Establishment of erythropoiesis following bone marrow transplantation in a patient with congenital hypoplastic anemia (Diamond-Blackfan syndrome)

Marrow transplantation was attempted in a 13-yr-old boy with congenital hypoplastic anemia who had never responded to corticosteroid therapy. Prior to the transplant, he had received 238 transfusions, at least 12 of which were from his father. He was prepared for grafting with antilymphocyte globulin, procarbazine, and total body irradiation (1000 rads). The patient, whose red cells were Group B, then received marrow cells from his Group O, histocompatible, sister. Thereafter, reticulocytes, Group O erythrocytes, and female leukocytes appeared in the peripheral blood. Erythroid precursors were seen in the patient's marrow for the first time in his life, and all lacked fluorescent Y chromosomes. Dividing cells were all female. After initially progressing well, the patient developed interstitial pneumonia and died 55 days after the transplant. The successful erythroid graft suggested that this patient's failure to produce red blood cells was due to a defective stem cell rather than to a humoral defect, plasma inhibitor, or abnormal marrow microenvironment. It suggested further that sibling marrow may be engrafted in patients who have received multiple transfusions, even from a parent.     

Proliferation of myeloid progenitor cells in human long-term bone marrow cultures is stimulated by interleukin-1 beta

To study the effect of interleukin-1 (IL-1) beta on the proliferation of hematopoietic progenitor cells (HPC) in long-term bone marrow cultures (LTBMC), stromal cell layers were established from normal human bone marrow. Autologous cryopreserved mononuclear phagocyte- and T-lymphocyte-depleted bone marrow cells were reinoculated on the stromal layers in fresh culture medium, with or without the addition of human IL-1 beta (30 U/mL). Once a week, half of the culture supernatant was replaced with fresh culture medium with or without IL-1, and all nonadherent cells were returned to the flasks. At weekly intervals during a period of 5 weeks, one culture was sacrificed to determine the total number of cells and hematopoietic progenitor cells, present in the adherent and the nonadherent cell fractions. In IL-1-stimulated cultures, the number of cells recovered during a period of 5 weeks exceeded the number of cells in unstimulated control cultures by 1.5 times. This difference was attributed to a twofold increase in the number of adherent cells. The number of HPC recovered from IL-1- stimulated cultures was not different from that recovered from controls. The levels of colony-stimulating activity (CSA) in supernatants from IL-1-stimulated cultures were significantly higher than those in supernatants from control cultures. These results indicate that IL-1 enhances the recovery of cells in LTBMC by stimulating the proliferation of HPC with the concurrent release of CSA from stromal cells, without diminishing the number of HPC.     

Hematopoietic growth factors for graft failure after bone marrow transplantation: a randomized trial of granulocyte-macrophage colony- stimulating factor (GM-CSF) versus sequential GM-CSF plus granulocyte- CSF

Delay in hematologic recovery after bone marrow transplantation (BMT) can extend and amplify the risks of infection and hemorrhage, compromise patients' survival, and increase the duration and cost of hospitalization. Because current studies suggest that granulocyte- macrophage (GM) colony-stimulating factor (CSF) may potentiate the sensitivity of hematopoietic progenitor cells to G-CSF, we performed a prospective, randomized trial comparing GM-CSF (250 micrograms/m2/d x 14 days) versus sequential GM-CSF x 7 days followed by G-CSF (5 micrograms/kg/d x 7 days) as treatment for primary or secondary graft failure after BMT. Eligibility criteria included failure to achieve a white blood cell (WBC) count > or = 100/microL by day +21 or > or = 300/microL by day +28, no absolute neutrophil count (ANC) > or = 200/microL by day +28, or secondary sustained neutropenia after initial engraftment. Forty-seven patients were enrolled: 23 received GM-CSF (10 unrelated, 8 related allogeneic, and 5 autologous), and 24 received GM- CSF followed by G-CSF (12 unrelated, 7 related allogeneic, and 5 autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC > or = 500/microL) occurred between 2 and 61 days (median, 8 days) after therapy, while those receiving GM-CSF+G-CSF recovered at a similar rate of 1 to 36 days (median, 6 days; P = .39). Recovery to red blood cell (RBC) transfusion independence was slow, occurring 6 to 250 days (median, 35 days) after enrollment with no significant difference between the two treatment groups (GM-CSF: median, 30 days; GM-CSF+G- CSF; median, 42 days; P = .24). Similarly, platelet transfusion independence was delayed until 4 to 249 days (median, 32 days) after enrollment, with no difference between the two treatment groups (GM- CSF: median, 28 days; GM-CSF+G-CSF: median, 42 days; P = .38). Recovery times were not different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone. Only 1 of 23 patients treated with GM-CSF died versus 7 of 24 treated with GM-CSF+G-CSF who died 16 to 84 days (median, 38 days) after enrollment, yielding Kaplan-Meier 100-day survival estimates of 96% +/- 8% for GM-CSF versus 71% +/- 18% for GM-CSF+G-CSF (P = .026). These data suggest that sequential growth factor therapy with GM-CSF followed by G-CSF offers no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assessment of myocardial salvage after ischemia and reperfusion using magnetic resonance imaging and spectroscopy

To test the hypothesis that contrast-enhanced magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) can differentiate reversible from irreversible myocardial injury, these modalities were used to study ischemia and reperfusion in a rat model. The presence of ischemia and reperfusion were confirmed with radiolabeled microspheres (n = 6). Groups of animals were subjected to either 16 (n = 17), 30 (n = 14), 60 (n = 11), or 90 (n = 14) minutes of left coronary artery (LCA) occlusion and 60 minutes reperfusion. After albumin-gadolinium (Gd)-DTPA injection, contrast-enhanced, T1-weighted, spin-echo proton images were acquired at baseline and every 16 minutes during LCA occlusion and reperfusion. In separate experiments, 31phosphorus (31P) spectra were acquired at similar time points during ischemia and reperfusion. After 16 minutes occlusion, normally perfused myocardium enhanced significantly compared with ischemic myocardium on MRI (104 +/- 7.9% vs. 61 +/- 11.0%, p less than 0.05, n = 5, mean +/- SEM, % of baseline value). MRS showed reduced phosphocreatine (PCr) and adenosine triphosphate (ATP) (58.8 +/- 2.4%, p less than or equal to 0.01; 81.4 +/- 2.4, p less than or equal to 0.01, n = 12). After 16 or 30 minutes ischemia, reflow resulted in uniform MRI signal intensity of the ischemic zone compared with normal myocardium (93.5 +/- 11.3 vs. 80.9 +/- 7.0, p = NS, n = 11, % of baseline value at 30 minutes reperfusion) and PCr recovery on MRS (94.3 +/- 4.0%, p = NS, n = 20, % baseline value at 30 minutes reflow). After 60 and 90 minutes ischemia, reflow resulted in marked enhancement of reperfused compared with normal myocardium on MRI (254.0 +/- 30.0 vs. 78.3 +/- 9.2, p less than or equal to 0.01, n = 10) and no recovery of PCr on MRS (64.1 +/- 3.0, p = NS, n = 14). Triphenyltetrazolium chloride (TTC) staining revealed transmural myocardial infarction (MI) in all hearts subjected to 60 or 90 minutes ischemia and reflow, and small nontransmural MIs in only 2/11 hearts subjected to 16 or 30 minutes ischemia and reperfusion. Thus, 1) MRI with albumin-Gd-DTPA is useful for identifying myocardial ischemia by enhancing the contrast between normally perfused and ischemic myocardia; 2) MRI with albumin-Gd-DTPA is useful for identifying reperfusion after myocardial ischemia; and 3) after reperfusion, reversible can be distinguished from irreversible myocardial injury by characteristic findings on MRI and MRS.     

Exploiting pre‐rRNA processing in Diamond Blackfan anemia gene discovery and diagnosis

Diamond Blackfan anemia (DBA), a syndrome primarily characterized by anemia and physical abnormalities, is one among a group of related inherited bone marrow failure syndromes (IBMFS) which share overlapping clinical features. Heterozygous mutations or single‐copy deletions have been identified in 12 ribosomal protein genes in approximately 60% of DBA cases, with the genetic etiology unexplained in most remaining patients. Unlike many IBMFS, for which functional screening assays complement clinical and genetic findings, suspected DBA in the absence of typical alterations of the known genes must frequently be diagnosed after exclusion of other IBMFS. We report here a novel deletion in a child that presented such a diagnostic challenge and prompted development of a novel functional assay that can assist in the diagnosis of a significant fraction of patients with DBA. The ribosomal proteins affected in DBA are required for pre‐rRNA processing, a process which can be interrogated to monitor steps in the maturation of 40S and 60S ribosomal subunits. In contrast to prior methods used to assess pre‐rRNA processing, the assay reported here, based on capillary electrophoresis measurement of the maturation of rRNA in pre‐60S ribosomal subunits, would be readily amenable to use in diagnostic laboratories. In addition to utility as a diagnostic tool, we applied this technique to gene discovery in DBA, resulting in the identification of RPL31 as a novel DBA gene. Am. J. Hematol. 89:985–991, 2014. © 2014 Wiley Periodicals, Inc.     

B1 but not B2 bradykinin receptor agonists promote DU145 prostate cancer cell proliferation and migration

Background

The kallikrein-kinin system (KKS) is an endogenous pathway involved in angiogenesis and tumourigenesis, both vital for cancer growth and progression.

Objectives

To investigate the effect of two bradykinin receptor (B1R and B2R) agonists on growth and motility of prostate tumour (DU145) and micro-vascular endothelial cells (dMVECs).

Methods

Increasing concentrations of selective B1R and B2R agonists were added to cultured cells. Cell proliferation and migration were assessed using the 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and modified Boyden Chamber assays, respectively. Where significant stimulation was found, the influence of an antagonist was also investigated.

Results

Neither growth nor motility of endothelial cells was affected by either agonist. In DU145 cells, while the B2R agonist was without any significant effect, the B1R agonist stimulated proliferation and migration at concentrations of 10nM and 50nM respectively. Further, this effect was abrogated when cells were pre-incubated with a B1R antagonist.

Conclusions

Unlike the physiologically-active B2R, the pathologically-inducible B1R may be implicated in prostate tumourigenic events. The involvement of the KKS in malignant prostate pathology supports on-going exploration of bradykinin receptor antagonists as target candidates in the development of alternate approaches to cancer therapy.