Evaluation of erythropoiesis in long-term hamster bone marrow suspension cultures: absence of a requirement for adherent monolayer cells

In long-term hamster bone marrow cultures, proliferation and differentiation of hemopoietic stem cells occurs for several months without need for hydrocortisone or adherent stromal elements, which are requirements for bone marrow growth in all other species studied. Only the most primitive erythroid progenitors (BFU-E) are produced in the cultures. Following treatment of the cells with erythropoietin, these progenitor cells undergo differentiation into mature hemoglobinized red blood cells. Concomitant addition of erythropoietin (Epo) and prostaglandin-E1 (PGE1) results in the production of large numbers of maturing red blood cells. In cultures stimulated with Epo and PGE1, as many as 70% of the cells are benzidine-positive, while Epo alone stimulated as many as 45% of the cells to become erythroid. Epo and PGE1 do not have any apparent deleterious effect on the continuous hemopoiesis occurring in these cultures. Under identical conditions, syngeneic adherent cell cultures do not produce any erythroid elements. The development of mature red blood cells from primitive erythroid precursors occurs in the presence of Epo alone and without any apparent need for adherent stromal elements. These cultures provide a useful in vitro model for dissecting the positive and negative signals that regulate erythropoiesis.     

Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors

Genomic DNA copy number alterations are key genetic events in the development and progression of human cancers. Here we report a genome-wide microarray comparative genomic hybridization (array CGH) analysis of DNA copy number variation in a series of primary human breast tumors. We have profiled DNA copy number alteration across 6,691 mapped human genes, in 44 predominantly advanced, primary breast tumors and 10 breast cancer cell lines. While the overall patterns of DNA amplification and deletion corroborate previous cytogenetic studies, the high-resolution (gene-by-gene) mapping of amplicon boundaries and the quantitative analysis of amplicon shape provide significant improvement in the localization of candidate oncogenes. Parallel microarray measurements of mRNA levels reveal the remarkable degree to which variation in gene copy number contributes to variation in gene expression in tumor cells. Specifically, we find that 62% of highly amplified genes show moderately or highly elevated expression, that DNA copy number influences gene expression across a wide range of DNA copy number alterations (deletion, low-, mid- and high-level amplification), that on average, a 2-fold change in DNA copy number is associated with a corresponding 1.5-fold change in mRNA levels, and that overall, at least 12% of all the variation in gene expression among the breast tumors is directly attributable to underlying variation in gene copy number. These findings provide evidence that widespread DNA copy number alteration can lead directly to global deregulation of gene expression, which may contribute to the development or progression of cancer.     

Fc gamma receptor II (CD32) on malignant B cells influences modulation induced by anti-CD19 monoclonal antibody

Antigenic modulation is one of many factors determining the effectiveness of monoclonal antibody (MoAb)-mediated therapy. To select the isotype of a CD19 MoAb most suitable for radioimmunotherapy of patients with B-cell malignancies, we studied the influence of MoAb isotype on modulation, after binding of the MoAb to different cell-line cells. The CD19-IgG1 MoAb was found to induce modulation of CD19 antigens on Daudi cell line cells more rapidly than did its IgG2a switch variant. We provide evidence that this difference in modulation rate is caused by the expression of Fc gamma receptor II (Fc gamma RII) on these cells. Experiments aimed at elucidating the mechanism of Fc gamma RII involvement in modulation induction by CD19-IgG1 showed that Fc gamma RII did not comodulate with CD19 MoAbs. However, cocrosslinking of CD19 and Fc gamma RII with CD19-IgG1 MoAb resulted in enhanced calcium mobilization in Daudi cells. This increased signal induction accompanies the enhanced capping and subsequent modulation of CD19 antigens. Because Fc gamma RII is expressed in varying densities on malignant B cells in all differentiation stages, our results have implications for the MoAb isotype most suitable for use in MoAb-based therapy of patients with B-cell malignancies.     

Diagnosis of intra-abdominal and mediastinal sarcoidosis with EUS-guided FNA

BACKGROUND: In the presence of a compatible clinical picture, the diagnosis of sarcoidosis requires pathologic confirmation of noncaseating epithelioid granuloma in affected tissues. The standard procedure of choice for most patients is a bronchoscopy with transbronchial biopsy (TBB), which has a diagnostic yield of 40% to 90%. The lowest yield with TBB is in cases that present with predominant mediastinal or intra-abdominal lymphadenopathy (LN) and minimal parenchymal lung involvement. OBJECTIVE: To study the diagnostic yield of EUS-guided FNA in diagnosing sarcoidosis with predominant LN or masses. DESIGN: Retrospective chart review. SETTING: Teaching university hospital. PATIENTS: Analysis of 21 consecutive patients with sarcoidosis and predominant mediastinal and/or intra-abdominal LN or masses who underwent EUS-guided FNA. RESULTS: EUS-guided FNA diagnosed sarcoidosis in 18 of 21 patients (86%). In 3 patients, EUS-guided FNA was either not diagnostic or inconclusive, and patients underwent mediastinoscopy with lymphadenectomy, which established the diagnosis of sarcoidosis. Seven of the 21 patients (33%) had intra-abdominal LN and/or masses, and EUS-guided FNA of the intra-abdominal pathology was diagnostic of sarcoidosis in 4 of the 7 patients (57%). Four of the 21 patients (19%) had a history of malignancy, and use of EUS-guided FNA helped in ruling out the recurrence of malignancy in 3 of the 4 patients (75%). LIMITATIONS: Mycobacterial and fungal culture was not obtained in all patients. CONCLUSIONS: EUS-guided FNA offers a practical, minimally invasive technique for the diagnosis of sarcoidosis in patients who present with predominant mediastinal and/or intra-abdominal LN or masses.     

Mechanism of inositol monophosphatase, the putative target of lithium therapy.

myo-Inositol monophosphatase (myo-inositol-1-phosphate phosphohydrolase, EC 3.1.3.25) is an attractive target for mechanistic investigation due to its critical role in the phosphatidylinositol signaling pathway and the possible relevance of its inhibition by Li+ to manic depression therapy. The x-ray crystallographic structure of human inositol monophosphatase in the presence of the inhibitory metal Gd3+ showed only one metal bound per active site, whereas in the presence of Mn2+, three ions were present with one being displaced upon phosphate binding. We report here modeling, kinetic, and mutagenesis studies on the enzyme, which reveal the requirement for two metal ions in the catalytic mechanism. Activity titration curves with Zn2+ or Mn2+ in the presence or absence of Mg2+ are consistent with a two-metal mechanism. Modeling studies based on the various x-ray crystallographic structures (including those with Gd3+ and substrate bound) further support a two-metal mechanism and define the positions of the two metal ions relative to substrate. While the first metal ion may activate water for nucleophilic attack, a second metal ion, coordinated by three aspartate residues, appears to act as a Lewis acid, stabilizing the leaving inositol oxyanion. In this model, the 6-OH group of substrate acts as a ligand for this second metal ion, consistent with the reduced catalytic activity observed with substrate analogues lacking the 6-OH. Evidence from Tb3+ fluorescence quenching and the two-metal kinetic titration curves suggests that Li+ binds at the site of this second metal ion.     

Onset of ovulation after menarche in girls: a longitudinal study

INTRODUCTION: Hypothalamic-pituitary axis maturity has been believed to be the rate-limiting step in the development of ovulatory menstrual cycles. We hypothesized that, given current nutritional conditions, hypothalamic-pituitary axis maturation would be relatively rapid in menarcheal girls. METHODS: Daily urine and menstrual records were collected for 2 yr each from 10 girls aged 11-13 yr at study entry. Urinary excretion of LH, FSH, estradiol (E1c), and progesterone (Pdg) metabolites was measured using established ELISAs. An objective algorithm detected rises of LH, FSH, E1c, and Pdg consistent with follicular maturation and/or ovulation. RESULTS: Nine of 10 girls enrolled into the study experienced the onset of menarche prior to or during the 2-yr collection period. LH and FSH surges, as well as small amplitude Pdg increments, were observed prior to menarche. Regular, ovulatory-appearing cycles with LH surges and gradually increasing and more sustained Pdg rises were observed over time after menarche, although duration of Pdg elevations remained shorter than in adult women (8.9 +/- 1.0 vs. 12.1 +/- 0.8 d, P = 0.043). E1c levels leading to LH/FSH surges were lower in perimenarcheal girls than adult controls, and bleeding episodes did not uniformly correlate with hormone patterns. Progressive increases in FSH and Pdg, but not LH or E1c, were observed in association with menarche. CONCLUSION: Mature hormone patterns are established within several months of and even prior to menarche in normal-weight perimenarcheal girls. Factors determining menstrual bleeding in perimenarcheal girls may not be solely dependent on reproductive hormones or the neuroendocrine axis.     

Validation of data from electronic data warehouse in diabetic ketoacidosis: Caution is needed

Aims

This study validated enterprise data warehouse (EDW) data for a cohort of hospitalized patients with a primary diagnosis of diabetic ketoacidosis (DKA).

Life Years Lost and Lifetime Health Care Expenditures Associated With Diabetes in the U.S., National Health Interview Survey, 1997–2000

OBJECTIVEThis study analyzed the lifetime health care expenditures and life years lost associated with diabetes in the U.S.RESULTSPredicted life expectancy for patients with diabetes and without diabetes demonstrated an inverted U shape across most BMI classifications, with highest life expectancy being for the overweight. Lifetime health care expenditures were higher for whites than blacks and for females than males. Using U.S. adults aged 50 years as an example, we found that diabetic white females with a BMI >40 kg/m² had 17.9 remaining life years and lifetime health expenditures of $185,609, whereas diabetic white females with normal weight had 22.2 remaining life years and lifetime health expenditures of $183,704.CONCLUSIONSOur results show that diabetes is associated with large decreases in life expectancy and large increases in lifetime health care expenditures. In addition to decreasing life expectancy by 3.3 to 18.7 years, diabetes increased lifetime health care expenditures by $8,946 to $159,380 depending on age-race-sex-BMI classification groups.