Identification of residues in the first and fourth helices of human granulocyte-macrophage colony-stimulating factor involved in biologic activity and in binding to the alpha- and beta-chains of its receptor

Residues within the first and fourth helices of human granulocyte- macrophage colony-stimulating factor (hGM-CSF) were analyzed for their role in biologic activity and interaction with the alpha- and beta- chains of the hGM-CSF receptor. Within the first helix substitution of the surface residues Glu14, Asn17, Gln20, Arg23, Arg24, and Asn27 or the buried residues Ala18, Leu25, and Leu28 did not significantly impair bioactivity or receptor binding. Substitutions at the buried residues Ala22 and Leu26 had intermediate bioactivity. However, substitutions of the surface residue Glu21 or the buried residue Ile19 reduced the relative bioactivity of the analogues to as little as 0.45% and 0.3%, respectively. Substitution of the charged surface residues of the fourth helix showed that substitution at Glu104, Lys107, and Lys111 had no significant effect on bioactivity, but substitution at Glu108 and Asp112 reduced the potency of the analogues to 34% and 7%, respectively. Receptor binding studies showed that, whereas Glu21 is the critical residue for binding to the hGM-CSF-receptor beta-chain, Asp112 is likely to be involved in binding to the GM-CSF-receptor alpha- chain. These results establish the relative contribution of residues in the first and fourth helices for GM-CSF bioactivity and receptor binding, and support a model where the fourth helix of GM-CSF interacts with the alpha-chain, and the first helix with the beta-chain of the GM- CSF receptor.     

Evidence that several high-frequency human blood group antigens reside on phosphatidylinositol-linked erythrocyte membrane proteins

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder associated with absence of expression of phosphatidylinositol (PI)- linked membrane proteins from circulating hematopoietic cells of multiple lineages. Recent work demonstrated that decay accelerating factor, one such PI-linked protein, bears the Cromer-related blood group antigens. This study demonstrated that other high incidence antigens, including Cartwright (Yta/Ytb), Holley-Gregory (Hy/Gya), John Milton Hagen (JMH), and Dombrock (Doa/Dob), are absent from the complement-sensitive (PNH III) erythrocytes of patients with PNH. The relatively normal, complement-insensitive erythrocytes from the same patients express these antigens normally. Therefore, these antigens most likely reside on PI-linked proteins absent from PNH III, but not PNH I, erythrocytes.     

Transformation-associated alterations in interactions between pre-B cells and fibronectin

Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2-10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2-10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co- cultured with M2-10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.     

Rapid and efficient selection of human hematopoietic cells expressing murine heat-stable antigen as an indicator of retroviral-mediated gene transfer

Recombinant retroviruses offer many advantages for the genetic modification of human hematopoietic cells, although their use in clinical protocols has thus far given disappointing results. There is therefore an important need to develop new strategies that will allow effectively transduced primitive hematopoietic target populations to be both rapidly characterized and isolated free of residual nontransduced but biologically equivalent cells. To address this need, we constructed a murine stem cell virus (MSCV)-based retroviral vector containing the 228-bp coding sequence of the murine heat-stable antigen (HSA) and generated helper virus-free amphotropic MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells. Light density and, in some cases, lineage marker-negative (lin-) normal human marrow or mobilized peripheral blood cells preactivated by exposure to interleukin-3 (IL- 3), IL-6, and Steel factor in vitro for 48 hours were then infected by cocultivation with these MSCV-HSA producer cells for a further 48 hours in the presence of the same cytokines. Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours later showed 21% to 41% (mean, 27%) of those that were still CD34+ to have acquired the ability to express HSA. The extent of gene transfer to erythroid and granulopoietic progenitors (burst-forming unit-erythroid and colony- forming unit-granulocyte-macrophage), as assessed by the ability of these cells to form colonies of mature progeny in the presence of normally toxic concentrations of G418, averaged 11% and 12%, respectively, in 6 experiments. These values could be increased to 100% and 77%, respectively, by prior isolation of the CD34+HSA+ cell fraction and were correspondingly decreased to an average of 2% and 5%, respectively, in the CD34+HSA- cells. In addition, the extent of gene transfer to long-term culture-initiating cells (LTC-IC) was assessed by G418 resistance. The average gene transfer to LTC-IC-derived colony- forming cells in the unsorted population was < or = 7% in 4 experiments. FACS selection of the initially CD34+HSA+ cells increased this value to 86% and decreased it to 3% for the LTC-IC plated from the CD34+HSA- cells. Transfer of HSA gene expression to a phenotypically defined more primitive subpopulation of CD34+ cells, ie, those expressing little or no CD38, could also be shown by FACS analysis of infected populations 24 hours after infection. These findings underscore the potential use of retroviral vectors encoding HSA for the specific identification and non-toxic selection immediately after infection of retrovirally transduced populations of primitive human hematopoietic cells. In addition, such vectors should facilitate the subsequent tracking of their marked progeny using multiparameter flow cytometry.     

Patterns of disease in patients at a tertiary referral centre requiring reoperative parathyroidectomy

Introduction

Reoperative parathyroidectomy is required when there is persistent or recurrent hyperparathyroidism following the initial surgery (at least 5% of parathyroidectomies nationally). By convention, ‘persistent disease’ is defined as the situation where the patient has not been cured by the first operation. The term ‘recurrent hyperparathyroidism’ is used when the patient was confirmed to be biochemically cured for six months from the first operation but has hyperparathyroidism after this date. Reoperative surgery is associated with higher rates of postoperative complications as well as a greater rate of failure to cure. The aim of our study was to review our departmental experience of reoperative parathyroidectomy, with a view to identify patterns of disease persistence and recurrence.

Methods

Using a departmental database, patients were identified who had undergone reoperative parathyroidectomy between 2006 and 2014. All the pre, intra and postoperative information was documented including the operative note so as to record the location of the abnormal parathyroid gland found at reoperation.

Results

Almost two-thirds (63%) of patients had negative, equivocal or discordant conventional imaging so secondary investigative tools were required frequently. The majority of abnormal glands were found in eutopic locations. The most common locations for ectopic glands were intrathyroidal, mediastinal and intrathymic. A third (33%) of the patients had multigland disease and over a quarter (28%) had coexisting thyroid disease.

Conclusions

Persistent hyperparathyroidism represents a challenging patient subgroup for which access to all radiological modalities and intraoperative parathyroid hormone monitoring are required. Patient selection for reintervention is a key determinant in the reoperation cure rate.     

Effective Transport for Trauma Patients under Current Circumstances in Korea: A Single Institution Analysis of Treatment Outcomes for Trauma Patients Transported via the Domestic 119 Service

In Korea, which still lacks a well-established trauma care system, the inability to transport patients to adequate treatment sites in a timely manner is a cause of low trauma patient survival. As such, this study was conducted to serve as a basis for the establishment of a future trauma transport system. We performed a comparative analysis of the transport time, and treatment outcomes between trauma victims transported by ground ambulance (GAMB) and those transported via the helicopter emergency medical service (HEMS) through the National Emergency Management Agency''s 119 reporting system, which is similar to the 911 system of the United States, from March 2011 to May 2014. The HEMS-transported patients received treatment instructions, by remote communication, from our trauma specialists from the time of accident reporting; in certain instances, members of the trauma medical staff provided treatment at the scene. A total of 1,626 patients were included in the study; the GAMB and HEMS groups had 1,547 and 79 patients, respectively. The median transport time was different between 2 groups (HEMS, 60 min vs. GAMB, 47 min, P<0.001) but for all patients was 49 min (less than the golden hour). Outcomes were significantly better in the HEMS compared to the GAMB, using the trauma and injury severity score (survival rate, 94.9% vs. 90.5%; Z score, 2.83 vs. -1.96; W score, 6.7 vs. -0.8). A unified 119 service transport system, which includes helicopter transport, and the adoption of a trauma care system that allows active initial involvement of trauma medical personnel, could improve the treatment outcome of trauma patients.

Graphical Abstract

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Autografting with cultured marrow in chronic myeloid leukemia: results of a pilot study [see comments]

Incubation of chronic myeloid leukemia (CML) marrow for 10 days in vitro causes a marked and selective loss of very primitive Philadelphia chromosome (Ph)+ as compared with Ph- progenitors. We have autografted 22 patients with CML (16 in first chronic phase [group 1] and 6 with more advanced disease [group 2]) with marrow treated in this way to facilitate restoration of Ph- hematopoiesis after intensive therapy. Hematologic recovery to greater than 0.5 x 10(9)/L neutrophils occurred in 16 patients, and to greater than 20 x 10(9)/L platelets in 15 of 21 evaluable patients at a median of 29 and 48 days postautograft, respectively. Regenerating marrow cells were 100% Ph- in 13 patients and 75% to 94% Ph- in 3. Between 4 and 36 months (median 12) postautograft, Ph+ cells became detectable in all but 1 (who died in remission) of the 13 patients who achieved complete cytogenetic remission. Four of 7 evaluable patients treated with low-dose interferon alpha were returned to complete cytogenetic remission. Thirteen group 1 patients (81%) are alive 1.0 to 5.7 years (median 2.6) after autografting: 4 in complete cytogenetic remission, 2 in hematologic remission, 6 in chronic phase, and 1 in myeloid blast phase. Three group 2 patients (50%) are alive at 2.6, 3.8, and 4.3 years after autografting: 1 in partial cytogenetic remission, 1 in chronic phase, and 1 in accelerated phase. Thus, autografts of cultured marrow can result in prolonged restoration of Ph- hematopoiesis for some patients with CML.     

Use of a sensitive bioimmunoabsorbent assay to isolate and characterize monoclonal antibodies to biologically active human erythropoietin

At present, one of the most sensitive assays for human erythropoietin (Ep) is a bioassay that measures the Ep-dependent proliferation of spleen cells from phenylhydrazine-treated mice after 24 hours in culture. We describe how this assay can be used as the basis of a very sensitive method for detecting mouse antibodies to biologically active human Ep. In this procedure, microtiter wells are first coated with goat anti-mouse Ig antibody, then treated with mouse antibodies (serum or hybridoma culture supernatants), and finally incubated with a fixed amount of pure human Ep. Specific binding of anti-Ep antibodies is detected by adding spleen cells from phenylhydrazine-treated mice to the wells and measuring the ability of the cells to incorporate 3H- thymidine 24 hours later. This bioimmunosorbent assay (BISA) revealed the presence of anti-EP antibodies in sera from mice immunized with either pure human urinary Ep or a synthetic dodecapeptide corresponding to the aminoterminal region of Ep and in the culture supernatants from three of eight stable anti-Ep antibody-producing hybridoma cell lines that we have isolated. The three monoclonal antibodies showed similar reactivities in the BISA, but showed different affinities for Ep, with Kd values of approximately 0.7, 8, and 240 nmol/L, respectively. Further studies showed that all antibodies were capable of neutralizing Ep bioactivity and of binding 125I-labeled Ep in a radioimmunosorbent assay (RIA) but were virtually unreactive to Ep adsorbed to the bottom of enzyme-linked immunosorbent assay (ELISA) wells. Our results suggest that the BISA strategy may be an important complement to conventional RIA and ELISA techniques for identification of monoclonal antibodies specific for biologically active growth factors.     

Molecular analysis of clonality and bcr rearrangements in Philadelphia chromosome-positive acute lymphoblastic leukemia

Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) patients consistently show a rearrangement in a 5.8-kilobase length of chromosome 22, referred to as the breakpoint cluster region (bcr). In Ph1-positive acute lymphoblastic leukemia (ALL), the breakpoint in chromosome 22 is more heterogeneous and, in some instances, does not occur within this region. In such cases the cell of origin of the neoplastic clone and the relationship of the disease to CML has remained obscure. We have analyzed the bcr rearrangement in the malignant cells from three patients who presented with Ph1-positive ALL and who in cytogenetic studies had shown evidence of variable involvement of myeloid cells in the Ph1-positive clone. Rearrangements in bcr typical of most cases of CML were detected in purified granulocyte preparations from two of the ALL patients (nos. 1 and 2) and in the blasts from patient 3 at the time of her terminal relapse. In the same analysis the simultaneously obtained granulocytes from patient 3, however, did not show any evidence of bcr rearrangement. Patient 3 was also heterozygous for the BamHI polymorphism in the X- linked hypoxanthine phosphoribosyltransferase (HPRT) gene, thus permitting a different method of clonal analysis based on methylation differences in active and inactive alleles. When DNA from her granulocytes that had shown no bcr rearrangement was hybridized to an HPRT probe, a pattern typical of a polyclonal population was seen. A similar pattern was exhibited by her marrow fibroblasts. In marked contrast, her simultaneously isolated blasts showed an unambiguous monoclonal pattern. These findings demonstrate the origin of the disease in the first two patients in a cell with myelopoietic as well as lymphopoietic potential and confirm the restricted lymphoid cell origin of the neoplastic clone in the third Ph1-positive ALL patient. Furthermore, they indicate that different target cells for transformation within the hematopoietic system may be affected by very similar bcr rearrangements.