Clonal hematopoiesis related TET2 loss-of-function impedes IL1ß-mediated epigenetic reprogramming in hematopoietic stem and progenitor cells.

Clonal hematopoiesis (CH) is defined as a single hematopoietic stem/progenitor cell (HSPC) gaining selective advantage over a broader range of HSPCs. When linked to somatic mutations in myeloid malignancy-associated genes, such as TET2-mediated clonal hematopoiesis of indeterminate potential or CHIP, it represents increased risk for hematological malignancies and cardiovascular disease. IL1ß is elevated in patients with CHIP, however, its effect is not well understood. Here we show that IL1ß promotes expansion of pro-inflammatory monocytes/macrophages, coinciding with a failure in the demethylation of lymphoid and erythroid lineage associated enhancers and transcription factor binding sites, in a mouse model of CHIP with hematopoietic-cell-specific deletion of Tet2. DNA-methylation is significantly lost in wild type HSPCs upon IL1ß administration, which is resisted by Tet2-deficient HSPCs, and thus IL1ß enhances the self-renewing ability of Tet2-deficient HSPCs by upregulating genes associated with self-renewal and by resisting demethylation of transcription factor binding sites related to terminal differentiation. Using aged mouse models and human progenitors, we demonstrate that targeting IL1 signaling could represent an early intervention strategy in preleukemic disorders. In summary, our results show that Tet2 is an important mediator of an IL1ß-promoted epigenetic program to maintain the fine balance between self-renewal and lineage differentiation during hematopoiesis.

Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are characterized by their ability to differentiate into all hematopoietic cell lineages while retaining their capacity for self renewal. One of the predictions of this model is the existence of a heterogeneous pool of HSCs, some members of which are destined to become lineage restricted progenitor cells while others function to renew the stem cell pool. To test whether HSCs are heterogeneous with respect to cell cycle status, we determined the fraction of phenotypically defined murine HSCs (Thy1.1lo Lin-/lo Sca-1+) that contain > 2n amount of DNA as measured by propidium iodide staining, Hoechst dye uptake and [3H]thymidine labeling; that fraction is 18-22%. In contrast, in the developing fetal liver, 40% of HSCs are in the S/G2/M phases of the cell cycle. Those HSCs which exhibit a low level of staining with rhodamine 123 are almost exclusively in G0/G1 (97%) whereas only 70% of HSCs which stain brightly for rhodamine 123 are in G0/G1. The injection of 100 G0/G1 HSCs rescued 90% of lethally irradiated mice in contrast to 100 S/G2/M HSCs, which protected only 25% of lethally irradiated recipients. Enhanced long-term donor-derived multilineage reconstitution of the peripheral blood was observed in recipients of 100 G0/G1 HSCs compared to recipients of 100 S/G2/M cells. These data indicate that a significant proportion of HSCs are actively proliferating during steady state hematopoiesis and that this subpopulation of cells exhibits reduced stem cell activity.

Heterogeneity of hematopoietic stem cells.

Hematopoietic stem cells are capable of multi-lineage differentiation to all blood cell types as well as self-renewal and radioprotection. Thy-1.1lo Lin-/lo Sca-1+ cells are a heterogeneous mixture of quiescent and self-renewing hematopoietic stem cells as well as multi-lineage expanding cells.

Thymic origin of the prolactin-dependent Nb2 lymphoma cell line.

Blast cells in acute leukemia and lymphoma appear to be "frozen" at various stages of lymphoid cell differentiation. The enzymatic and antigenic phenotypes expressed by these cells often correspond to the gene products of their normal precursors. We have used various immunocytochemical and enzymatic techniques to identify membrane, nuclear, and cytoplasmic markers associated with the prolactin-dependent Nb2 lymphoma cell line. The Nb2 cells, whether stationary or in log-phase growth, did not express any surface immunoglobulin. However, 100% of the Nb2 cells bound both a monoclonal antibody raised to rat thymocyte W3/25-HLK, which specifically binds an antigenic determinant on rat T-helper cells, and second monoclonal antibody OX8-HL, which identifies rat nonhelper T-cells. Transmission electron microscopy showed no evidence of phagocytic vacuoles, and activity of the lysosomal enzyme muramidase was also absent. There was no evidence of the DNA polymerase enzyme terminal deoxynucleotidyl transferase. alpha-Naphthyl acetate esterase activity was indicated in about 50% of the Nb2 cells by a faint particulate cytoplasmic staining similar to that found in thymocytes. Rosette formation with guinea pig erythrocytes, a property of mature rat thymocytes, was not observed with Nb2 cells. The data suggest that the Nb2 tumor may have arisen from a thymocyte at an intermediate stage of differentiation. The presence of Thy-like alpha-naphthyl acetate esterase pattern and the binding of both W3/25-HLK and OX8-HL support the thymic origin and relative immaturity of these lymphoid cells. It is becoming increasingly apparent that a significant proportion of lymphomas and leukemias also originate in undifferentiated thymic cels.

Prevalence of multidrug resistance related to activation of the mdr1 gene in human sarcoma mutants derived by single-step doxorubicin selection.

Fluctuation analysis experiments were performed in the human sarcoma cell line MES-SA to assess whether selection or induction mechanisms determine resistance to doxorubicin (DOX), mutation rates, and the nature of the surviving clones. Thirteen flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 3.3 x 10(6) cells. After reseeding in 96-well plates, each population was treated with 40 nM DOX for 2 weeks. Surviving colonies were scored and harvested. Clones were propagated and analyzed for drug resistance phenotype. Expression of the mdr1, mrp, and topoisomerase II alpha and II beta genes was analyzed by reverse transcription-polymerase chain reaction. Accumulation of the P-glycoprotein substrate rhodamine-123 was measured by flow cytometry, with and without the cyclosporin D analogue SDZ PSC 833. Cellular glutathione levels were measured by flow cytometry, and M(r) 110,000 vesicular protein (p110) expression was detected by immunohistochemistry. Analysis of variance supported the hypothesis of spontaneous mutations rather than induction conferring DOX resistance. At this stringent level (5-6 log cell killing) of drug exposure, the mutation rate was estimated at 1.8 x 10(-6) per cell generation. All 30 propagated clones demonstrated cross-resistance to vinblastine, etoposide, and paclitaxel (Taxol), but not to cisplatin or bleomycin. Increased mRNA levels of mdr1 were observed in all 27 clones tested, including at least 1 from each of the 13 populations. No alterations were found in expression or level of topoisomerase II alpha or II beta, mrp, glutathione, and p110. Expression of P-glycoprotein was confirmed by flow cytometry using the monoclonal antibody UIC2. In almost all tested clones, decreased intracellular rhodamine-123 accumulation was modulated by 2 microM SDZ PSC 833, and the vinblastine resistance in all examined clones was completely reversed by SDZ PSC 833 and verapamil. Our study demonstrates that survival of cells exposed to DOX in a single step occurs as a result of a stochastic process consistent with mutational events. Activation of the mdr1 gene is the predominant mechanism selected by DOX in these resistant clones.

Expression of the c-myc protooncogene in human prostatic carcinoma and benign prostatic hyperplasia.

We have examined the level of c-myc transcripts in prostate tissue obtained from patients with both benign prostatic hyperplasia and adenocarcinoma of the prostate. A significantly higher level of c-myc transcripts is observed in patients with adenocarcinoma (P less than 0.05). In addition, a subset of patients with adenocarcinoma had levels of c-myc transcripts 2-fold higher than the mean level for this group. These preliminary results indicate that the investigation of c-myc levels as a prognostic indicator in prostatic carcinoma is warranted.

Postnatal atresia of extraparenchymal pulmonary veins, fulminant necrotizing pulmonary arteritis and elevated circulating immune complexes.

An infant with operatively corrected total anomalous pulmonary venous connection developed postnatal atresia of the extraparenchymal left pulmonary veins with secondary arteritis of the ipsilateral intraparenchymal pulmonary arteries. Atresia of the right or left main pulmonary veins or of the common pulmonary vein is a rare occurrence and it is believed that association of such with necrotizing pulmonary arteritis has never been reported. This case illustrates the potential consequences of severe pulmonary venous obstruction in the absence of a left to right shunt.

Progenipoietin-1: a multifunctional agonist of the granulocyte colony-stimulating factor receptor and fetal liver tyrosine kinase-3 is a potent mobilizer of hematopoietic stem cells.

OBJECTIVE: Progenipoietin-1 is an agonist of both the granulocyte colony-stimulating factor and fetal liver tyrosine kinase-3 receptors capable of inducing the proliferation of multiple hematopoietic cell lineages. The potential of progenipoietin-1 to mobilize transplantable hematopoietic stem cells into the peripheral blood was evaluated. METHODS: Cohorts of donor mice were treated with either progenipoietin-1, fetal liver tyrosine kinase-3 ligand, granulocyte colony-stimulating factor, or a vehicle control. Hematopoietic progenitor/stem-cell activity in donor blood was assayed by radioprotection, multilineage reconstitution, secondary transplantation, and competitive repopulation. RESULTS: Only 1 microL of peripheral blood from progenipoietin-1-treated donors was required to protect 80% of lethally irradiated mice, while in contrast 1 microL of peripheral blood from granulocyte colony-stimulating factor-treated donors failed to protect any recipients. The radioprotected recipients of progenipoietin-1-treated donor cells showed donor-derived (Ly5.2) multilineage hematopoietic reconstitution for up to 6 months. Serial transplantation studies using bone marrow from radioprotected, chimeric recipients demonstrated long-term donor-derived hematopoiesis, indicating the successful transplantation of multipotent hematopoietic stem cells. The engraftment potential of progenipoietin-1 donor-derived cells was directly compared with donors treated with granulocyte colony-stimulating factor or fetal liver tyrosine kinase-3 ligand alone or in combination. Both spleen colony-forming activity and competitive repopulating activity was highest in the blood from progenipoietin-1-treated donors. CONCLUSIONS: These studies demonstrate that progenipoietin-1 is a potent mobilizer of transplantable hematopoietic stem cells and indicate that this dual-receptor agonist has greater biologic activity than its constituent molecules.

Human growth hormone induces and maintains c-myc gene expression in Nb2 lymphoma cells.

The effect of human GH (hGH) on c-myc gene expression and cell proliferation in the Nb2 lymphoma cell line was examined. The addition of hormone to stationary cultures of Nb2 cells resulted in an increased accumulation of c-myc transcripts that was detectable within 15 min, reached a maximum induction of 25-fold in 3 h, and then gradually declined. Although maximal accumulation of c-myc transcripts occurred within 3 h after hGH addition, removal of the hormone even after 4 h of treatment resulted in a failure of the cells to proliferate. This loss of proliferative capacity following hormone removal was accompanied by a 20-fold reduction in the level of c-myc transcripts. These results indicate that hGH induced proliferation of Nb2 cells is associated with the induction and maintenance of c-myc gene expression.

Ligation of patent ductus arteriosus in premature infants: importance of accurate anatomic definition.

The aortic arch anatomy of the premature infant may not be as simple to determine as the anatomy of an older child. A case of a premature infant who had inadvertent ligation of the left pulmonary artery during attempted ligation of a patent ductus arteriosus is described. On the fourth day following the initial operation, the patient underwent successful surgery to remove the pulmonary artery ligature and to ligate the persistent patent ductus arteriosus. A lung scan on the seventh day following removal of the pulmonary artery ligature showed normal perfusion of both lungs. Six months later, the child's growth, development, and chest roentgenogram were normal.

Critical aortic coarctation: patch aortoplasty in infants less than age 3 months.

Twenty-three infants less than age 3 months (mean age 31 days) underwent patch aortoplasty for relief of coarctation of the aorta. All had intractable congestive heart failure, despite aggressive medical therapy. Each infant had other cardiac anomalies, including patent ductus arteriosus (83 percent) and ventricular septal defect (74 percent). All patients underwent closure of the ductus arteriosus and patch angioplasty of the aorta to produce a luminal diameter of at least 16 mm. In addition, 9 of the 17 patients (53 percent) with a large shunt ventricular septal defect underwent pulmonary arterial banding. There was one hospital death 42 days after operation secondary to bowel perforation and sepsis. Hospitalization beyond 21 days postoperatively was always due to other unrepaired cardiac lesions. The three late deaths at 3, 9 and 18 months after operation were associated with additional major anomalies. Fourteen patients have had postoperative catheterization. No gradient was found across the site of coarctation repair, but one patient had a gradient between the left carotid and left subclavian arteries. Surgical repair of critical coarctation of the aorta in infants can safely be offered despite the presence of other cardiac anomalies.

Sustained ventricular tachycardia after repair of tetralogy of Fallot: new electrophysiologic findings.

Ventricular arrhythmia originating from the outflow tract of the right ventricle is a presumed cause of late sudden death in patients after repair of tetralogy of Fallot. Exercise testing has been shown to enhance detection, and phenytoin has been shown to control ventricular arrhythmias in these patients. This study reports new findings in 3 patients who underwent electrophysiologic studies at postoperative cardiac catheterization; in each, sustained ventricular tachycardia was induced and found to originate from the inflow-septal area of the right ventricle. Using serial studies, the same sustained ventricular tachycardia was induced during therapeutic serum concentrations of phenytoin but not after propranolol. No patient had ventricular arrhythmia during a 24-hour electrocardiogram or during exercise. Although no patient had normal hemodynamic function, only 1 patient had right ventricular pressure greater than two-thirds systemic pressure. Each patient had undergone initial intracardiac repair at a relatively late age (3, 9, and 9 years).

Retraction of bioprosthetic heart valve cusps: a cause of wide-open regurgitation in right-sided heart valves.

Most failures of bioprosthetic heart valves in children are due to stenosis secondary to thrombus, calcific deposits, or tissue ingrowth. Valve failures due to regurgitation typically involve cuspal detachment, tears, or perforations. We present four cases of prosthetic valve regurgitation in children caused by cuspal retraction without stenosis and describe the morphologic findings related to the valves at autopsy or explantation. A mononuclear cell and giant cell response to the cusps of the valve was a striking finding in one patient.

Kindness pays dividends: the medical benefits of intercostal nerve block following thoracotomy.

Postoperative pain is an important factor in the management of children undergoing thoracotomy. Intercostal nerve block has been used in adult patients, but its applicability in the pediatric age group has not been previously evaluated. Eighty-nine children (85 girls and 31 boys) aged 6 months to 16 years (mean age 4.7 years) underwent ligation of a patent ductus arteriosus (PDA) through a left thoracotomy. Twenty-nine children received intercostal blocks with bupivacaine from the level of the second to sixth thoracic vertebrae. Sixty cases constituted the control group. The patients with intercostal block had fewer doses of pain medication postoperatively, 2.7 mean (0 to 9), than did the control patients, 3.9 mean (0 to 21). The mean hospital stay was shortened in the patients with nerve block, 5.1 days versus 7.3 days for the control group. No ill effects of bupivacaine were noted. We conclude that intercostal nerve block is a valuable procedure reducing the need for postoperative analgesia and shortening hospital stay.

Echocardiographic identification of a pericardial cyst.

The echocardiogram of a woman with an anterior mediastinal mass showed an echo-free space between the chest wall and the right ventricle. The interface of mass with pericardium was outlined by a narrow echo. These findings suggested a homogeneous fluid-filled, thin-walled sac. Postoperation, echocardiography was normal.

Prostaglandin E1 therapy. Is it associated with a higher incidence of wound infection in the cyanotic neonate?

Prostaglandin (PGE1) may be used to maintain ductal patency in the infant with cyanotic congenital heart disease, but the risk of infection may be increased. Between October, 1976 and December, 1982, 38 neonates with complex cyanotic congenital heart disease required operations creating systemic-to-pulmonary artery shunts. Of 13 patients who did not receive PGE1 therapy, none developed a wound infection. Of 25 patients who did receive PGE1 therapy, four (16 percent) developed a significant wound infection. The two patient groups were similar when compared by age and weight at operation, by severity of heart disease and by the presence of other congenital anomalies. Pathogenic Staphylococcus epidermidis was recovered from all infected wounds, all of which responded favorably over a period of two to four weeks with a short course of antibiotics and wound debridement.

Pulmonary resection for invasive Aspergillus infections in immunocompromised patients.

Standard antifungal medical therapy of invasive pulmonary aspergillosis that occurs in immunocompromised patients with hematologic diseases with neutropenia or in liver transplant recipients results in less than a 5% survival. In view of these dismal mortality rates, we adopted an aggressive approach with resection of the involved area of lung along with systemic antifungal therapy when localized invasive pulmonary aspergillosis developed in these patients. Between January 1987 and December 1993, 14 patients with hematologic diseases and 2 liver transplant recipients underwent resection of acute localized pulmonary masses suggestive of invasive pulmonary aspergillosis a median of 7.5 days (range 1 to 45 days) after the diagnosis was clinically suggested and confirmed by chest computed tomographic scans. Operative procedures done included two pneumonectomies, one bilobectomy with limited thoracoplasty, nine lobectomies, and five wedge resections (one patient with hematologic disease had two procedures). All patients were treated before and after the operation with antifungal agents. Nine (64%) of 14 patients with hematologic disease and 2 (100%) of 2 liver transplant recipients survived the hospitalization with no evidence of recurrent Aspergillus infection after a median 8 months of follow-up (range 3 to 82 months). The five hospital deaths (all patients with hematologic diseases) occurred a median of 20 days after operation from diffuse alveolar hemorrhage in three, graft-versus-host disease in one, and multiple organ system failure with presumed disseminated Aspergillus infection in one. Four of the five deaths were in patients with allogeneic bone marrow transplants. Two of the three patients requiring resection of multiple foci of infection died, as did the only patient who was preoperatively ventilator dependent. In immunocompromised patients with hematologic diseases or liver transplantation with invasive pulmonary aspergillosis, early pulmonary resection should be strongly considered when the characteristic clinical and radiographic pictures appear.

Combined prostaglandin therapy and ductal formalin infiltration in neonatal pulmonary oligemia.

Prostaglandins and ductal formalin infiltration, singly and together, have been used in efforts to improve pulmonary flow in very ill newborn infants with right ventricular outflow tract obstruction. To evaluate the efficacy of concurrent use of prostaglandins and ductal formalin infiltration, we have reviewed our experience with 25 infants with right ventricular outflow tract obstruction and prostaglandin-ductal formalin infiltration therapy. Prostaglandin therapy was begun 22 +/- 21 hours (range 20 to 93 hours) before and was continued 20 +/- 18 hours (range 0 to 62 hours) following ductal formalin infiltration; prostaglandin administration was initiated at a dose of 0.05 to 0.1 microgram/kg/min and tapered postoperatively. Clinical cyanosis was diminished in 20 of 25 infants (80%) postoperatively. Systemic arterial pH and oxygen saturation both improved following prostaglandin-ductal formalin infiltration therapy from 7.35 to 7.41 (p less than 0.001) and from 35.7 to 50.3 (p less than 0.001), respectively. Persistent ductal patency (mean 219 +/- 191 days) was observed in 17 survivors of the early postoperative period (more than 14 days). Two of five infants who died within 14 days of operation had a widely patent ductus with resultant progressive congestive heart failure. The other three infants died as a result of operative technical problems, dysrhythmias, and thrombotic ductal closure, respectively. No correlation was observed between duration of ductus patency and operatively determined size of ductus, total prostaglandin dose, or duration of prostaglandin infusion. Secondary operative intervention was delayed by 92 +/- 74 days with prostaglandin-ductal formalin infiltration therapy; thus prostaglandin-ductal formalin infiltration therapy may have a role in selected neonates with right ventricular outflow tract obstruction.

Serum concentrations of prostacyclin and thromboxane in children before, during, and after cardiopulmonary bypass.

Twenty-six consecutive pediatric patients undergoing reparative procedures necessitating cardiopulmonary bypass were prospectively studied to determine changes in serum levels of 6-keto-prostaglandin F1 alpha and thromboxane B2. Cardiac lesions included acyanotic lesions (five patients), obstructive lesions (10 patients), and right-to-left shunts (11 patients). There was a significant (p less than 0.05) increase in 6-keto-prostaglandin F1 alpha from preoperative levels measured at the time of arterial and venous cannula insertion. This concentration was maintained throughout cardiopulmonary bypass and remained significantly elevated (p less than 0.001) in the recovery room, but returned to preoperative levels by the morning after the operation. Preoperative levels of thromboxane B2 varied widely and were not significantly different from intraoperative levels. The postoperative levels of thromboxane B2, however, were significantly different (p less than 0.05) from the intraoperative levels. In the pediatric age group undergoing cardiopulmonary bypass, 6-keto-prostaglandin F1 alpha and thromboxane B2 change during bypass but do not significantly differ when preoperative levels are compared to postoperative values.

Toxic megacolon: a life-threatening complication of high-dose therapy and autologous stem cell transplantation among patients with AL amyloidosis.

AL amyloidosis is a plasma cell disorder in which tissue deposition of immunoglobulin light chains leads to organ dysfunction. Recent reports of high-dose therapy with autologous stem cell transplantation for amyloidosis suggest higher response rates and extended survival compared to those seen with conventional chemotherapy. However, substantial treatment-related toxicity has been observed. This case series describes our institutional experience with autologous transplantation in four patients with amyloidosis with an emphasis on unique gastrointestinal toxicities, including toxic megacolon.