Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector

Retroviral-mediated gene transfer is the most attractive modality for gene transfer into hematopoietic stem cells. However, transduction efficiency has been low using amphotropic Moloney murine leukemia virus (MoMLV) vectors. In this study, we investigated modifications of gene transfer using amphotropic MoMLV vectors in cell-free supernatant for their ability to increase the currently low transduction of both committed hematopoietic progenitors, granulocyte-macrophage colony- forming units (CFU-GMs), and their precursors, long-term culture- initiating cells (LTC-IC). First, based on the observation that bone marrow cells express more gibbon ape leukemia virus (GALV) receptor (Glvr-1) than amphotropic receptor (Ram-1), PG13/LN, which is a MoMLV vector pseudotyped with the GALV envelope, was compared with the analogous amphotropic envelope vector (PA317/LN). Second, progenitor cell transduction efficiency was compared between CD34 enriched and nonenriched progenitor populations. Third, the duration of transduction in vitro was extended to increase the proportion of progenitor cells that entered cell cycle and could thereby integrate vector cDNA. In 20 experiments, 1 x 10(6) marrow or peripheral blood mononuclear cells (PBMCs)/mL were exposed to identical titers of pseudotyped PG13/LN vector or PA317/LN vector in the presence of recombinant human interleukin-1 (IL-1), IL-3, IL-6, and stem cell factor (SCF; c-kit ligand) for 5 days. 50% of fresh vector supernatant was refed daily. Hematopoietic progenitor cells as measured by G418-resistant granulomonocytic colony (CFU-GM) formation were transduced more effectively with PG13/LN (19.35%) than with PA317/LN (11.5%, P = .012). In 11 further experiments, enrichment of CD34 antigen positive cells significantly improved gene transfer from 13.9% G418-resistant CFU-GM in nonenriched to 24.9% in CD34-enriched progenitor cells (P < .01). To analyze gene transfer after extended growth factor-supported long-term culture, 1 x 10(6) marrow cells/mL were cultured with IL-1, IL-3, IL-6, and SCF (50 ng/mL each) for 1, 2, and 3 weeks. Fifty percent of PG13/LN supernatant with growth factors was refed on 5 days per week. Five percent of marrow CFU-GM and 67% of LTC-IC were G418 resistant at 1 week (n = 4), 60% of CFU-GM and 100% of LTC-IC were resistant at 2 weeks (n = 2) and 74% of CFU-GM (n = 4) and 82% of LTC-IC (n = 2) were resistant at three weeks.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cranioplasty after decompressive hemicraniectomy: Underestimated surgery-associated complications?

Objective

Decompressive hemicraniectomy (DC) and duroplasty after malignant brain infarction or traumatic brain injury is a common surgical procedure. Usually, preserved bone flaps are being reimplanted after resolution of brain swelling. Alloplast cranioplasties are seldom directly implanted due to the risk of wound healing disorders. While numerous studies deal with DC, little is known about the encountered problems of bone flap reimplantation. Thus, aim of the study was to identify surgery-associated complications after bone flap reimplantation.

Methods

We performed a retrospective chart analysis of patients that underwent DC and subsequent bone flap reimplantation between 2001 and 2011 at our institution. We registered demographic data, initial clinical diagnosis and surgery-associated complications.

Results

We identified 136 patients that underwent DC and subsequent reimplantation. Forty-one patients (30.1%) had early or late surgery-associated complications after bone flap reimplantation. Most often, bone flap resorption and postoperative wound infections were the underlying causes (73%, n = 30/41). Multivariate analysis identified age (p = 0.045; OR = 16.30), GOS prior to cranioplasty (p = 0.03; OR = 2.38) and nicotine abuse as a prognostic factor for surgery-associated complications (p = 0.043; OR = 4.02). Furthermore, patients with early cranioplasty had a better functional outcome than patients with late cranioplasty (p < 0.05).

Conclusions

Almost one-third of the patients that are operated on for bone flap reimplantation after DC suffer from surgery-associated complications. Most often, wound healing disorders as well as bone flap resorption lead to a second or even third operation with the need for artificial bone implantation. These results might raise the question, if subsequent operations can be avoided, if an artificial bone is initially chosen for cranioplasty.     

Muscle Activation during Push-Ups with Different Suspension Training Systems

The purpose of this study was to analyze upper extremity and core muscle activation when performing push-ups with different suspension devices. Young fit male university students (n = 29) performed 3 push-ups each with 4 different suspension systems. Push-up speed was controlled using a metronome and testing order was randomized. Average amplitude of the electromyographic root mean square of Triceps Brachii, Upper Trapezius, Anterior Deltoid, Clavicular Pectoralis, Rectus Abdominis, Rectus Femoris, and Lumbar Erector Spinae was recorded. Electromyographic signals were normalized to the maximum voluntary isometric contraction (MVIC). Electromyographic data were analyzed with repeated-measures analysis of variance with a Bonferroni post hoc. Based upon global arithmetic mean of all muscles analyzed, the suspended push-up with a pulley system provided the greatest activity (37.76% of MVIC; p < 0.001). Individually, the suspended push-up with a pulley system also provided the greatest triceps brachii, upper trapezius, rectus femoris and erector lumbar spinae muscle activation. In contrast, more stable conditions seem more appropriate for pectoralis major and anterior deltoid muscles. Independent of the type of design, all suspension systems were especially effective training tools for reaching high levels of rectus abdominis activation.

Key Points

• Compared with standard push-ups on the floor, suspended push-ups increase core muscle activation.
• A one-anchor system with a pulley is the best option to increase TRICEP, TRAPS, LUMB and FEM muscle activity.
• More stable conditions such as the standard push-up or a parallel band system provide greater increases in DELT and PEC muscle activation.
• A suspended push-up is an effective method to achieve high muscle activity levels in the ABS.

Key words: EMG, unstable, core, trunk, exercise     

Knowledge of Repetitions Range Affects Force Production in Trained Females

Most studies have examined pacing strategies with cyclical activities (running and cycling). It has been demonstrated that males employ different pacing strategies during repeated maximal voluntary contractions (MVCs) dependent upon a known endpoint. Since different fatiguing mechanisms have been identified between the genders, it is not known if females use comparable pacing strategies. The purpose of this study was to examine if informing female subjects regarding the number of MVCs to perform would affect force and electromyography (EMG). Twenty well-trained females completed 3 fatiguing protocols in a randomized order. In the control condition participants were informed they would perform twelve MVCs and then actually completed twelve. In the unknown condition they were not told how many MVCs to perform but were stopped after twelve. In the deception condition they were initially informed to perform 6 MVCs, but after the 6^th MVC they were asked to perform a few more MVCs and were stopped after twelve. During the first 6 MVCs, forces in the deception condition were greater compared to the unknown (p = 0.021, ES = 0.65, 5%) and control (p = 0.022, ES = 0.42, 3%) conditions. No differences were found between conditions in the last 6 MVCs. A main effect for repetitions showed force deficits during the first 6 MVCs (p = 0.000, ES = 1.81, 13%) and last 6 MVCs (p = 0.05, ES = 0.34, 3%). No differences were found between conditions in biceps and triceps EMG. However, EMG decreased during the first 6 MVCs for biceps (p = 0.001, ES = 1.0, 14%) and triceps (p = 0.001, ES = 0.76, 14%) across conditions. No differences were found in the last 6 MVCs. The anticipation of performing fewer MVCs led to increased force, whereas no endpoint led to decreased force production.

Key points

• Pacing strategies occur during repeated (fatiguing) MVCs as a function of end point expectations.
• Females use similar pacing strategies as previously published results with males.
• Without a known end point, females will tend to pace themselves by decreasing force output even when asked to perform maximal contractions.

Key words: Fatigue, electromyography, deception, pacing     

Myelosuppressive conditioning improves autologous engraftment of genetically marked hematopoietic repopulating cells in dogs

We have studied the role of different conditioning regimens for engraftment of genetically marked hematopoietic repopulating cells in dogs. Peripheral blood (PB) and/or marrow cells collected after treatment with recombinant canine stem cell factor (rcSCF) or cyclophosphamide were transduced in a vector-containing long-term culture system. Three different vector-producing cell lines with similar viral titers were used. In two of them, the neo-containing LN vector was packaged either in the PA317 cell line with an amphotropic murine retrovirus envelope or the PG13 cell line with the gibbon ape leukemia virus (GALV) envelope. The MFG/GC vector produced in PA317 cells contained the human glucocerebrosidase gene. Nineteen dogs received either no conditioning (group A, n = 5), irradiation to both humeri with 1,000 cGy (group B, n = 5), a sublethal dose of cyclophosphamide 40 mg/kg (group C, n = 4), a sublethal dose of 200 or 300 cGy total body irradiation (TBI) (group D, n = 3), or an otherwise lethal dose of 920 cGy TBI (group E, n = 3) before intravenous (groups A, C, D, E) or intramedullary (group B) infusion of the transduced autologous hematopoietic cells. Transduction efficiency of hematopoietic cells at the time of infusion into the animals was similar among the different conditioning groups. Dogs were observed for at least 6 months. PB granulocytes were obtained at least every 3 weeks after transplant and analyzed by polymerase chain reaction for the presence of the transduced genes. The percentages of positive results in dogs more than 4 weeks after transplantation were 0% without conditioning, 5% with local irradiation, 18% with sublethal cyclophosphamide, 33% with sublethal TBI, and 17% with otherwise lethal TBI. Analyzing the influence of conditioning regimens by a generalized estimating equation (GEE) technique, which considered the use of different retrovirus vectors and the number of mononuclear cells infused as potential confounding variables, we found that engraftment of genetically marked repopulating cells was significantly improved (P < .001) in dogs receiving systemic conditioning with either otherwise lethal TBI, sublethal TBI, or sublethal cyclophosphamide compared to dogs with local irradiation only or no conditioning. Within the limitation of the experimental design, these data suggest that myeloablative or myelosuppressive conditioning improves engraftment of genetically marked hematopoietic repopulating cells.     

DLA-identical bone marrow grafts after low-dose total body irradiation: the effect of canine recombinant hematopoietic growth factors

Previous studies found that bone marrow (BM) allografts from DLA- identical littermates resulted in survival of two thirds of recipient dogs after otherwise lethal doses of 450 to 600 cGy of total body irradiation (TBI) because of successful allografts or autologous recovery after rejection of the allografts. The current study asked whether survival could be further improved by treating allograft recipients with recombinant canine granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), or G-CSF/SCF. Of 21 dogs, 14 (67%) receiving allografts but no growth factors survived, 10 with successful allografts (including 5 mixed chimeras) and 4 with autologous recovery; whereas 7 animals died, 5 from infections during BM aplasia and 2 from acute graft-versus-host disease. By comparison, 30 of 34 dogs (88%) receiving hematopoietic growth factors in addition to the BM graft survived, 17 with successful allografts (including 10 mixed chimeras) and 13 with autologous recovery; whereas 4 died, all with infection related to BM aplasia after rejection of the allograft. Survival was similar for recipients of G-CSF, SCF, or the combination of G-CSF and SCF. Logistic regression analyses, which accounted for possible effects of TBI dose, showed a trend for improved survival in dogs receiving growth factors (P = .09), no change in allogeneic engraftment (P = .74), and a slight increase in autologous recovery (P = .22). In agreement with previous data, we found that grafts of BM from DLA-identical littermates improved survival of recipient dogs exposed to low but otherwise lethal doses of TBI. A further improvement in survival could be achieved by additional treatment with G-CSF, SCF, or G-CSF/SCF. Results suggest that treatment by hematopoietic growth factors along with BM grafts should be considered for victims of radiation accidents.     

Hyperdiploid (47-50) acute lymphoblastic leukemia in children.

Among ploidy groups in childhood acute lymphoblastic leukemia (ALL), hyperdiploidy 47 to 50 is perhaps the least well known. From December 1979 to December 1990, we successfully studied banded karyotypes in 598 cases of newly diagnosed ALL, of which 86 (14.4%) had modal chromosome numbers of 47 to 50. In this group, the most frequently acquired numerical abnormalities were +21 (n = 34), +X (18), +8 (8), and +10 (7). The chromosomal regions most often affected by structural abnormalities were 1q (n = 13), 6q (12), 12p (18), and 19p (9). Analysis of event-free survival (EFS) for Studies X and XI among patients with hyperdiploid (47 to 50) ALL showed no significant differences in outcome according to the presence (n = 36) or absence (n = 35) of chromosomal translocations (P = .81) or the gain of specific chromosomes (P = .40). Patients with hyperdiploid (47 to 50) ALL treated in a contemporary program of multiagent chemotherapy had a significantly better outcome than did those in an earlier study using less intensive therapy (4-year EFS = 75% [95% confidence interval, 55% to 86%] v 41% [22% to 59%]; P = .006 by the logrank test). Our findings indicate that the adverse prognosis previously attributed to hyperdiploidy 47 to 50 improves significantly with more effective chemotherapy.