ventricular activation was heterogeneous, with latest activation in the lateral LV base in three patients and in the anterolateral, midlateral, or inferior LV in the remainder of patients. Anterior LV was susceptible to block and slow conduction. Right ventricular pacing improved electrical synchrony in two of six patients. LV pacing in three of four patients involved fusion with intrinsic excitation resulting in electrical resynchronization similar to biventricular pacing. Although generally electrical synchrony improved significantly with biventricular pacing, it was not always accompanied by clinical benefit. CONCLUSION: Results suggest that (1) when accompanied by fusion, LV pacing alone can be as effective as biventricular pacing for electrical resynchronization; (2) right ventricular pacing is not effective for resynchronization; and (3) efficacy of CRT depends strongly on the patient-specific electrophysiologic substrate.     

Ultrastructural and phenotypic analysis of in vitro erythropoiesis from human cord blood CD34<Superscript>+</Superscript> cells

Erythropoietin (EPO) induces erythropoiesis in vitro as well as in vivo, and the process of erythroid differentiation has been explored phenotypically and morphologically. However, morphological analysis of in vitro erythropoiesis of human hematopoietic progenitor cells at the ultrastructural level has not been reported before. In the present study, we have traced the ultrastructural changes of erythroid differentiation during ex vivo expansion of human cord blood (CB) CD34(+) cells in the presence of EPO by electron microscopy (EM), along with concurrent phenotypic analysis. CD34(+) cells purified from ten CBs by immunomagnetic selection were cultured in serum-free essential media in the presence of a combination of the several cytokines including EPO, thrombopoietin, flt3-ligand (FL), stem cell factor (SCF), granulocyte colony-stimulating factor, interleukin (IL)-3 and/or IL-11. Phenotypic analysis was performed by flow cytometric analysis for erythroid markers, including glycophorin C (GPC), Kell-related, glycophorin A (GPA), band 3, Lu(b), and RhD. Ultrastructural analysis was performed by electron-microscopic examination of the cultured cells stained with uranyl acetate and lead citrate. Phenotypic analysis revealed that in the absence of EPO, genuine erythroid fraction expressing the typical pattern of erythroid markers did not appear. The order of the above markers expressed in the cultured cells in the presence of EPO was GPC, Kell-related, GPA, band 3, Lu(b), and RhD, irrespective of the type of cytokine added. Of the cytokines used in combination with EPO, FL + IL-3 was the most efficient in inducing erythroid differentiation, which was followed by SCF + IL-3. EM examination demonstrated complete process of erythroid development from pronormoblasts to reticulocytes with nuclei having been extruded and mature erythrocytes. These results suggest that morphologically intact erythrocytes could be produced by ex vivo expansion of CB CD34(+) cells using EPO.     

In vivo expression of the B7 costimulatory molecule by subsets of antigen-presenting cells and the malignant cells of Hodgkin's disease

The B-lymphocyte/accessory-cell activation antigen B7 (BB1) has been shown in vitro to stimulate T-lymphocyte proliferation and cytokine production via CD28 present on the latter cells. In this study, benign lymphoid tissues, lymphomas, and extralymphoid inflammatory sites were examined immunohistochemically using anti-B7 and other relevant monoclonal antibodies. B7 was expressed by benign transformed germinal center B cells, as it was by B cells of follicular lymphomas. B7 was also expressed by a subpopulation (a mean of 31% to 65%) of macrophages and dendritic cells in a variety of lymphoid tissues. It was present in abundance on all macrophages constituting sarcoid granulomas in lymph nodes. In extralymphoid inflammation, 17% to 35% of macrophages expressed B7 only weakly. Cases of Hodgkin's disease showed expression of B7 by the majority of Reed-Sternberg cells or malignant mononuclear variants, a phenomenon that potentially contributes to the lymphocytic accumulation that is a feature of this condition. CD28+ T cells were seen in all areas where T cells were present. B7+ and CD28+ cells colocalized in, for example, lymphoid follicles, lymph node paracortex, sarcoid granulomas, and Hodgkin's disease tissue, indicating a potential for cellular interaction via these molecules at these sites.     

Clonal dysregulation of the antibody response to tetanus-toxoid after bone marrow transplantation

After bone marrow transplantation (BMT), a prolonged dysregulation of humoral immunity can be observed. In the present study, we investigated whether this is reflected in an abnormal production of specific antibodies (Ab) to the T-cell-dependent recall antigen tetanus-toxoid (TT). The study group consisted of children receiving transplants of an unmodified allogeneic graft and of adults receiving either a T-cell- depleted allogeneic or an unmodified autologous BM graft. Findings were compared with those in healthy controls. In pediatric graft recipients, who were routinely revaccinated early after BMT, the Ab response was quantitatively superior to that in adult graft recipients who did not receive early revaccination. In the majority of graft recipients, the time period after vaccination required to reach the peak level of antibodies was prolonged and the number of responding TT-specific B- cell clones was markedly decreased in comparison with controls. In controls, a low frequency of dominant B-cell clones may produce low quantities of homogeneous Ab components (H-Ab) against a heterogeneous background. However, in BM graft recipients, "overshooting" of Ab production by separate B-cell clones was observed, resulting in the development of H-Ab at a relatively high concentration. These abnormalities were present up to 10 years after BMT, irrespective of either the age of the recipient, the modulation of the graft, or the vaccination schedule used. It is hypothesized that the dysregulated Ab production is the consequence of activation of a restricted number of resting memory B cells, present in germinal centers, repopulating gradually after BMT. Our data show that routine revaccination early after BMT improves the humoral immune response. However, because of a clonally dysregulated Ab production, long-lasting qualitative defects may be present even after normalization of Ab titers.     

Assessing the delivery of neutrophils to tissues in neutropenia

Studies of neutrophil kinetics in neutropenic individuals, as well as clinical observations of variability in the occurrence of infection among patients with neutropenia, have suggested that blood neutrophil counts may not uniformly reflect the effective delivery of neutrophils to extravascular tissues where the cells perform their principal host defense functions. To evaluate this possibility we developed a sensitive, reproducible method of measuring the extravascular delivery of neutrophils to a normal mucosal site of neutrophil turnover. This method is based upon the quantification of neutrophils recoverable from saline mouth wash specimens. Twenty-five mL specimens, obtained in a controlled manner from neutropenic patients and normal subjects, were centrifuged and the sediments resuspended in 1.0 mL Hank's buffer with 2 micrograms acridine orange, incubated at 37 degrees C for 15 minutes, and then examined in a hemocytometer chamber by fluorescence microscopy. Neutrophils could be clearly distinguished by their characteristic fluorescence and were counted. With this method as few as 1,500 neutrophils were detected reliably in mouth wash specimens. Mucosal neutrophil counts varied less than 10% with repeated sampling of individual subjects over 5-day periods and were consistently greater than 1.3 X 10(5)/specimen in non-neutropenic individuals. Although profound neutropenia was generally reflected by lower than normal oral mucosal neutrophil counts, these counts were significantly higher in individuals with chronic severe neutropenia (blood neutrophils less than 300/mm3) than in patients with acute neutropenia of comparable severity that had developed following chemotherapy. Also, in individuals recovering from profound neutropenia, neutrophils usually reappeared earlier in mouth wash specimens than in blood, and oral mucosal neutrophil counts attained recovery levels more rapidly than did blood counts. This phenomenon was particularly evident in an individual with cyclic neutropenia. Moreover, mucosal neutrophils could occasionally be detected in profoundly neutropenic patients when neutrophils were not present in blood samples. These findings indicate that mucosal neutrophil counts in individuals with neutropenia provide information about the delivery of neutrophils to tissues that may not be apparent from blood neutrophil counts alone.     

Whole-pelvic volumetric-modulated arc therapy for high-risk prostate cancer: treatment planning and acute toxicity

The objectives of this study were to evaluate dosimetric quality and acute toxicity of volumetric-modulated arc therapy (VMAT) and daily image guidance in high-risk prostate cancer patients. A total of 100 consecutive high-risk prostate cancer patients treated with definitive VMAT with prophylactic whole-pelvic radiotherapy (WPRT) were enrolled. All patients were treated with a double-arc VMAT plan delivering 52 Gy to the prostate planning target volume (PTV), while simultaneously delivering 46.8 Gy to the pelvic nodal PTV in 26 fractions, followed by a single-arc VMAT plan delivering 26 Gy to the prostate PTV in 13 fractions. Image-guided RT was performed with daily cone-beam computed tomography. Dose–volume parameters for the PTV and the organs at risk (OARs), total number of monitor units (MUs) and treatment time were evaluated. Acute toxicity was assessed using the Common Terminology Criteria for Adverse Events, version 4.0. All dosimetric parameters met the present plan acceptance criteria. Mean MU and treatment time were 471 and 146 s for double-arc VMAT, respectively, and were 520 and 76 s for single-arc VMAT, respectively. No Grade 3 or higher acute toxicity was reported. Acute Grade 2 proctitis, diarrhea, and genitourinary toxicity occurred in 12 patients (12%), 6 patients (6%) and 13 patients (13%), respectively. The present study demonstrated that VMAT for WPRT in prostate cancer results in favorable PTV coverage and OAR sparing with short treatment time and an acceptable rate of acute toxicity. These findings support the use of VMAT for delivering WPRT to high-risk prostate cancer patients.     

Utility of Neutrophil-to-Lymphocyte Ratio (NLR) as a Predictor of Acute Infarction in New-Onset Acute Vertigo Patients Without Neurologic and Computed Tomography Abnormalities

Background

Neutrophil-to-lymphocyte ratio (NLR) has been used as a predictive marker for various conditions. However, there are no previous studies about NLR as a prognostic marker for acute infarction.