Armand B. Glassman

The Sox4/Tcf7l1 axis promotes progression of BCR-ABL-positive acute lymphoblastic leukemia

The transcription factor Sox4 plays an indispensable role in the development of early progenitor B cells from hematopoietic stem cells. However, its role in B-cell acute lymphoblastic leukemia, a malignant counterpart of normal progenitor B cells, is not fully understood. Here we show that SOX4 is highly expressed in human acute lymphoblastic leukemia cells. To systematically study the function of Sox4 in acute lymphoblastic leukemia, we established a genetically defined mouse leukemia model by transforming progenitor B cells carrying a floxed Sox4 allele and inducing deletion of the allele by the self-excising Cre recombinase. This model allowed us to work with two groups of leukemic cells that had either one copy or both copies of Sox4 deleted. We found that depletion of Sox4 in transformed cells in vitro reduced cell growth in vitro and the progression of leukemia in vivo. Moreover, depletion of Sox4 in leukemic cells in vivo prolonged the survival of the mice, suggesting that it could be a potential target in acute lymphoblastic leukemia therapy. Our microarray and bioChIP studies revealed that Tcf7l1 was the key gene directly regulated by Sox4. Knockdown of Tcf7l1 reduced cell proliferation, just as did knockout of Sox4, and ectopic expression of Tcf7l1 could reverse the effect of Sox4 knockout on cell proliferation. These data suggest that Sox4 and Tcf7l1 form a functional axis that promotes the progression of BCR-ABL-positive acute lymphoblastic leukemia.

Trace metal levels in commercially prepared tissue culture media

Four trace elements, lead, copper, tin and zinc, in addition to certain electrolytes, were measured in 11 commercially prepared tissue culture media. Glass media bottles and plastic tissue culture dishes and flasks were treated with a HCl acid solution to determine the amounts of trace metals leached from their surfaces. Zinc, lead and copper were detected in all media. Tin was detected only in RPMI Medium 1640, fetal bovine serum, minimum essential medium and penicillin-streptomycin. It is possible that a major cause of variability in tissue culture experimental results may be due to effects on growth caused by fluctuation in trace element contamination from batch to batch. Variability in establishing primary cultures of corneal endothelial cells was traced to high lead levels in commercially prepared tissue culture media. A strong case is made for continued diligent efforts to expand analytical horizons and our definition of substances in culture media.

Antibodies to A19 and Bw35 complexes of human leukocyte antigens are present in infertile subjects with sperm antibodies

Sera and secretions from 100 couples with unexplained infertility were tested for sperm antibodies by cytotoxicity and passive hemagglutination and also for antibodies to human leukocyte antigen (HLA) by cytotoxicity assays. Lymphocytes of the study subjects were typed for 61 HLA-A and B alleles. Thirteen of 30 (43%) men with sperm autoimmunity also had HLA antibodies in their serum and/or seminal plasma samples, in contrast to 2 of 35 (6%) nonautoimmune males (P = 0.0003). Twenty-five of 35 (71%) sperm antibody-positive infertile women had HLA antibodies in their sera and/or secretions, while only 7 of 31 (23%) women without sperm antibodies were positive (P = 0.00007). Antibodies to HLA-A19 (A26, A29, AW30, AW31, AW32, AW33, and AW34) and Bw35 (B5, B15, B17, and B18) complexes were present in 19 of 22 (86%) infertile men and 44 of 48 (92%) infertile women positive for HLA antibodies (P less than 0.01). The presence of antibodies to HLA-A19 and/or Bw35 in the infertile subjects did not correlate with the presence of HLA-A19 and/or Bw35 in the husbands. The presence of antibodies to HLA-A19 and/or Bw35 in the cervical mucus of the infertile women correlated with their presence in the seminal plasma of their husbands. It is suggested that antibodies to sperm antigens cross-reactive with HLA-A19 and/or Bw35 may be relevant to infertility.

Bactericidal capacity of polymorphonuclear leukocytes from patients receiving theophylline therapy

The capacity of polymorphonuclear leukocytes (PMNs) to kill Staphylococcus aureus strains 502 A was studied in 25 patients receiving theophylline therapy and in normal healthy controls. A significant difference in PMN bactericidal capacity was found between controls and patients with serum theophylline levels higher than 8 micrograms/ml, as determined by high-pressure liquid chromatography. The bactericidal capacity of PMNs from both the patient and control populations was reduced in the presence of theophylline levels above 8 micrograms/ml. In addition, a transient but significant drug-independent reduction was found in the bactericidal capacity of transient but significant drug-independent reduction was found in the bactericidal capacity of PMNs from patients receiving theophylline.

Corneal endothelium: A modified method for cultivation

SummaryA modified method for establishing cultures of rabbit corneal cells is described. The new technique utilized a Lucite disc in combination with a Tygon ring for growth of pure cell cultures and was compared with an explant method for growing cells. Each method provided adequate cell cultures for biochemical or ultrastructure studies of rabbit corneal cells, but the ring and disc method described here allowed the isolation of specific cell types without the interference of stromal cell contamination.

Male-To-Female Sex Ratios of Abnormalities Detected by Fluorescence in Situ Hybridization in a Population of Chronic Lymphocytic Leukemia Patients

Distorted sex ratios occur in hematologic disorders. For example, chronic lymphocytic leukemia (CLL) displays disproportionate sex ratios with a large male excess. However, the underlying genetics for these disparities are poorly understood, and gender differences for specific cytogenetic abnormalities have not been carefully investigated. We sought to provide an initial characterization of gender representation in genetic abnormalities in CLL by using fluorescence in situ hybridization (FISH). We confirm the well known skewed male-tofemale (M/F sex ratio) of ~1.5 in our CLL study population, but also determine the genotypic M/F sex ratio values corresponding to specific FISH DNA probes. Genetic changes in CLL detectable by four FISH probes were statistically compared with respect to gender. Initial FISH evaluations of 4698 CLL patients were retrospectively examined and new findings of the genotypic M/F sex ratios for these probes are reported. This study represents the largest CLL survey conducted in the United States using FISH probes. The CLL database demonstrated that FISH abnormalities (trisomy 12, 13q14.3 deletion and 17p13.1 deletion) probes had skewed M/F ratios of ~1.5. Also, by statistical analysis it was shown that ATM gene loss (11q22.3q23.1 deletion) solely or with other abnormalities was considerably higher in males with an M/F ratio of 2.5 and significantly different from M/F ratios of 1.0 or 1.5. We hypothesize that interactions involving these autosomal abnormalities (trisomy 12, and deletions of 11q22.3, 13q14.3, and 17p13.1), and the sex chromosomes may provide the genetic basis for the altered phenotypic M/F ratio in CLL.

Incidence of neutrophil antigens on human cord neutrophils

ABSTRACT: Neutrophils isolated from cord blood of healthy newborns (33 blacks and 21 whites) were investigated by EDTA‐microagglutination for their expression of neutrophil specific antigens that have been associated with isoimmune neonatal or autoimmune neutropenia. Equal volumes of various neutrophil anti‐sera (2 μ) and cord neutrophils (3–5 × 106/ml) were mixed in tissue typing microplates under oil and were incubated at room temperature for 6–8 hr, following which the degree of agglutination was noted. Our data revealed that all the currently recognized neutrophil antigens are readily demonstrable by antineutrophil antibodies in cord blood, (NA1, 52–54%; NA2, 81–85%; NB1, 95–96%; NC1, 90%; 9A, 29–30%) suggesting that neutrophil antigens are fully expressed at birth.

DNA and protein synthesis in normal and transformed lymphocytes exposed to abrin

The dose dependent effects of abrin, a toxic D-galactose binding plant lectin, on 3H-TdR and 14C-leucine uptake are studied in normal and Epstein Barr Virus (EBV) transformed lymphocyte cultures. Results show that while abrin is highly toxic to both the DNA and protein synthesis in EBV lymphocytes, some toxicity to the normal cells is also seen. It is postulated that lymphocyte DNA synthesis is affected by ribosomal shutdown induced by the abrin.

Studies on the Toxicity and Binding Kinetics of Abrin in Normal and Epstein Barr Virus-Transformed Lymphocyte Culture-I: Experimental Results – 3

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Using data on EBV-transformed lymphocyte cell density as a function of time and dose of abrin, one can demonstrate that the mean number of sites bound/EBV-lymphocyte needed to exert a biological influence upon the cell DNA synthesis lies between 59,264 and 370,000 sites/cell. Using a simple packing model, one can demonstrate that a theoretical estimate places the number of binding sites between 57,600 and 360,000 sites/cells.

On the dynamics of abrin binding to receptor sites in normal and Epstein Barr Virus transformed lymphocyte cell cultures.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA an protein synthesis of normal and Epstein Barr Virus transformed lymphocytes has been investigated. Activation, stimulation, and relative toxicity factor indices are studied, as well as possible relationships between DNA and protein synthesis rates, as measured by simultaneous tritiated thymidine (3H-TdR) and 14C-leucine uptake. Studies of the two new indices, the metabolic self and cross coupling indices lead to the prediction that there are three morphologically distinct subpopulations of EBV-transformed lymphocytes with different abrin receptor site concentrations. This prediction is supported by SEM morphological differences. Using data on EBV-transformed lymphocyte cell density as a function of time and dose of abrin, one can demonstrate that the mean number of receptors bound-EBV-lymphocyte needed to exert a biological influence lies in the interval 59,264 receptors/cell to 370.040 receptors/cell. Using a simple packing model, one can demonstrate that a theoretical estimate places the number of binding sites between 57,600 receptors/cell and 360,000 receptors/cell.