Haakon B. Benestad

Vitamin C and E supplementation hampers cellular adaptation to endurance training in humans: a double-blind, randomised, controlled trial

Recent studies have indicated that antioxidant supplementation may blunt adaptations to exercise, such as mitochondrial biogenesis induced by endurance training. However, studies in humans are sparse and results are conflicting. Isolated vitamin C and E supplements are widely used, and unravelling the interference of these vitamins in cellular and physiological adaptations to exercise is of interest to those who exercise for health purposes and to athletes. Our results show that vitamin C and E supplements blunted the endurance training‐induced increase of mitochondrial proteins (COX4), which is important for improving muscular endurance. Training‐induced increases in V̇O2 max and running performance were not detectably affected by the supplementation. The present study contributes to understanding of how antioxidants may interfere with adaptations to exercise in humans, and the results indicate that high dosages of vitamins C and E should be used with caution.

A COX‐2 inhibitor reduces muscle soreness, but does not influence recovery and adaptation after eccentric exercise

The aim of this study was to investigate the effect of a cyclooxygenase (COX)‐2 inhibitor on the recovery of muscle function, inflammation, regeneration after, and adaptation to, unaccustomed eccentric exercise. Thirty‐three young males and females participated in a double‐blind, placebo‐controlled experiment. Seventy unilateral, voluntary, maximal eccentric actions with the elbow flexors were performed twice (bouts 1 and 2) with the same arm, separated by 3 weeks. The test group participants were administered 400 mg/day of celecoxib for 9 days after bout 1. After both bouts 1 and 2, concentric and isometric force‐generating capacity was immediately reduced (∼40–50%), followed by the later appearance of muscle soreness and increased serum creatine kinase levels. Radiolabelled autologous leukocytes (detected by scintigraphy) and monocytes/macrophages (histology) accumulated in the exercised muscles, simultaneously with increased satellite cell activity. These responses were reduced and recovery was faster after bout 2 than 1, demonstrating a repeated‐bout effect. No differences between the celecoxib and placebo groups were detected, except for muscle soreness, which was attenuated by celecoxib. In summary, celecoxib, a COX‐2 inhibitor, did not detectably affect recovery of muscle function or markers of inflammation and regeneration after unaccustomed eccentric exercise, nor did the drug influence the repeated‐bout effect. However, it alleviated muscle soreness.

Endostatin reduces vascularization, blood flow, and growth in a rat gliosarcoma

Endostatin, the 20-kDa C-terminal fragment of collagen XVIII, has previously been shown to inhibit growth and induce regression of different experimental tumors in rodents. In this study, we show that recombinant murine and human endostatin, produced in 293 EBNA cells and yeast, respectively, inhibit ectotopic as well as orthotopic growing BT4Cn gliosarcomas in BD-IX rats. In rats in which s.c. gliomas were grown for a total of 29 days, systemic treatment with recombinant murine endostatin induced about 50% reduction of intratumoral blood flow and tumor size after only 10 days of therapy. In contrast, the blood flow to irrelevant organs was unaffected by endostatin, indicating its specificity of action. Tumors were not observed to increase in size or regrow after cessation of therapy. Furthermore, endostatin-treated rats with i.c. tumors had significantly longer survival time than did untreated controls. In the treated rats, endostatin therapy resulted in a reduced tumor blood vessel volume and an increased tumor cell density with an increased apoptotic index within a given tumor volume, as verified by flow cytometry and by staining with deoxynucleotidyltransferase-mediated dUTP nick-end labeling. This work verifies the general anti-angiogenic and antitumor effects of endostatin and indicates that the protein may also be considered as a treatment strategy for malignant brain tumors.

Recruitment of alloreactive natural killer cells to the rat peritoneum by a transfected cell line secreting rat recombinant interleukin-2

In order to obtain large numbers of natural killer (NK) cells from single rats for functional studies, we have devised a method for the generation of IL-2-activated NK cells in vivo. Rats were implanted intraperitoneally with cell-impermeable diffusion chambers (DC) containing cultures of transfected Chinese hamster ovary (CHO) cells secreting rat recombinant interleukin-2 (rIL-2). This resulted in a dramatic increase in the peritoneal exudate cell (PEC) number with a peak (300-1000 x 10(6)) 1 week after implantation. The majority were mononuclear cells of which a large proportion were CD3-NKR-P1+ NK cells, but with substantial numbers of macrophages (M phi) and CD3+8+NKR-P1+ T cells also. The NK activity against standard tumor target cells was high among PEC from six different inbred rat strains tested. However, the NK cell-mediated reactivity against concanavalin A (ConA)-activated T cell blasts from a panel of major histocompatibility complex (MHC) congenic strains differed widely. PEC from some strains (PVG, LOU/C, and AO) efficiently lysed all the MHC-disparate lymphoblasts. In other strains (BN and LEW) more restricted allorecognition repertoires were observed, whereas PEC from one strain (DA) were unresponsive. The secretion of rat rIL-2 intraperitoneally did not lead to a significant increase in the IL-2 level in the blood or in the total number or activity of NK cells in blood and spleen. The present method represents a most potent technique for generating large numbers of functional rat NK cells and shows the high efficiency with which IL-2 can induce NK cell recruitment in vivo.

Inhibitors of angiogenesis selectively reduce the malignant cell load in rodent models of human myeloid leukemias

Angiogenesis is essential for growth and metastasis of solid tumors and probably also for hematological malignancies. Angiogenic inhibitors, like endostatin (ES) and PI-88, retard cancer growth. We tested these in mice with juvenile myelomonocytic leukemia (JMML), and in rats with acute myeloid leukemia (BNML). Eight weeks after transplantation and with a continuous drug treatment for the last 4 weeks, the leukemic cell mass decreased from almost 90% of all bone marrow cells to about 15 and 45% with ES, to about 35 and 55% with PI-88, and to about 10 and 25% with ES + PI-88 in the leukemic mice and rats, respectively. The numbers of normal human bone marrow cells transplanted into mice were unchanged by the treatments. The microvessel density in leukemic animals given ES or PI-88 was 10–50% of that in untreated animals. Notably, simultaneous treatment with ES and PI-88 led to a reduction of about 95% in JMML mice and 85% in BNML rats. In vitro proliferation of either JMML or BNML cells was not significantly altered by either drug, demonstrating the selectivity of ES and PI-88 as angiogenic inhibitors. In conclusion, anti-angiogenic therapy may be a valuable adjunct to conventional treatment of leukemia.

Macrophage‐dependent regulation of neutrophil mobilization and chemotaxis during development of sterile peritonitis in the rat

Abstract: Pro‐inflammatory cytokines attract leukocytes to inflamed tissues and activate them. Few attempts have been made to identify the sources of cytokines in vivo. We examined the importance of peritoneal macrophages in the mobilization and homing of neutrophils to a sterile peritonitis in the rat, with emphasis on their cytokine production. Macrophages, present in virtually all tissues, are known to be easily activated and to serve as an important source of cytokines. Flow cytometric analysis of cells stained intracellularly with tagged antibodies against various cytokines revealed that the peritoneal macrophages were stimulated to produce the following cytokines: interleukin (IL)‐1β, macrophage inflammatory protein‐2 (MIP‐2), and keratinocyte‐derived cytokine (KC). High numbers of neutrophils, activated on arrival into the peritoneal cavity, also produced IL‐1β, whereas lower numbers contained interleukin‐6, tumor necrosis factor‐α, MIP‐2, KC, and MIP‐1α. This marked activation of peritoneal neutrophils was also reflected by increased surface expression of CD11b. On the other hand, peritoneal macrophages expressed high basal levels of CD11b, which were reduced 24 h after the onset of inflammation. In rats selectively depleted of macrophages by i.p. injection of liposome‐containing clodronate, the massive influx of neutrophils to the peritoneal cavity was markedly reduced, as was the rapid mobilization of mature bone marrow neutrophils. Local macrophages are important both for the accumulation of neutrophils in the inflamed peritoneal cavity and for the early mobilization of neutrophils from the bone marrow. Macrophage‐derived IL‐1β, MIP‐2, and KC are possible mediators of neutrophil homing to inflamed tissues.

Adrenaline-induced leucocytosis: recruitment of blood cells from rat spleen, bone marrow and lymphatics

It is well known that adrenaline causes leucocytosis, but the sources and the mechanisms of this have not been clarified. We investigated the contributions of subpopulations of white blood cells to this leucocytosis and the importance of the spleen, bone marrow and lymphatics in releasing leucocytes into the blood stream following an injection of adrenaline. We studied possible effects of adrenaline on blood flow to the spleen and bone marrow to see if any contribution to leucocytosis from these organs could be perfusion dependent. In intact awake rats, total blood leucocytes increased within 5 min to about 220% of baseline concentration, the increases of lymphocytes and neutrophilic granulocytes being about 250% and 160%, respectively. The T and B lymphocytes and natural killer cells were all mobilized, to about 230% to 250% of baseline concentrations. The leucocytosis was short-lasting, so that the cell concentrations returned to baseline within 25 min after adrenaline injection. The bone marrow, spleen, and efferent lymphatics all contributed substantially to this leucocytosis, since band-nucleated granulocytes increased upon adrenaline injection, and splenectomized or thoracic duct drained rats showed a markedly reduced leucocytosis in response to adrenaline. Supplementary data were obtained with bone marrow depleted (with 89Sr irradiation) rats. The release of leucocytes from these organs was apparently not blood-flow dependent in the control rats since organ perfusion remained unaltered after adrenaline injection. Adrenaline was found to stimulate the release of both mono- and polymorphonuclear cells in the awake rat and the release of leucocytes from the spleen, bone marrow and efferent lymphatics to contribute significantly to the leucocytosis.

Sequestration patterns of transfused rat neutrophilic granulocytes under normal and inflammatory conditions.

Abstract: The fate of polymorphonuclear neutrophilic granulocytes (PMN) after their mobilization from the bone marrow of healthy individuals is not clearly understood. It has been suggested that there is a continuous utilization of these cells in widespread, subclinical inflammatory foci, where they are ultimately degraded. The goal of the present experiments was to determine whether an alternative ecotaxis (“homing”) exists, namely sequestration and degradation of PMN by mononuclear phagocytes exposed to the bloodstream in the liver, spleen and bone marrow. Blood PMN were collected from donor rats, labelled with 51Cr, and injected i.v. into 2 syngeneic rats, one of them having an induced sterile peritonitis. After various time intervals up to 18 h, the rats were killed and exsanguinated. As expected, we found cell‐bound radioactivity in the inflamed peritoneal cavities, and also a high amount of radioactivity in liver, spleen, and bone marrow. The bone marrow uptake of PMN appeared to be much lower in the inflammation rats than in the normal controls. These findings were confirmed in PMN transfer experiments using PVG rats congenic for the RT7 alloantigenic system. Here, transfused blood leukocytes were traced with fluorescent, monoclonal HIS41 antibodies and flow cytometry. A possible corticosteroid effect on the bone marrow sequestration could not be substantiated. Uptake and degradation of PMN takes place in organs containing phagocytes exposed to the bloodstream. Sequestration of PMN in the bone marrow is apparently down‐regulated in inflammatory states, perhaps increasing the PMN availability to inflamed tissue.

Decreased blood flow to rat bone marrow, bone, spleen, and liver in acute leukemia

An acute promyelocytic leukemia in the rat (BNML) has been used in model studies on pathogenesis and therapy of human acute myeloid leukemia. The blood supply to bone marrow during BNML development has hitherto not been examined, even though in general, blood flow to hematopoietic tissues might affect drug treatment and marrow transplantation regimes. We measured the perfusion of various organs during the course of the disease in untreated rats and in rats given one injection of cyclophosphamide treatment. Organ perfusion was measured with radioactive microspheres. Blood flow per gram tissue to the bone marrow, bone, spleen, and liver declined gradually during the leukemic progression, thus paralleling the growth of leukemic deposits. Cyclophosphamide treatment retarded, but did not reverse, the decreasing perfusion of these tissues.

A Second Prophylactic Mhc-mismatched Bone Marrow Transplantation Protects Against Rat Acute Myeloid Leukemia (bnml) Without Lethal Graft-versus-host Disease

Background. We have employed a rat model for human acute myeloid leukemia, a promyelocytic leukemia in the BN rat strain (BNML), to develop new protocols for immunotherapy in combination with allogeneic bone marrow transplantation (alloBMT). The status of mixed chimerism in allotransplanted rats provided an opportunity for immunotherapy using alloreactive donor cells. In addition to T or natural killer (NK) cells, we introduced a second infusion of bone marrow cells as prophylactic donor lymphocyte infusions (DLI) to test whether an effective graft-versus-leukemia (GVL) response could be obtained without clinical graft-versus-host disease (GVHD). Methods. BN rats were sublethally irradiated and transplanted with T-cell depleted bone marrow cells from either fully major histocompatibility complex (MHC)-mismatched (PVG) donor rats or MHC-matched (PVG.1N) as controls. Seven days after transplantation, rats were given 500 leukemic cells to mimic minimal residual disease. Additional cellular therapy was given at day +7. The efficiency of DLI was monitored by chimerism analysis in peripheral blood. Results. Rats receiving infusions of NK cells succumbed to leukemia. T-DLI induced complete donor T-cell chimerism and lethal GVHD. A second alloBMT protected against leukemia. This effect was dependent on an MHC incompatibility between the donor and host and also on the presence of alloreactive T cells in the second bone marrow inoculum, resulting in an increased, mixed donor T-cell chimerism. Conclusion. A second prophylactic transplantation influenced the degree of T-cell chimerism to balance favorably between GVL and GVHD. If applicable to humans, repeated alloBMT may provide a novel approach to leukemia therapy.