Pilot dose-finding and safety study of bivalirudin in infants <6 months of age with thrombosis

BACKGROUND: Thrombosis is not uncommon in children with serious medical conditions. Unfractionated heparin, the most commonly used anticoagulant in the acute management of thrombosis, has significant pharmacologic limitations, especially in infants. Newer anticoagulants have improved properties relative to heparin, and this may enhance the outcome in children. OBJECTIVE: To determine dosing, and to assess the safety and efficacy of bivairudin for infants with thrombosis. METHODS: Infants <6 months old were chosen for this pilot study as they may most benefit from a direct thrombin inhibitor because of their physiologically low antithrombin levels. This was an open label, dose-finding and safety study. Patients received one of three bolus doses and one of two initial infusion doses with subsequent dosing adjusted utilizing the activated partial thromboplastin time. Safety was assessed by specific bleeding endpoints. Efficacy was determined by reassessing the initial imaging study at 48-72 h and by measurement of molecular markers of thrombin generation. RESULTS: Sixteen patients completed the study. All three bolus doses resulted in therapeutic anticoagulation, as did both initial infusion doses. A dose-response effect was noted for the continuous infusion but not the bolus dosing. Two patients met the study criteria for major bleeding, both with gross hematuria, which resolved with a reduction in the bivalirudin infusion rate. In terms of efficacy, 37.5% of patients had complete or partial resolution of their thrombosis by 48-72 h. There was a significant decrease in all three molecular markers of thrombin generation. CONCLUSION: This study demonstrates the potential utility of bivalirudin in the pediatric population.     

Factor XIII deficiency

Summary. Inherited factor XIII (FXIII) deficiency is a rare bleeding disorder that can present with umbilical bleeding during the neonatal period, delayed soft tissue bruising, mucosal bleeding and life‐threatening intracranial haemorrhage. FXIII deficiency has also been associated with poor wound healing and recurrent miscarriages. FXIII plays an integral role in haemostasis by catalysing the cross‐linking of fibrin, platelet membrane and matrix proteins throughout thrombus formation, thus stabilizing the blood clot. The molecular basis of FXIII deficiency is characterized by a high degree of heterogeneity, which contributes to the different clinical manifestations of the disease. There have been more than 60 FXIII mutations identified in the current literature. In addition, single nucleotide polymorphisms have been described, some of which have been shown to affect FXIII activity, contributing further to the heterogeneity in patient presentation and severity of clinical symptoms. Although there is a lifelong risk of bleeding, the prognosis is excellent when current prophylactic treatment is available using cryoprecipitate or plasma‐derived FXIII concentrate.     

Similar rearrangements of T-cell receptor beta gene in cell lines and uncultured cells from patients with large granular lymphocyte leukemia

We established interleukin-2-(IL-2) dependent cell lines from three patients with large granular lymphocyte (LGL) leukemia. Phenotypic analysis demonstrated retention of the CD3+, CD8+ phenotype that was observed in the original leukemic LGL. Unique rearrangements of T-cell receptor beta gene occurring in uncultured leukemic LGL, were also found in cell lines, which suggests that the cell lines were derived from the original leukemic LGL clone in each case.     

Antibodies to CD40 prevent Epstein-Barr virus-mediated human B-cell lymphomagenesis in severe combined immune deficient mice given human peripheral blood lymphocytes

CD40 is expressed on both normal and neoplastic B lymphocytes. Signal transduction through CD40 in vitro has been shown to exert stimulatory effects on normal B cells and inhibitory effects on Epstein-Barr virus (EBV)-induced B-cell lymphoma lines and some other cell lines derived from patients with aggressive histology lymphoma. The transfer of normal human peripheral blood lymphocytes (huPBL) from EBV-seropositive donors into severe combined immune deficient (SCID) mice has been previously shown to result in the generation of human B-cell lymphomas. These tumors are similar to the highly aggressive EBV-induced lymphomas that can arise clinically after transplantation or in the setting of immunodeficiency. Treatment of huPBL-SCID chimeric mice with anti-CD40 or anti-CD20 monoclonal antibodies (MoAb) significantly delayed the development of EBV-induced B-cell lymphoma. However, the effects of the two MoAb were mechanistically distinct. Anti-CD40 treatment prevented lymphoma generation, while still allowing for functional human B-cell engraftment in the huPBL-SCID mice compared with mice receiving no treatment, all of which succumbed to lymphoma. By contrast, treatment with anti-CD20 significantly inhibited total human B-cell engraftment in the SCID recipients, which accounted for the absence of lymphomas. In vitro assays examining the transformation of human B cells by EBV also indicated that anti-CD40 could directly inhibit EBV- transformation, whereas anti-CD20 antibodies had no effect. Thus, anti- CD40 exerts selective effects to allow for the engraftment of normal human B cells and prevent the emergence of EBV lymphomas. Stimulation of CD40 by antibodies or its physiologic ligand may, therefore, be of significant clinical use in the prevention of EBV-induced B lymphomas that may arise when EBV-seropositive individuals receive immunosuppressive regimens after transplantation or in immune deficiency states, such as acquired immune deficiency syndrome.     

Direct synergistic effects of interleukin-7 on in vitro myelopoiesis of human CD34+ bone marrow progenitors

Interleukin-7 (IL-7) is an important growth factor in B and T lymphopoiesis in mouse and human, whereas IL-7 has been regarded to lack proliferative effects on cells within the myeloid lineage. However, we have recently reported that IL-7 potently can enhance colony stimulating factor (CSF)-induced myelopoiesis from primitive murine hematopoietic progenitors, showing a novel role of IL-7 in early murine myelopoiesis. Using CD34+ human hematopoietic progenitor cells, we show here a similar role of IL-7 in human myelopoiesis, although interesting differences between the two species were found as well. Although purified recombinant human (rh)IL-7 alone did not induce any proliferation of CD34+ cells, IL-7 in a concentration-dependent manner enhanced the colony formation induced by all four CSFs up to threefold. Furthermore, stem cell factor (SCF)-induced granulocyte-macrophage (GM) colony formation was increased fourfold in the presence of IL-7. Single- cell cloning assays showed that these synergistic effects of IL-7 were directly mediated on the targeted progenitors, and that IL-7 increased the number, as well as the size of the colonies formed. Morphological examination showed that IL-7 affected the progeny developed from CD34+ cells stimulated by G-CSF or IL-3, increasing the number of CFU-M (colony forming unit-macrophage) and CFU-granulocyte-macrophage, whereas the number of CFU-granulocyte were unaltered.     

Human T-lymphocyte growth factor: regulation of growth and function of T lymphocytes

The discovery of T-cell growth factor (TCGF) has made it possible to now routinely grow in tissue culture normal and neoplastic human T cells for long periods and in large amounts. TCGF has been recently purified. It is a small protein released by a subset of mature T cells following lectin-antigen activation, which in turn acts upon other T- cell subsets that have developed specific receptors for TCGF after lectin-antigen stimulation. Thus, release of TCGF and development of receptors for it appear to be obligatory for the clonal expansion of all activated T cells. Unlike normal T cells, neoplastic T cells respond directly to TCGF, requiring no prior in vitro lectin-antigen activation. This has led to the development of several new cell lines from patients with T-cell leukemias and lymphomas. In some cases, these cells become independent of exogenous TCGF by producing their own growth factor, implying a role for TCGF in the continuous proliferation of these cells. These developments necessitate a reevaluation of some concepts of immunoregulation of T-cell activities in terms of production and response to TCGF. In addition, this information has clinical implications. Recent results have shown that a major defect of the athymic nude mouse is the inability to produce TCGF and that some immunosuppressive agents, such as glucocorticosteroids and cyclosporin- A, exert their effects on T cells by disrupting the TCGF-T-cell interaction. Some human immune deficiencies might be due to a failure to respond to or to produce TCGF, which in some cases might be corrected by exogenous TCGF.     

Distinct and overlapping direct effects of macrophage inflammatory protein-1 alpha and transforming growth factor beta on hematopoietic progenitor/stem cell growth

Both transforming growth factor beta (TGF beta) and macrophage inflammatory protein 1 alpha (MIP-1 alpha) have been shown to be multifunctional regulators of hematopoiesis that can either inhibit or enhance the growth of hematopoietic progenitor cells (HPC). We report here the spectrum of activities of these two cytokines on different hematopoietic progenitor and stem cell populations, and whether these effects are direct or indirect. MIP-1 alpha enhances interleukin-3 (IL- 3)/and granulocyte-macrophage colony-stimulating factor (GM- CSF)/induced colony formation of normal bone marrow progenitor cells (BMC) and lineage-negative (Lin-) progenitors, but has no effect on G- CSF or CSF-1/induced colony formation. Similarly, TGF beta enhances GM- CSF/induced colony formation of normal BMC and Lin- progenitors. In contrast, TGF beta inhibits IL-3/ and CSF-1/induced colony formation of Lin- progenitors. The effects of MIP-1 alpha and TGF beta on the growth of Lin- progenitors were direct and correlate with colony formation in soft agar. Separation of the Lin- cells into Thy-1 and Thy-1lo subsets showed that the growth of Thy-1lo Lin- cells is directly inhibited by MIP-1 alpha and TGF beta regardless of the cytokine used to stimulate growth (IL-3), GM-CSF, or CSF-1). In contrast, two other stem cell populations (0% to 15% Hoechst 33342/Rhodamine 123 [Ho/Rh123] and Lin- Sca-1+ cells) were markedly inhibited by TGF beta and unaffected by MIP- 1 alpha. Furthermore, MIP-1 alpha has no effect on high proliferative potential colony-forming cells 1 or 2 (HPP-CFC/1 or /2) colony formation in vitro, whereas TGF beta inhibits both HPP-CFC/1 and HPP- CFC/2. Thus, MIP-1 alpha and TGF beta are direct bidirectional regulators of HPC growth, whose effects are dependent on other growth factors present as well as the maturational state of the HPC assayed. The spectrum of their inhibitory and enhancing activities shows overlapping yet distinct effects.     

Inhibitory cytokine circuits involving transforming growth factor-beta, interferon-gamma, and interleukin-2 in human monocyte activation

We have previously reported that transforming growth factor-beta 1 (TGF- beta 1) inhibits interleukin-6 (IL-6) induction by IL-2 and IL-1 in fresh human monocytes. We investigated the effects of TGF-beta 1 on the expression of tumoricidal activity induced by IL-2 or interferon-gamma (IFN-gamma) in human monocytes. We showed that TGF-beta 1 specifically inhibited, in a dose-dependent manner, IL-2-induced but not IFN-gamma- induced monocyte tumoricidal activity. The inhibitory effects of TGF- beta 1 on IL-2-activated monocytes were not caused by down-modulation of the IL-2 receptor beta (IL-2R beta) because the treatment of monocytes with IL-2 and TGF-beta 1 increased IL-2R beta mRNA expression. However, we found that TGF-beta 1 down-modulated IL-2- induced IL-2R gamma mRNA, which may be responsible for the TGF-beta 1 inhibition of monocyte activation by IL-2. The resistance of the IFN- gamma-induced activation to the inhibitory effects of TGF-beta 1 could be caused by the ability of IFN-gamma to decrease TGF-beta 1 receptor expression, as shown by cross-linking experiments. Overall, these results showed that TGF-beta 1 is a powerful inhibitor of IL-2- but not of IFN-gamma-induced activation of monocytes to a cytotoxic stage. This differential effect may be attributed to modulation of cytokine receptor expression.     

Celecoxib in the treatment of haemophilic synovitis, target joints, and pain in adults and children with haemophilia

Joint haemorrhage and subsequent haemophilic arthropathy are significant complications of haemophilia. The pathophysiology involves inflammation and angiogenesis. Cyclo-oxygenase 2 (COX-2) inhibitors have anti-inflammatory, anti-angiogenic and analgesic properties yet do not affect platelet function in the manner of traditional non-steroidal anti-inflammatory drugs. We have previously reported the successful use of rofecoxib in the management of haemophilic arthropathy and currently report our use of celecoxib. A retrospective chart review was conducted of all patients with haemophilia A or B seen at the Children's Hospital of Orange County and treated with celecoxib for chronic synovitis, target joint or pain. Efficacy in chronic synovitis and pain reduction was judged subjectively as effective, partially effective or ineffective. Efficacy in resolution of target joints was judged as effective if the target joint resolved or ineffective if it did not resolve. Twelve patients between 9 and 54 years old were treated for a total of 14 courses of celecoxib treatment. All courses were evaluated for safety and efficacy. Celecoxib was used in eight patients with chronic synovitis, three patients with pain, and in one patient with a target joint on three occasions. A response was noted in seven of eight for synovitis, three of three with pain, but no response in the target joint patient. No serious adverse events including hypertension were observed. This is the first study evaluating celecoxib as adjunctive therapy in haemophilia and suggests that celecoxib is safe and effective in treating chronic synovitis and joint pain similar to the previous study of rofecoxib.     

Vascular remodeling underlies rebleeding in hemophilic arthropathy

Hemophilic arthropathy is a debilitating condition that can develop as a consequence of frequent joint bleeding despite adequate clotting factor replacement. The mechanisms leading to repeated spontaneous bleeding are unknown. We investigated synovial, vascular, stromal, and cartilage changes in response to a single induced hemarthrosis in the FVIII‐deficient mouse. We found soft‐tissue hyperproliferation with marked induction of neoangiogenesis and evolving abnormal vascular architecture. While soft‐tissue changes were rapidly reversible, abnormal vascularity persisted for months and, surprisingly, was also seen in uninjured joints. Vascular changes in FVIII‐deficient mice involved pronounced remodeling with expression of α‐Smooth Muscle Actin (SMA), Endoglin (CD105), and vascular endothelial growth factor, as well as alterations of joint perfusion as determined by in vivo imaging. Vascular architecture changes and pronounced expression of α‐SMA appeared unique to hemophilia, as these were not found in joint tissue obtained from mouse models of rheumatoid arthritis and osteoarthritis and from patients with the same conditions. Evidence that vascular changes in hemophilia were significantly associated with bleeding and joint deterioration was obtained prospectively by dynamic in vivo imaging with musculoskeletal ultrasound and power Doppler of 156 joints (elbows, knees, and ankles) in a cohort of 26 patients with hemophilia at baseline and during painful episodes. These observations support the hypothesis that vascular remodeling contributes significantly to bleed propagation and development of hemophilic arthropathy. Based on these findings, the development of molecular targets for angiogenesis inhibition may be considered in this disease. Am. J. Hematol. 90:1027–1035, 2015. © 2015 Wiley Periodicals, Inc.