Antoinette Rabel

LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo

Reactivation of fetal hemoglobin (HbF) holds therapeutic potential for sickle cell disease and β-thalassemias. In human erythroid cells and hematopoietic organs, LIN28B and its targeted let-7 microRNA family, demonstrate regulated expression during the fetal-to-adult developmental transition. To explore the effects of LIN28B in human erythroid cell development, lentiviral transduction was used to knockdown LIN28B expression in erythroblasts cultured from human umbilical cord CD34+ cells. The subsequent reduction in LIN28B expression caused increased expression of let-7 and significantly reduced HbF expression. Conversely, LIN28B overexpression in cultured adult erythroblasts reduced the expression of let-7 and significantly increased HbF expression. Cellular maturation was maintained including enucleation. LIN28B expression in adult erythroblasts increased the expression of γ-globin, and the HbF content of the cells rose to levels >30% of their hemoglobin. Expression of carbonic anhydrase I, glucosaminyl (N-acetyl) transferase 2, and miR-96 (three additional genes marking the transition from fetal-to-adult erythropoiesis) were reduced by LIN28B expression. The transcription factor BCL11A, a well-characterized repressor of γ-globin expression, was significantly down-regulated. Independent of LIN28B, experimental suppression of let-7 also reduced BCL11A expression and significantly increased HbF expression. LIN28B expression regulates HbF levels and causes adult human erythroblasts to differentiate with a more fetal-like phenotype.

CT of sclerotic bone lesions: imaging features differentiating tuberous sclerosis complex with lymphangioleiomyomatosis from sporadic lymphangioleiomymatosis.

PURPOSE To determine if sclerotic bone lesions evident at body computed tomography (CT) are of value as a diagnostic criterion of tuberous sclerosis complex (TSC) and in the differentiation of TSC with lymphangioleiomyomatosis (LAM) from sporadic LAM. MATERIALS AND METHODS Informed consent was signed by all patients in this HIPAA-compliant study approved by the institutional review board. Retrospective analysis was performed of the body CT studies of 472 patients: 365 with sporadic LAM, 82 with TSC/LAM, and 25 with TSC. The images were reviewed by using a picture archiving and communication system workstation with bone settings (window width, 1500 HU; window level, 300 HU) and fit-to-screen option. CT image characteristics assessed included shape, size, and distribution of sclerotic bone lesions with subsequent calculation of differences in the frequency of these lesions. RESULTS Most commonly the sclerotic bone lesions were round, measured 0.3 cm (range, 0.2-3.2), and were distributed throughout the spine. The frequencies differed among the three patient groups Four or more sclerotic bone lesions were detected in all 25 (100%) of those with TSC, with a sensitivity of .89 (72 of 82) and specificity of .97 (355 of 367) in the differentiation of sporadic LAM from TSC/LAM (P < .01). CONCLUSION The number of sclerotic bone lesions at body CT is of potential value in the diagnosis of TSC and in the differentiation of patients with sporadic LAM from those with TSC/LAM. (c) RSNA, 2010.

Reversible Airflow Obstruction in Lymphangioleiomyomatosis

BACKGROUND We previously reported that approximately one-fourth of patients with lymphangioleiomyomatosis (LAM) may respond to therapy with bronchodilators. However, the validity of those observations has been questioned. The aims of the present study were to determine the prevalence of reversible airflow obstruction in patients with LAM and to identify associated clinical and physiologic parameters. METHODS First, the clinical and physiologic characteristics of 235 patients were analyzed to determine the frequency of the response to albuterol during a total of 2,307 visits. Second, we prospectively evaluated the response to albuterol (2.5 mg) and ipratropium (500 mug) in 130 patients, and correlated their responses with their clinical and physiologic characteristics. RESULTS In the retrospective study, 51% of the patients responded at least once to bronchodilators; of these, 12% responded >/= 50% of the time. A higher frequency of positive bronchodilator responses was associated with greater rates of decline in FEV(1) and diffusing capacity of the lung for carbon monoxide (Dlco). In the prospective study, 39 patients (30%) responded to bronchodilators, including 12 to ipratropium, 9 to albuterol, and 18 to both. The prevalence of asthma and smoking in the 39 responders was not different from that seen in the 91 nonresponders. Patients who responded to ipratropium, albuterol, or both had significantly (p < 0.02) lower FEV(1) and Dlco, and a greater rate of FEV(1) decline (p = 0.044) and Dlco decline (p = 0.039) than patients who did not respond to these bronchodilators. After adjusting for FEV(1)/FVC ratio, Dlco decline also was greater in responders than in nonresponders (p = 0.009). CONCLUSIONS Patients with LAM may have partially reversible airflow obstruction. A positive response to bronchodilators is associated with an accelerated rate of decline in pulmonary function.

Expression of growth differentiation factor 15 is not elevated in individuals with iron deficiency secondary to volunteer blood donation.

BACKGROUND: Low serum hepcidin levels provide a physiologic response to iron demand in patients with iron deficiency (ID). Based on a discovery of suppressed hepcidin expression by a cytokine named growth differentiation factor 15 (GDF15), it was hypothesized that GDF15 may suppress hepcidin expression in humans with ID due to blood loss.

HEPCIDIN, ANAEMIA, AND PROSTATE CANCER

Sir, Anaemia is a common haematological abnormality in patients with cancer. The anaemia of chronic disease or inflammation is thought to be a result of induction of the iron-regulatory hormone, hepcidin, by inflammatory cytokines, especially interleukin 6 (IL-6) [1]. Elevation of hepcidin by IL-6 leads to hypoferraemia associated with anaemia and iron-restricted erythropoiesis [2]. A recent study showed that patients with certain early-stage malignancies are less prone to developing anaemia of chronic disease [3]. Among more than 1400 men with prostate cancer, the odds of developing anaemia before diagnosis were not increased. We hypothesized that the paucity of significant anaemia during the early stages of prostate cancer may be due to potentially balanced effects of hepcidin-regulating cytokines produced by prostate cancer cells. Like IL-6, growth differentiation factor 15 (GDF15), also a cytokine, is overexpressed in prostate cancer tissues [4,5]. However, unlike IL-6, GDF15 suppresses hepcidin expression [6]. Hepcidin, IL-6 and GDF15, as well as iron and haematological variables were therefore examined to determine the potential role of iron-restricted anaemia in men with prostate cancer. In all, 29 men with prostate cancer were recruited for this preliminary study and grouped according to the presence or absence of metastatic bone disease. All studies were conducted at the National Institutes of Health (NIH) and were approved by both the Office of Human Subjects Research and the Institutional Review Board. Mild anaemia (defined here as a haemoglobin [Hb] level of 10.0–13.7 g/dL) was detected in >80% of men in both groups (Fig. 1). As expected, Hb levels were lower in men with metastatic bone disease, with a mean (SD) Hb of 11.6 (1.4) g/dL (P = 0.009) and haematocrit (Hct) of 36.5 (4.1)% (P = 0.025), than in men without metastasis, with a mean (SD) Hb of 12.8 (0.8) g/dL and Hct of 39.4 (2.1)%. None of the 29 men had received erythropoietin or transfused blood, and only one had received iron supplements within the 6 weeks prior to the study. The iron parameters were within the normal range in >80% of the men. FIG. 1 Hepcidin, IL-6, and GDF15 levels in men with prostate cancer with or with no bone metastasis. Serum concentrations of (A) hepcidin, (B) IL-6, and (C) GDF15 from men with prostate cancer, without (−, 14 men) or with (+, 15 men) bone metastasis. ... ELISA-based assay systems were used to examine the serum levels of hepcidin, GDF15 and IL-6 [6, 7]. As shown in the attached Figure 1A, 23 of 29 men studied had measurable hepcidin within the assay’s normal range (10–298 ng/mL) [7]. Five men had hepcidin levels at or near the lower limit of the assay (5.0 ng/mL). Serum hepcidin level increased beyond the normal range only in one man. In the absence of bone metastasis, hepcidin levels clustered in the low normal range, with a mean value of 42 ng/mL. Even with bone metastasis, over half of the men maintained hepcidin levels of <100 ng/mL, but values were more broadly distributed within the normal range. Statistical analyses showed that the mean (SD) hepcidin level was significantly increased, at 121 (111) ng/mL (P = 0.028) in men with bone metastasis compared with men with no metastasis, at 42.0 (64.2) ng/mL. Further comparison showed that IL-6 levels increased in men with bone metastasis (Fig. 1B), while GDF15 levels increased in both groups. However, increases in GDF15 were more robust and consistent in the men with metastatic disease (Fig. 1C). These data support the notion that hepcidin regulation, and its contribution to subsequent development of anaemia, is complex in men with prostate cancer. In addition to IL-6, serum GDF15 may play a role in hepcidin regulation. Increased serum GDF15 is associated with disease progression in men with prostate cancer, and expression of GDF15 is significantly higher in cancerous prostatic tissue than in normal tissue [4, 5]. Larger studies are needed to determine if the high level of GDF15 combined with mild elevation of serum IL-6 have balanced effects upon serum hepcidin levels in men with prostate cancer.

CT of Pleural Abnormalities in Lymphangioleiomyomatosis and Comparison of Pleural Findings After Different Types of Pleurodesis

OBJECTIVE The objective of our article was to describe the spectrum and frequency of pleural abnormalities on CT in patients with lymphangioleiomyomatosis (LAM) and the pleural findings associated with different types of pleurodesis (talc, mechanical, and chemical) performed to treat the complications of pleural disease in these patients. MATERIALS AND METHODS Two hundred fifty-eight patients with LAM underwent CT of the chest. Pleural abnormalities assessed included pleural thickening, presence of a pleural mass, areas of high attenuation, effusion, and pneumothorax. In patients who had had pleurodesis, the CT findings were correlated with the type of procedure performed. RESULTS One hundred thirty-three (52%) of 258 patients had pleurodesis (unilateral, 68/133; bilateral, 65/133). Pleural abnormalities were more common in patients who had pleurodesis (101/133, 76%) than in those who had not (47/125, 38%) and were more prevalent on the operated side than on the unoperated side of those 68 patients who had unilateral pleurodesis. The frequencies of findings for the group without pleurodesis versus the group with pleurodesis were pleural thickening (26% vs 65%), effusion (10% vs 13%), loculated effusion (2.4% vs 11%), pneumothorax (1.6% vs 10%), areas of high attenuation (1.6% vs 23%), and mass (0.8% vs 14%), respectively. Areas of high attenuation in the pleura were present in all types of pleurodesis (mechanical, 8%; chemical, 13%; talc, 40%) and in two patients who had had repeated thoracentesis or pleurectomy. Pleural masses were present in patients who had had all types of pleurodesis (mechanical, 10%; chemical, 9%; talc, 24%) and in one patient who had had thoracentesis and thoracostomy; the masses commonly enhanced and did not change in size over time. CONCLUSION Pleural abnormalities are common in patients with LAM as complications of the disease itself and as sequelae of pleurodesis and other pleura manipulations. Pneumothorax and pleural effusion result from the underlying pathophysiology of LAM, whereas areas of high attenuation and masses develop after all types of pleurodesis and other manipulations of the pleura (i.e., thoracentesis, thoracostomy).

Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts.

Increasing fetal hemoglobin (HbF) levels in adult humans remains an active area in hematologic research. Here we explored erythroid-specific LIN28A expression for its effect in regulating gamma-globin gene expression and HbF levels in cultured adult erythroblasts. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes. Transgene expression of LIN28A with a linked puromycin resistance marker was restricted to the erythroid lineage as demonstrated by selective survival of erythroid colonies (greater than 95% of all colonies). Erythroblast LIN28A over-expression (LIN28A-OE) did not significantly affect proliferation or inhibit differentiation. Greater than 70% suppression of total let-7 microRNA levels was confirmed in LIN28A-OE cells. Increases in gamma-globin mRNA and protein expression with HbF levels reaching 30–40% were achieved. These data suggest that erythroblast targeting of LIN28A expression is sufficient for increasing fetal hemoglobin expression in adult human erythroblasts.

LIN28A Expression Reduces Sickling of Cultured Human Erythrocytes

Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with sickle cell disease (SCD) and the beta-thalassemias. It was recently reported that increased expression of LIN28 proteins or decreased expression of its target let-7 miRNAs enhances HbF levels in cultured primary human erythroblasts from adult healthy donors. Here LIN28A effects were studied further using erythrocytes cultured from peripheral blood progenitor cells of pediatric subjects with SCD. Transgenic expression of LIN28A was accomplished by lentiviral transduction in CD34(+) sickle cells cultivated ex vivo in serum-free medium. LIN28A over-expression (LIN28A-OE) increased HbF, reduced beta (sickle)-globin, and strongly suppressed all members of the let-7 family of miRNAs. LIN28A-OE did not affect erythroblast differentiation or prevent enucleation, but it significantly reduced or ameliorated the sickling morphologies of the enucleated erythrocytes.

A synthetic model of human beta-thalassemia erythropoiesis using CD34+ cells from healthy adult donors

Based upon the lack of clinical samples available for research in many laboratories worldwide, a significant gap exists between basic and clinical studies of beta-thalassemia major. To bridge this gap, we developed an artificially engineered model for human beta thalassemia by knocking down beta-globin gene and protein expression in cultured CD34+ cells obtained from healthy adults. Lentiviral-mediated transduction of beta-globin shRNA (beta-KD) caused imbalanced globin chain production. Beta-globin mRNA was reduced by 90% compared to controls, while alpha-globin mRNA levels were maintained. HPLC analyses revealed a 96% reduction in HbA with only a minor increase in HbF. During the terminal phases of differentiation (culture days 14–21), beta-KD cells demonstrated increased levels of insoluble alpha-globin, as well as activated caspase-3. The majority of the beta-KD cells underwent apoptosis around the polychromatophilic stage of maturation. GDF15, a marker of ineffective erythropoiesis in humans with thalassemia, was significantly increased in the culture supernatants from the beta-KD cells. Knockdown of beta-globin expression in cultured primary human erythroblasts provides a robust ex vivo model for beta-thalassemia.

IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

Significance Fetal hemoglobin (HbF) expression is a tissue- and stage-specific marker of ontogeny in large mammals, which also has therapeutic importance for beta hemoglobinopathies. The heterochronic let-7 miRNAs, which regulate the time and sequence of stage-specific developmental events, have also been shown to regulate HbF in adult human erythroblasts. Here we provide a focused investigation of a let-7 target named “insulin-like growth-factor 2 mRNA-binding protein 1” (IGF2BP1), for its potential role in reactivating HbF in adult cells. IGF2BP1 overexpression caused robust increases of HbF and a reversal from the adult toward a fetal-like globin phenotype. IGF2BP1 effects are partially mediated by posttranscriptional regulation of the known HbF regulator BCL11A. These results suggest a novel mechanism for the regulation of BCL11A and HbF in humans. Here we investigated in primary human erythroid tissues a downstream element of the heterochronic let-7 miRNA pathway, the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), for its potential to affect the hemoglobin profiles in human erythroblasts. Comparison of adult bone marrow to fetal liver lysates demonstrated developmental silencing in IGF2BP1. Erythroid-specific overexpression of IGF2BP1 caused a nearly complete and pancellular reversal of the adult pattern of hemoglobin expression toward a more fetal-like phenotype. The reprogramming of hemoglobin expression was achieved at the transcriptional level by increased gamma-globin combined with decreased beta-globin transcripts resulting in gamma-globin rising to 90% of total beta-like mRNA. Delta-globin mRNA was reduced to barely detectable levels. Alpha-globin levels were not significantly changed. Fetal hemoglobin achieved levels of 68.6 ± 3.9% in the IGF2BP1 overexpression samples compared with 5.0 ± 1.8% in donor matched transduction controls. In part, these changes were mediated by reduced protein expression of the transcription factor BCL11A. mRNA stability and polysome studies suggest IGF2BP1 mediates posttranscriptional loss of BCL11A. These results suggest a mechanism for chronoregulation of fetal and adult hemoglobin expression in humans.