Thrombopoietin, but not erythropoietin promotes viability and inhibits apoptosis of multipotent murine hematopoietic progenitor cells in vitro

The recently cloned c-mpl ligand, thrombopoietin (Tpo), has been extensively characterized with regard to its ability to stimulate the growth, development, and ploidy of megakaryocyte progenitor cells and platelet production in vitro and in vivo. Primitive hematopoietic progenitors have been shown to express c-mpl, the receptor for Tpo. In the present study, we show that Tpo efficiently promotes the viability of a subpopulation of Lin-Sca-1+ bone marrow progenitor cells. The ability of Tpo to maintain viable Lin-Sca-1+ progenitors was comparable to that of granulocyte colony-stimulating factor and interleukin-1, whereas stem cell factor (SCF) promoted the viability of a higher number of Lin-Sca-1+ progenitor cells when incubated for 40 hours. However, after prolonged (> 40 hours) preincubation, the viability- promoting effect of Tpo was similar to that of SCF. An increased number of progenitors surviving in response to Tpo had megakaryocyte potential (37%), although almost all of the progenitors produced other myeloid cell lineages as well, suggesting that Tpo acts to promote the viability of multipotent progenitors. The ability of Tpo to promote viability of Lin-Sca-1+ progenitor cells was observed when cells were plated at a concentration of 1 cell per well in fetal calf serum- supplemented and serum-depleted medium. Finally, the DNA strand breakage elongation assay showed that Tpo inhibits apoptosis of Lin-Sca- 1+ bone marrow cells. Thus, Tpo has a potent ability to promote the viability and suppress apoptosis of primitive multipotent progenitor cells.     

Regulation of erythropoietin production in a human hepatoblastoma cell line

Production of immuno and biologically active erythropoietin was documented to occur in the human hepatoblastoma cell line HepG-2. The expression of the erythropoietin gene was further verified by Northern blot analysis using a single stranded RNA probe. In vitro studies showed that erythropoietin production by these cells was not stimulated by hypoxia or cobalt chloride, but was related to the proliferative activity of the cells in culture. In addition it was found that the secretion of erythropoietin was almost completely abrogated by tunicamycin, an inhibitor of N-linked glycosylation. This effect of tunicamycin was also observed in a permanently transfected cell line that secretes erythropoietin in large quantities.     

Thrombopoietin, but not erythropoietin, directly stimulates multilineage growth of primitive murine bone marrow progenitor cells in synergy with early acting cytokines: distinct interactions with the ligands for c-kit and FLT3

Thrombopoietin (Tpo), the ligand for c-mpl, has been shown to be the principal regulator of megakaryocytopoiesis and platelet production. The ability of Tpo to potently stimulate the growth of committed megakaryocyte (Mk) progenitor cells has been studied in detail. Murine fetal liver cells, highly enriched in primitive progenitors, have been shown to express c-mpl, but little is known about the ability of Tpo to stimulate the growth and differentiation of primitive multipotent bone marrow (BM) progenitor cells. Here, we show that Tpo alone and in combination with early acting cytokines can stimulate the growth and multilineage differentiation of Lin- Sca-1+ BM progenitor cells. In particular, Tpo potently synergized with the ligands for c-kit (stem cell factor [SCF]) and flt3 (FL) to stimulate an increase in the number and size of clones formed from Lin- Sca-1+ progenitors. When cells were plated at 1 cell per well, the synergistic effect of Tpo was observed both in fetal calf serum-supplemented and serum-depleted medium and was decreased if the addition of Tpo to cultures was delayed for as little as 24 hours, suggesting that Tpo is acting directly on the primitive progenitors. Tpo added to SCF + erythropoietin (Epo)-supplemented methylcellulose cultures potently enhanced the formation of multilineage colonies containing granulocytes, macrophages, erythrocytes, and Mks. SCF potently enhanced Tpo-stimulated production of high-ploidy Mks from Lin- Sca-1+ progenitors, whereas the increased growth response obtained when combining Tpo with FL did not translate into increased Mk production. The ability of Tpo and SCF to synergistically enhance the growth of Lin- Sca-1+ progenitors was predominantly observed in the more primitive rhodamine 123(lo) fraction. Tpo also enhanced growth of Lin- Sca-1+ progenitors when combined with interleukin-3 (IL-3) and IL-11 but not with IL-12, granulocyte colony-stimulating factor, granulocyte-macrophage colony- stimulating factor, or Epo. Epo, which has high homology to Tpo, was unable to stimulate the growth of Lin- Sca-1+ progenitors alone or in combination with SCF or FL, suggesting that c-mpl is expressed on more primitive stages of progenitors than the Epo receptor. Thus, the present studies show the potent ability of Tpo to enhance the growth of primitive multipotent murine BM progenitors in combination with multiple early acting cytokines and documents its unique ability to synergize with SCF to enhance Mk production from such progenitors.     

Increased incidence of hepatitis B markers in children with sickle-cell anemia

Summary Screening of 143 children 6 months to 12 years of age with sickle-cell anemia showed that 39.2% were HBsAg-positive as compared with 19.3% of the 161 control children of the same age group, who had Hb genotype AA (²=14.7383; P<0.001). Fifty percent of the HbSS children under the age of 1 year were HBsAg-positive as opposed to 4.3% of the control group (²=9.1955; P<0.001), while 28.6% of patients were HBsAg-positive at the age of 4 years compared with only 7.4% of the controls at the same age. The incidence of anti-HBc in both groups was similar. Markers of HBV infection (HBsAg + anti-HBc) were, however, on the whole higher in the patients with sickle-cell anemia [88/118 (74.6%)] than in the controls [54/88 (61.3%; P<0.005)].     

Distinct ongoing Ig heavy chain rearrangement processes in childhood B- precursor acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is thought to arise from the clonal expansion of a single transformed precursor cell. However, an oligoclonal Ig heavy chain (IgH) rearrangement pattern has been observed in 30% of ALL patients and was shown to be the result of ongoing rearrangement events. The extent and nature of these ongoing rearrangement processes in individual patients has so far remained obscure. We performed a detailed analysis of leukemic VHDJH rearrangements in three children with B-precursor ALL at diagnosis and one B-lymphoid blast crisis of a child with Ph+ chronic myeloid leukemia at diagnosis and relapse. The children were selected because they presented with multiple IgH rearrangements on Southern blot analysis. Polymerase chain reaction analysis of leukemic cells from two B-precursor ALL patients showed exclusively two groups of related sequences resulting from VH gene replacement events. Most VH gene replacements involved 3' located acceptor VH genes. Analysis of cells from the other B-precursor ALL patient showed exclusively related sequences as a result of VH gene joinings to a pre-existing DJH rearrangement. In the B-lymphoid blast crisis, a single germline precursor cell had generated multiple unrelated rearrangements and additional groups of related rearrangements resulting from VH to DJH joinings. Direct proof for the VH to DJH joining mechanism was obtained by amplification of the expected preexisting DJH rearrangements. Our findings suggest that the pattern of ongoing rearrangements in an individual patient reflects the IgH rearrangement status of the precursor cell at the time of malignant transformation. Sequence analysis of VHDJH rearrangements at diagnosis may therefore allow a prediction of the reliability of complementarity determining region 3 probes for the detection of minimal residual disease.     

Regulation of inflammation by lipid mediators in oral diseases

Lipid mediators (LM) of inflammation are a class of compounds derived from ω‐3 and ω‐6 fatty acids that play a wide role in modulating inflammatory responses. Some LM possess pro‐inflammatory properties, while others possess proresolving characteristics, and the class switch from pro‐inflammatory to proresolving is crucial for tissue homeostasis. In this article, we review the major classes of LM, focusing on their biosynthesis and signaling pathways, and their role in systemic and, especially, oral health and disease. We discuss the detection of these LM in various body fluids, focusing on diagnostic and therapeutic applications. We also present data showing gender‐related differences in salivary LM levels in healthy controls, leading to a hypothesis on the etiology of inflammatory diseases, particularly Sjögren's syndrome. We conclude by enumerating open areas of research where further investigation of LM is likely to result in therapeutic and diagnostic advances.     

Ophthalmic manifestations and risk factors for mortality of HIV patients in the post‐highly active anti‐retroviral therapy era

Background: To evaluate the ophthalmic manifestations and risk factors for mortality in HIV patients in the post‐highly active anti‐retrovirus therapy (HAART) era. Design: Retrospective study. Samples: 151 patients with HIV infection. Methods: Review of all HIV patients who have attended the Hong Kong Eye Hospital between 2000 and 2007. Main Outcome Measures: Ocular findings especially opportunistic infections and medical information including mortality during follow up. Results: At presentation, 139 (92.1%) patients were already diagnosed with HIV and 58 (41.7%) had an AIDS indicator condition. Fifty‐one (33.8%) patients had HIV‐related eye disease on presentation and the leading manifestations were cytomegalovirus (CMV) retinitis and HIV microangiopathy. Low baseline CD4 cell count <100 cells/L was significantly related with HIV‐related ophthalmic manifestations and CMV retinitis at presentation (P < 0.013). 105 patients were followed for 6 months or more and the mean follow‐up was 4.8 years. There was no significant change in visual acuity compared with baseline (P = 0.13). 20 (19.0%) patients had one eye with final visual acuity of 20/200 or worse and the leading cause for poor vision was CMV retinitis. 11 (10.5%) patients died during the follow‐up due to complications of HIV/AIDS. The presence of HIV retinal microangiopathy was significantly associated with mortality (P = 0.005). Conclusions: CMV retinitis remains the main HIV‐related ocular disease in the post‐HAART era. HIV retinal microangiopathy might be an important prognostic factor for mortality. Appropriate ophthalmic monitoring is justified to detect for ophthalmic complications in HIV patients regardless of HAART use in order for prompt initiation of treatment.     

Ultrasonographic evaluation of breech presentation as a risk factor for hip dysplasia

In order to gain more information of breech position as a risk factor for congenital hip dysplasia or dislocation, the hips of 408 newborns delivered in the breech position were examined by ultrasound. Clinical examination was performed by both experienced paediatricians and orthopaedic surgeons. The infants were re-examined by ultrasound at 2–3 months of age. Twenty-five newborns (6.1%) had neonatal hip instability. Breech presentation as a risk factor was confirmed, with first borns, breech position with extended knees, and high birthweight as special high-risk groups. Ultrasound showed subluxation in most of the unstable hips. The main benefit of using ultrasound was that direct visualization permitted more reliable evaluation, especially when the clinical findings were uncertain. Normal ultrasound findings in false positive and uncertain Ortolani tests reduced the frequency of unnecessary treatment. Because ultrasound was used in follow-up, the need of radiography was reduced. There were no late-detected cases of hip dysplasia or dislocation, indicating that routine follow-up is not necessary in breech infants with normal hips at birth, provided that the neonatal screening is optimal.