Phenotyping of acute myelocytic leukemia (AML) progenitors: an approach for tracing minimal numbers of AML cells among normal bone marrow

Previous studies have shown that the phenotypes of progenitors of human AML (AML-CFU) are variable, reflecting arrests at different stages of maturation. We were interested to seek discrepancies between the surface properties of AML precursors and normal bone marrow colony formers in order to detect minimal numbers of AML cells among normal bone marrow cells in remission bone marrow. Therefore, we selected two surface markers, the MoAb CD34, reactive with blast cells, and Vim-2, a surface marker reactive with mature myeloid cells, and determined the antigen density of these markers (relative fluorescence intensity using fluorescence-activated cell sorting) for normal marrow and AML progenitors. While these markers defined an identical phenotype (CD34++/Vim-2-/+) for a broad spectrum of normal progenitors, i.e., CFU-GEMM, BFU-e, day 15 CFU-GM, and day 7 CFU-GM, referred to as the "normal" progenitor phenotype, AML progenitors frequently exhibited different phenotypes. In 12 of 20 cases the phenotypes of the majority of AML progenitors were discrepant from the normal surface profile, i.e., according to one marker in 8 cases (CD34-/+/Vim-2-/+ or CD34++/Vim-2++) and two markers in 4 cases (CD34-/+/Vim-2++). Since these data indicate that AML and normal progenitors were frequently distinguishable, we then determined the potential utility of these phenotypic dissimilarities for detection of minimal disease. Artificial mixtures of normal bone marrow and minimal numbers (0.1-1%) of AML cells were prepared. Based upon the phenotypic discrepancies, AML metaphases were successfully demonstrated in these mixtures following cell sorting and culture. Thus, it appears that minimal numbers of AML mitoses can be identified with an approximate 10(-2) to 10(-3) sensitivity by taking advantage of differential coexpression of surface antigens.     

Impact of United States 2017 Immigration Policy changes on missed appointments at two Massachusetts Safety-Net Hospitals

Introduction

Studies have shown mixed findings regarding the impact of immigration policy changes on immigrants’ utilization of primary care.

Methods

We used a difference-in-differences analysis to compare changes in missed primary care appointments over time across two groups: patients who received care in Spanish, Portuguese, or Haitian Creole, and non-Hispanic, white patients who received care in English.

Results

After adjustment for age, sex, race, insurance, hospital system, and presence of chronic conditions, immigration policy changes were associated with an absolute increase in the missed appointment prevalence of 0.74 percentage points (95% confidence interval: 0.34, 1.15) among Spanish, Portuguese and Haitian-Creole speakers. We estimated that missed appointments due to immigration policy changes resulted in lost revenue of over $185,000.

Conclusions

We conclude that immigration policy changes were associated with a significant increase in missed appointments among patients who receive medical care in languages other than English.

     

Stability of aztreonam in a portable pump reservoir used for home intravenous antibiotic treatment (HIVAT)

The stability of the monocyclic -lactam antibiotic aztreonam in portable pump reservoirs was studied during storage at temperatures of-20°C and +5°C and during drug delivery at 37°C. Three 100-ml drug reservoirs and three glass containers containing 60 mg/ml aztreonam were stored at-20°C and 2-ml samples were analysed in the freshly prepared solution and after thawing at days 7, 21, 28, 70 and after 6 months of storage. A separate triplicate batch of 100-ml prefilled drug reservoirs and glass containers containing a similar aztreonam concentration (60 mg/ml) were refrigerated and tested immediately after preparation and daily for 8 days and after 70 days. Solutions of aztreonam in duplicate freshly prepared reservoirs were tested for stability when the solution was pumped at 37°C over a 24-h period. All solutions were inspected for visual changes and tested for pH. Drug concentration was analysed by high-performance liquid chromatography. No colour changes or pH differences were observed in any of the solutions in the reservoirs or containers. No statistically significant decrease in aztreonam concentration could be detected after 6 months of storage at-20°C. Aztreonam was stable at 5°C for at least 8 days. A 24-h pumping period at 37°C showed a 3.6% decrease in aztreonam concentration. Aztreonam at a concentration of 60 mg/ml in a pump reservoir is sufficiently stable to be used in home intravenous antibiotic treatment programmes.     

Tryptophan 650 of human granulocyte colony-stimulating factor (G-CSF) receptor, implicated in the activation of JAK2, is also required for G- CSF-mediated activation of signaling complexes of the p21ras route

Granulocyte colony-stimulating factor (G-CSF) induces rapid phosphorylation of JAK kinases as well as activation of the p21ras route through interaction with its specific receptor (G-CSF-R). The cytoplasmic membrane-proximal region of G-CSF-R (amino acids 631 to 684) is necessary for proliferation induction and activation of JAK2. In contrast, activation of Shc and Syp, signaling molecules implicated in the p21ras signaling route, depends on the phosphorylation of tyrosine residues located in the membrane-distal region (amino acids 685 to 813) of G-CSF-R. We investigated whether G-CSF-induced activation of signaling complexes of the p21ras route depends on the function of the membrane-proximal cytoplasmic region of G-CSF-R. A G- CSF-R mutant was constructed in which tryptophan 650 was replaced by arginine and expressed in BAF3 cells (BAF/W650R). In contrast to BAF3 cell transfectants expressing wild-type G-CSF-R, BAF/W650-R cells did not proliferate and did not show activation of JAK2, STAT1, or STAT3 in response to G-CSF. Immunoprecipitations with anti-Shc and anti-Grb2 antisera showed that mutant W650R also failed to activate Syp and Shc. These data indicate that the membrane-proximal cytoplasmic domain of G- CSF-R is not only crucial for proliferative signaling and activation of JAK2 and STATs, but is also required for activation of the p21ras route, which occurs via the membrane-distal region of G-CSF-R.     

A functional single-nucleotide polymorphism of the G-CSF receptor gene predisposes individuals to high-risk myelodysplastic syndrome

The granulocyte colony-stimulating factor receptor (G-CSF-R) transmits signals for proliferation and differentiation of myeloid progenitor cells. Here we report on the identification of a rare single nucleotide polymorphism within its intracellular domain (G-CSF-R_Glu785Lys). Screening a cohort of 116 patients with primary myelodysplastic syndromes (MDS), de novo acute myeloid leukemia (AML) (84 patients), as well as 232 age- and sex-matched controls revealed a highly significant association of the G-CSF-R_785Lys allele with the development of high-risk MDS as defined by more than 5% bone marrow blasts (9.7% versus 0.9% in controls; P = .001; odds ratio [OR], 12.5; 95% confidence interval [CI], 2.4-58.9) or an International Prognostic Scoring System score of intermediate-2 or high (13.0% versus 0.9%; P < .001; OR, 14.0; 95% CI, 3.4-85.0). Functional analysis by retroviral transfer of G-CSF-R_785Lys into myeloid progenitor cells of G-CSF-R-deficient mice showed a significantly diminished colony-formation capacity after G-CSF stimulation as compared with cells transduced with the wild-type receptor. These results suggest that lifelong altered G-CSF response by the G-CSF-R_785Lys may render individuals susceptible to development of high-risk MDS.     

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.