Sequence comparison of putative regulatory DNA of the 5' flanking region of the myeloperoxidase gene in normal and leukemic bone marrow cells.

Myeloperoxidase (MPO) is an enzyme which is exclusively expressed in immature myeloid cells with downregulation of gene expression occurring during granulocytic maturation. Levels of MPO RNA, protein, and enzyme activity differ, usually in a concordant fashion, among the various classes of acute leukemia and among different cases within a particular class. One portion of the gene thought to be involved in regulation of MPO expression is the proximal 5' flanking region. To determine if mutations in this putatively regulatory region of the MPO gene might be responsible for some of the differences in level of MPO expression among different cases or classes of acute leukemia, we compared the nucleotide sequence of this part of the gene from 16 patients with acute leukemia, with DNA from normal human bone marrow cells and selected other neoplasms and cell lines. The sequence of this regulatory region was found to be identical in cases of acute myeloid leukemia (AML) with tha of normal DNA except for a dA to dG transition in the Alu region, 463 bases upstream from the transcription start site. This base substitution was seen in almost all cases of AML studied, regardless of the level of MPO which they expressed. It was absent from normal human DNA obtained from various tissues, and cases of acute and chronic lymphocytic leukemia, carcinoma of lung, and most cell lines examined. The base substitution was also absent in a remission blood sample from one of the cases which showed the dA to dG transition in leukemic marrow, suggesting that the base substitution is a mutation rather than a polymorphism. Our results suggest that mutations in promoter or enhancer DNA are not an important cause of the differences in level of MPO gene expression seen among different cases or different classes of AML. However, the base substitution we have detected could potentially serve as a useful marker for detection of residual disease in patients with AML following treatment.

The epidemiology of venous thromboembolism in Caucasians and African-Americans: the GATE Study.

The aim of this study was to assess, comprehensively, medical and genetic attributes of venous thromboembolism (VTE) in a multiracial American population. The Genetic Attributes and Thrombosis Epidemiology (GATE) study is an ongoing case-control study in Atlanta, Georgia, designed to examine racial differences in VTE etiology and pathogenesis. Between 1998 and 2001, 370 inpatients with confirmed VTE, and 250 control subjects were enrolled. Data collected included blood specimens for DNA and plasma analysis and a medical lifestyle history questionnaire. Comparing VTE cases, cancer, recent surgery, and immobilization were more common in caucasian cases, while hypertension, diabetes, and kidney disease were more prevalent in African-American cases. Family history of VTE was reported with equal frequency by cases of both races (28-29%). Race-adjusted odds ratios for the associations of factor V Leiden and prothrombin G20210A mutations were 3.1 (1.5, 6.7) and 1.9 (0.8, 4.4), respectively. Using a larger external comparison group, the odds ratio for the prothrombin mutation among Caucasians was a statistically significant 2.5 (1.4, 4.3). A case-only analysis revealed a near significant interaction between the two mutations among Caucasians. We found that clinical characteristics of VTE patients differed across race groups. Family history of VTE was common in white and black patients, yet known genetic risk factors for VTE are rare in African-American populations. Our findings underscore the need to determine gene polymorphisms associated with VTE in African-Americans.

The distribution of HLA antigens on human corneal tissue.

Frozen sections of human corneas, as well as cultured cells derived from the epithelial, stromal, and endothelial layers, were examined for class I (HLA-A, B, C) and class II (HLA-DR) histocompatibility antigens using mouse monoclonal antibodies in an indirect immunofluorescence assay. On frozen sections, class I antigens were readily detected on corneal epithelium and keratocytes. Class I antigens were not detected on endothelial cells on frozen sections of adult corneas, but were identified on endothelial cells from some individuals less than 2 years of age. Class II antigens were not detected on corneal epithelial, stromal or endothelial cells on frozen sections. However, HLA-DR-positive dendritic cells were seen in corneal epithelium and were more numerous near the limbus. HLA-DR was expressed by cuboidal cells in the basal layer of conjunctival epithelium from several infants. Cultured cells derived from corneal epithelium, stroma, and endothelium consistently expressed class I but not class II antigens.

Modulation of HLA antigen expression on corneal epithelial and stromal cells.

Human corneal epithelial cells and stromal fibroblasts in culture were incubated with gamma interferon or with medium conditioned by phytohemagglutinin (PHA)-stimulated mononuclear cells. The corneal cells were placed into suspension, assayed for class I (HLA-A,B,C) and class II (HLA-DR) antigens by indirect immunofluorescence, and analyzed with flow cytometry. Epithelial cells treated for 5 days with conditioned medium (CND-M) did not exhibit an increase in class I or an induction of class II antigen expression, although a trend toward increased class I antigen expression was present. Epithelial cells treated for 5 days with 250-500 U/ml of gamma interferon did not demonstrate an increase in class I but did show an induction of class II antigen expression; again, however, a trend toward increased class I antigen expression was present. Stromal fibroblasts treated for 3-5 days with CND-M exhibited an increase in class I antigen expression, but stromal fibroblasts treated for 1-5 days with CND-M did not show an induction of class II antigen expression. Stromal fibroblasts incubated for 1-5 days with 250-750 U/ml of gamma interferon demonstrated both an increase in class I and an induction of class II antigen expression. These data suggest that host lymphokines may intensify the process of corneal graft rejection by augmenting class I antigen expression on allogeneic cells. Moreover, the induction of class II antigen expression by host lymphokines on cells in transplanted corneal tissue may lead to host sensitization and subsequent allograft rejection.

Laser densitometric analysis of class I HLA antigen expression by corneal epithelium.

Laser densitometric analysis of immunoperoxidase stained tissue was used to quantitate class I HLA (HLA-A,B,C) antigen expression by human corneal epithelium. Frozen sections of human donor corneas stored in modified McCarey-Kaufman medium for less than 24 hr were evaluated for class I HLA antigen by an indirect immunoperoxidase technique using a monoclonal antibody reactive against a class I HLA antigen determinant. Photomicrographs of stained epithelium taken under standardized conditions were evaluated by laser densitometry. Measurements from peripheral and central corneal epithelium on the same tissue section were compared. Total stain, stain density, and stain intensity were higher for peripheral than for central corneal epithelium, indicating that class I HLA antigen expression is greater for peripheral than for central epithelium.

The relationship between polymorphisms in the endothelial cell nitric oxide synthase gene and the platelet GPIIIa gene with myocardial infarction and venous thromboembolism in African Americans.

STUDY OBJECTIVES: To determine whether the polymorphic dinucleotide repeats found in intron 4 of the endothelial cell nitric oxide synthase (ecNOS) gene and the platelet GPIIIa PLA(1)/A(2) polymorphism are associated with myocardial infarction (MI) and venous thromboembolism (VTE) in African Americans. Because these two genes may interact physiologically, the third objective was to determine if there was a relationship between the polymorphisms with respect to MI and VTE. DESIGN: A hospital-based case-control study. After informed consent was obtained, blood used for DNA extraction was drawn from the subjects. SETTING: The study was conducted in the Anticoagulant Clinic and the Cardiology Clinic at Grady Memorial Hospital in Atlanta Georgia. PATIENTS: Subjects were recruited from African-American patients with a reported history of MI (n = 110) or VTE (n = 91). Control subjects (n = 185) without a history of cardiovascular or venous disease were recruited from an outpatient clinic. MEASUREMENTS AND RESULTS: The 393 ecNOS allele was more common among MI cases (36%; p = 0.01) and VTE cases (35%; p = 0.04) than among control subjects (26%). There was no association between the GPIIIa genotypes and either MI or VTE. However, among the MI subjects, there was a strong association between the ecNOS 393/393 genotype and the Pl(A2) allele. It was also found that the frequency of the 393 allele was higher in African-American persons (0.26) compared with what has been reported for Australian Caucasians (0. 14) and Japanese (0.10). CONCLUSIONS: The 393 allele but not the Pl(A2) allele was significantly associated with both MI and VTE in African Americans. Homozygosity for the 393 allele was significantly associated to the diagnosis of MI prior to the age of 45. The combination of the 393 allele and a Pl(A2) allele was also highly associated with MI. The frequency of the 393 allele was significantly higher in African Americans than what has been reported for other populations. This study furthers not only extends the association of the 393 allele to VTE but has demonstrated an interaction with the Pl(A2) allele with respect to MI.

HLA typing of cultured amniotic fluid cells.

HLA typing was performed on 18 cultures of human amniotic fluid cells using cytotoxicity and absorption technics. Confirmation of antigen assignments was obtained in nine of ten instances, where HLA typing also was performed on cord blood. Three major problems were encountered in performing these studies: (1) complement cytotoxicity, (2) false-positive reactions, and (3) false-negative reactions. False-positive and false-negative reactions occurred more frequently with sera defining HLA-B locus specificities than with sera defining HLA-A locus specificities. Absorption studies were helpful in making antigen assignments when false reactions occurred. Preliminary studies suggest that the frequency of false-positive reactions can be decreased by absorbing HLA typing sera with antigen-negative amniotic fluid cultured cells, buffy coat, or platelets. Accurate antigen assignment is difficult when parental HLA types are unavailable.

Prolymphocytic leukemia of helper cell phenotype: report of a case and review of the scientific literature.

A case of prolymphocytic leukemia was studied by using light and electron microscopy, cytochemistry and multiple immunological markers for T and B lymphocytes. Cytochemical and immunological studies showed that the cells were T-lymphocytes. By using monoclonal antibodies to human T-cell subsets, we found that these cells had the T-helper cell phenotype. The usefulness of multiparameter studies in subclassifying T-cell malignancies is discussed and the literature on T-prolymphocytic leukemia reviewed.

The role of hematopoietic growth factors in transfusion medicine.

Hematopoietic growth factors have already had an enormous impact on transfusion practice by eliminating or reducing the need for red blood cell transfusions in a variety of anemic states characterized by an absolute or relative decrease in erythropoietin. In addition, GM-CSF and G-CSF have stimulated the production of autologous neutrophils in febrile neutropenic patients in whom granulocyte transfusions had been considered ineffective. With the discovery of c-Mpl ligand and the promising results obtained with IL-11 and IL-3, a combination of growth factors that successfully stimulate platelet production may soon be identified. This first era in the clinical application of hematopoietic growth factors has been characterized largely by treatment of the patient to stimulate production of autologous cells or to enhance the ability of transplanted hematopoietic progenitor cells to repopulate the patient. The use of G-CSF to increase the yield of granulocytes harvested by apheresis procedures and to mobilize peripheral blood stem cells in allogeneic donors has initiated a new era in which the cell donor is treated to enhance cell production and enhance the repopulating ability of hematopoietic progenitor cells. As our understanding of hematopoiesis grows, scientists will be able to identify growth factors to overcome or correct deficient hematopoiesis. Increasingly, component transfusions will be reserved for life-threatening situations in which endogenous cell production cannot be stimulated or cell production will be too slow to prevent life-threatening events.

The role of the t-PA I/D and PAI-1 4G/5G polymorphisms in African-American adults with a diagnosis of myocardial infarction or venous thromboembolism.

To determine whether or not the PAI-1 4G/5G and t-PA I/D polymorphisms in African-Americans were linked to cardiovascular disease, the association of these polymorphisms to disease expression was analyzed in a recently completed case-control study of myocardial infarction or venous thromboembolism among African-Americans. All African-Americans patients with a history of venous thromboembolism attending an anticoagulant clinic, and patients with a history of a MI attending a cardiology clinic at a large local urban public hospital were eligible for inclusion as cases in the study. In this study it was observed that there was a statistically significant association between the D allele of the t-PA I/D polymorphism and venous thromboembolism and a nonsignificant association between the D allele and myocardial infarction among African-Americans. t-PA antigen levels were statistically significantly higher among both myocardial infarction and venous thromboembolism cases compared with control subjects. The genotypes were unrelated to t-PA plasma levels. There was no association between either myocardial infarction or venous thromboembolism and the 4G/5G PAI-1 genotype. It was also found that genotype frequencies for both PAI-1 4G/5G and t-PA I/D polymorphisms in African-American adults were different from those reported for both U.S. Causcians and Europeans.

HLA antigen frequencies in paralytic dementia.

HLA typing was performed on 75 caucasian and 58 black patients with paralytic dementia hospitalized at 2 state mental institutions in New York. Serological testing was performed to confirm previous infection with T. pallidum. The caucasian patients showed an increase in B18(18.7% vs. 9.6% in controls). The black patients showed an increase in A2 (48.3% vs. 28.7% in controls) and an increase in B7 (27.6% vs. 15.7% in controls.

Donor origin Rh antibodies as a cause of significant hemolysis following ABO-identical orthotopic liver transplantation.

A group A, D-positive patient underwent orthotopic liver transplantation from a group A, D-negative (cde/cde) donor. Anti-D and -E were eluted from the recipient's red cells and were found in the recipient's serum 13 days later, at which time significant hemolysis developed. These Rh antibodies appear to he secondary to passive transfer of sensitized donor lymphocytes, a rare finding following liver transplantation.