Irradiation Attenuates Systemic Lupus Erythematosus-Like Morbidity in NZBWF1 Mice: Focusing on CD180-Negative Cells.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies that can induce systemic inflammation. Ultraviolet-A and X-ray irradiation have been reported to have therapeutic effects in patients with SLE. We previously demonstrated that CD180-negative cells, these are radiosensitive, contribute to the development of SLE-like morbidity in NZBWF1 mice. In this study, the effects of irradiation on SLE-like morbidity manifestations in NZBWF1 mice and on CD180-negative cells were investigated. Whole-body irradiation, excluding the head, attenuated SLE-like morbidity in vivo, as indicated by the prevention of the renal lesion development, inhibition of anti-dsDNA antibody production, reduction of urinary protein levels, and prolongation of the lifespan. Irradiation also reduced the proportion of CD180-negative cells in the spleen. Although other immune cells or molecules may be triggered because of the whole-body irradiation treatment, previous research, and the current results suggest a strong relationship between the radiation-induced decrease in CD180-negative cells and the amelioration of SLE-like morbidities. Clinical trials assessing CD180-negative cells as a therapeutic target for SLE have been hampered by the lack of validated cell markers; nonetheless, the present findings suggest that radiotherapy may be a new therapeutic strategy for managing SLE symptoms.

Reduced High-Dose Radiation-Induced Residual Genotoxic Damage by Induction of Radioadaptive Response and Prophylactic Mild Dietary Restriction in Mice.

Radioadaptive response (RAR) describes a phenomenon in a variety of in vitro and in vivo systems that a low-dose of priming ionizing radiation (IR) reduces detrimental effects of a subsequent challenge IR at higher doses. Among in vivo investigations, studies using the mouse RAR model (Yonezawa Effect) showed that RAR could significantly extenuate high-dose IR-induced detrimental effects such as decrease of hematopoietic stem cells and progenitor cells, acute radiation hematopoietic syndrome, genotoxicity and genomic instability. Meanwhile, it has been demonstrated that diet intervention has a great impact on health, and dietary restriction shows beneficial effects on numerous diseases in animal models. In this work, by using the mouse RAR model and mild dietary restriction (MDR), we confirmed that combination of RAR and MDR could more efficiently reduce radiogenotoxic damage without significant change of the RAR phenotype. These findings suggested that MDR may share some common pathways with RAR to activate mechanisms consequently resulting in suppression of genotoxicity. As MDR could also increase resistance to chemotherapy and radiotherapy in normal cells, we propose that combination of MDR, RAR, and other cancer treatments (i.e., chemotherapy and radiotherapy) represent a potential strategy to increase the treatment efficacy and prevent IR risk in humans.

Increased Hematopoietic Stem Cells/Hematopoietic Progenitor Cells Measured as Endogenous Spleen Colonies in Radiation-Induced Adaptive Response in Mice (Yonezawa Effect).

The existence of radiation-induced adaptive response (AR) was reported in varied biosystems. In mice, the first in vivo AR model was established using X-rays as both the priming and the challenge doses and rescue of bone marrow death as the end point. The underlying mechanism was due to the priming radiation-induced resistance in the blood-forming tissues. In a series of investigations, we further demonstrated the existence of AR using different types of ionizing radiation (IR) including low linear energy transfer (LET) X-rays and high LET heavy ion. In this article, we validated hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPCs) measured as endogenous colony-forming units-spleen (CFU-S) under AR inducible and uninducible conditions using combination of different types of IR. We confirmed the consistency of increased CFU-S number change with the AR inducible condition. These findings suggest that AR in mice induced by different types of IR would share at least in part a common underlying mechanism, the priming IR-induced resistance in the blood-forming tissues, which would lead to a protective effect on the HSCs/HPCs and play an important role in rescuing the animals from bone marrow death. These findings provide a new insight into the mechanistic study on AR in vivo.

Peripheral nerve block combined with general anesthesia for lower extremity amputation in hemodialysis patients: case series.

BACKGROUND: Anesthetic management of lower extremity amputation in chronic hemodialysis (HD) patients can be challenging because of their poor cardiovascular status. As previously reported, peripheral nerve block (PNB) may be beneficial in these complicated cases. We report the effects of PNB combined with general anesthesia on hemodynamic stability in HD patients undergoing elective lower extremity amputation. METHODS: We retrospectively analyzed 13 HD patients who underwent lower extremity amputation. Patients received general anesthesia (GA group, n = 7) or general anesthesia combined with PNB (GA with PNB group, n = 6), as decided by the anesthesiologists. Mean blood pressure (MBP), systolic blood pressure (SBP), lowest BP, heart rate (HR), blood loss, fluid and blood infusion volumes, and doses of vasopressors required were compared for hemodynamic assessment. The coefficient of variation ([Formula: see text]) of MBP (CVMBP) and SBP (CVSBP) was calculated to compare hemodynamic stability. Intraoperative opioid use and postoperative pain scores at rest using a numerical rating scale (NRS) on postoperative days 0 and 1 were compared for pain assessment. We also assessed 30-day mortality. RESULTS: CVMBP in the GA group was significantly higher than that in the GA with PNB group (0.15 ± 0.05 and 0.08 ± 0.04, respectively, p = 0.03). The CVSBP in the GA group was also significantly higher than that in the GA with PNB group (0.16 ± 0.02 and 0.09 ± 0.01, respectively, p = 0.03). No significant differences in other hemodynamic parameters were observed. Intraoperative fentanyl doses were significantly lower in the GA with PNB group (GA 210.7 ± 99.9 µg vs. GA with PNB 113.0 ± 75.6 µg, p = 0.04). There were no significant differences in other pain parameters and 30-day mortality between the groups. CONCLUSION: Our results suggest that PNB combined with general anesthesia contributes to intraoperative hemodynamic stability through better pain control in HD patients undergoing lower extremity amputation.

Antifibrotic response of cardiac fibroblasts in hypertensive hearts through enhanced TIMP-1 expression by basic fibroblast growth factor.

BACKGROUND: Cardiac fibroblasts (CFs) play a pivotal role in the development of myocardial fibrosis. We previously demonstrated that direct injection of basic fibroblast growth factor (bFGF) into the hypertensive Dahl salt-sensitive (DS) rat heart prevented systolic dysfunction and left ventricular dilation effectively. However, the precise role played by bFGF in fibrotic response of CFs remains unclear. We suggested potential effects of bFGF on the fibrotic response of CFs in vitro. METHODS AND RESULTS: Histopathologic assessment of cardiac fibrosis demonstrated a marked decline in the extent of perivascular and interstitial fibrosis in bFGF-injected hypertensive DS rat hearts. CFs harvested from the hearts of noninjected DS rats demonstrated a significantly increased messenger RNA (mRNA) expression of matrix metalloproteinase (MMP)-2, MMP-9, and both collagen I and III. In contrast, bFGF treatment in the CFs induced a marked increase in tissue inhibitor of MMP (TIMP)-1 expression and a marked decline in MMP-9 activation. bFGF also induced a decline in α-smooth muscle actin and collagen I and III mRNA expression in the CFs accompanied by inhibited differentiation of CFs into myofibroblasts. Small interfering RNA targeting FGF receptor 1 confirmed a specific interference of the mRNA expression changes elicited by bFGF. In vivo examination confirmed many TIMP-1-positive CFs in perivascular spaces of bFGF-injected hearts. CONCLUSIONS: Up-regulated TIMP-1 expression and down-regulated MMP-9 activation by bFGF in CFs could prevent excessive ECM degradation and collagen deposition in perivascular spaces effectively, leading to prevention of cardiac fibrosis during hypertensive heart failure. SUMMARY: Cardiac fibroblasts (CFs) play a pivotal role in myocardial fibrosis. The precise role of CFs in fibrotic response played by growth factors remains unclear. Our results indicates that basic fibroblast growth factor could up-regulate TIMP-1 expression and down-regulate MMP-9 activation in CFs in perivascular spaces, leading to inhibited progression of cardiac fibrosis during hypertensive heart failure.

Sodium orthovanadate (vanadate), a potent mitigator of radiation-induced damage to the hematopoietic system in mice.

Previous in vitro and in vivo studies have shown that sodium orthovanadate (vanadate), an inorganic vanadium compound, could effectively suppress radiation-induced p53-mediated apoptosis via both transcription-dependent and transcription-independent pathways. As a potent radiation protector administered at a dose of 20 mg/kg body weight (20 mg/kg) prior to total body irradiation (TBI) by intra-peritoneal (ip) injection, it completely protected mice from hematopoietic syndrome and partially from gastrointestinal syndrome. In the present study, radiation mitigation effects from vanadate were investigated by ip injection of vanadate after TBI in mice. Results showed that a single administration of vanadate at a dose of 20 mg/kg markedly improved the 30-day survival rate and the peripheral blood hemogram, relieved bone marrow aplasia and decreased occurrence of the bone marrow micronucleated erythrocytes in the surviving animals. The dose reduction factor was 1.2 when a single dose of 20 mg/kg was administered 15 min after TBI in mice using the 30-day survival test as the endpoint. Results also showed that either doubling the vanadate dose (40 mg/kg) in a single administration or continuing the vanadate treatment (after a single administration at 20 mg/kg) from the following day at a dose of 5 mg/kg per day for 4 consecutive days further significantly improved the efficacy for rescuing bone marrow failure in the 30-day survival test. Taken together, these findings indicate that vanadate would be a potent mitigator suppressing the acute lethality (hematopoietic syndrome) and minimizing the detrimental effects (anhematopoiesis and delayed genotoxic effects) induced by TBI in mice.

Pathogenesis of lupus-like nephritis through autoimmune antibody produced by CD180-negative B lymphocytes in NZBWF1 mouse.

Toll-like receptors appear to play an important role in the pathogenesis of lupus-like nephritis in mice. In human and mouse, CD180 is a homologue of TLR4. In SLE patients, the number of CD180-negative B cells in peripheral blood changes in parallel with disease activity. In the present study using NZBWF1 mice, the population of splenic CD180-negative B cells increased with progression of renal lesions and aging. These cells produced both anti-dsDNA and histone antibodies; the peripheral blood levels of anti-dsDNA antibody increased markedly with aging. B cells infiltrating into renal lesions were CD180-negative and produced anti-dsDNA antibody. Considered together, these findings indicate that CD180-negative B cells contribute significantly to development of SLE-like morbidity in NZBWF1 mice by autoantibody production.

Trp53 activity is repressed in radio-adapted cultured murine limb bud cells.

Understanding the effects of of ionizing radiation (IR) at low dose in fetal models is of great importance, because the fetus is considered to be at the most radiosensitive stage of the development and prenatal radiation might influence subsequent development. We previously demonstrated the existence of an adaptive response (AR) in murine fetuses after pre-exposure to low doses of X-rays. Trp53-dependent apoptosis was suggested to be responsible for the teratogenic effects of IR; decreased apoptosis was observed in adapted animals. In this study, in order to investigate the role of Trp53 in AR, we developed a new model of irradiated micromass culture of fetal limb bud cells, which replicated proliferation, differentiation and response to IR in murine embryos. Murine fetuses were exposed to whole-body priming irradiation of 0.3 Gy or 0.5 Gy at embryonic day 11 (E11). Limb bud cells (collected from digital ray areas exhibiting radiation-induced apoptosis) were cultured and exposed to a challenging dose of 4 Gy at E12 equivalent. The levels of Trp53 protein and its phosphorylated form at Ser18 were investigated. Our results suggested that the induction of AR in mouse embryos was correlated with a repression of Trp53 activity.

X-ray-induced radioresistance against high-LET radiations from accelerated heavy ions in mice.

Induction of an adaptive response by priming X rays in combination with challenge irradiations from high-LET accelerated heavy ions was attempted in young adult female C57BL/6J Jms mice using 30-day survival after the challenge irradiations as an index. Three kinds of accelerated heavy ions from monoenergetic beams of carbon, silicon and iron ions with LETs of about 15, 55 and 200 keV/µm, respectively, were examined. A priming low dose of 0.50 Gy X rays in combination with a challenging dose of 7.50 Gy was used in the animals serving as a positive control group to confirm the successful induction of an adaptive response. The priming low dose of 0.50 Gy X rays was also used in combination with accelerated heavy ions. The priming low dose of X rays significantly reduced the mortality from the high challenge doses of carbon or silicon particles but not from iron particles. These results indicate that an adaptive response could be induced by priming low-LET X rays in combination with subsequent challenge high-LET irradiations from certain kinds of accelerated heavy ions, and successful induction of an adaptive response would possibly be an event related to the LET and/or the type of heavy ions. This is the first time that the existence of an adaptive response induced by low-LET X rays against high-LET whole-body irradiation in mice has been demonstrated. These findings would provide new insight into the radiation-induced adaptive response in vivo.

The mechanisms underlying fibroblast apoptosis regulated by growth factors during wound healing.

While investigating the mechanisms underlying cell death during wound healing processes, we uncovered the pro-apoptotic effects of basic fibroblast growth factor (bFGF) on granulation tissue fibroblasts following pretreatment with transforming growth factor (TGF)-beta1 in vitro. bFGF induced caspase-3 activation and apoptosis in TGF-beta1-pretreated granulation tissue-derived fibroblasts (GF-1) following bFGF treatment for 48 and 96 h. In contrast, fibroblasts that had been treated in the same manner and that originated from the uninjured dermis did not display apoptosis, indicating that the mechanisms underlying apoptosis events in fibroblasts that originate from normal dermal and wound tissues differ. In this process, we also found that bFGF inhibited Akt phosphorylation at serine 473 and induced a rapid loss of phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 in pretreated GF-1 cells, an event that coincided with the dissociation of phosphorylated FAK from the focal adhesions. Therefore, inhibition of survival signals relayed via the disrupted focal adhesion structures and inactivated Akt following bFGF treatment may lead to apoptosis in GF-1 cells pretreated with TGF-beta1. Pretreatment of GF-1 with TGF-beta1 followed by the addition of bFGF resulted in significantly greater inhibition of phosphorylation of Akt and FAK compared to treatment with TGF-beta1 or bFGF alone. The combinatorial treatment also led to proteolysis of FAK and inhibition of FAK and Akt protein expression in GF-1 cells. These findings demonstrated a significant role for the two cytokines in apoptosis of granulation tissue fibroblasts during wound healing. In vivo studies also confirmed a marked decline in phosphorylation and protein expression of Akt and FAK in bFGF-injected skin wounds. These results led to the hypothesis that temporal activation of TGF-beta1 and bFGF at the injury site promotes apoptosis in granulation tissue fibroblasts, an event that is critical for the termination of proliferative granulation tissue formation.

Myocardial apoptosis associated with the expression of proinflammatory cytokines during the course of myocardial infarction.

To clarify the role of myocardial apoptosis associated with the expression of proinflammatory cytokines in human myocardial infarction (MI), we have analyzed the expression of apoptosis positive for single-stranded DNA (ss-DNA) antibody, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-8 in 147 samples of infarcted myocardial tissue from 65 patients. ss-DNA-positive apoptotic nuclei were found mainly in cardiomyocytes in the border zones and granulation tissue cells in the infarct foci. The ss-DNA index (SI) of cardiomyocytes (average 0.13%) peaked at stage II (established myocardial necrosis), the value being significantly higher than at stages III (macrophage infiltration), IV (granulation formation), and V (scar formation) (P<0.05), whereas the SI of granulation tissue (average 0.08%) at stages III, IV, and V showed no significant differences between the three stages. These results suggest that cardiomyocyte apoptosis in the border zone is responsible for cellular loss in the acute stage of MI, whereas granulation tissue apoptosis may not be involved in the process of ventricular remodeling. TNF-alpha was expressed in cardiomyocytes in the border zones of infarct foci, but no significant positive correlation was found between SI and TNF-alpha index in cardiomyocytes (r=0.08, P = 0.37), suggesting that TNF-alpha does not serve as a direct trigger of cardiomyocyte apoptosis in vivo. The number of IL-8-positive cells peaked at stage II, and IL-8-myeloperoxidase-double-positive neutrophils were frequently detected, indicating that infiltrating neutrophils are the predominant source of IL-8 in the infarcted myocardium. These results suggest that, in human MI, TNF-alpha produced by cardiomyocytes does not play a critical role in their apoptosis, and that IL-8 produced by neutrophils is responsible for the subsequent accumulation and activation of neutrophils, thus increasing the degree of myocardial damage.

Effects of prenatal irradiation with an accelerated heavy-ion beam on postnatal development in rats: I. Neurophysiological alterations.

Effects on postnatal neurophysiological development in offspring were studied after exposure of pregnant Wistar rats to accelerated carbon-ion beams with an LET of about 13 keV/ mum at doses ranging from 0.1 Gy to 2.5 Gy on the 15th day of gestation. The age at which four physiological markers appeared and five reflexes were acquired was examined prior to weaning. Gain in body weight was monitored until the offspring were 3 months old. Male offspring were evaluated as young adults using two behavioral tests. The effects of X rays estimated for the same biological end points were studied for comparison. For most of the end points at early age, no significant alterations were observed in offspring that received prenatal irradiation with 0.1 Gy of either accelerated carbon ions or X rays compared to the offspring of sham-irradiated dams. However, all offspring whose dams received 2.5 Gy died prior to weaning. Offspring from dams irradiated with accelerated carbon ions generally showed higher incidences of prenatal death and preweaning mortality, markedly delayed accomplishment in their physiological markers and reflexes, and gain in body weight compared to those exposed to X rays at doses of 0.5 to 2 Gy. Significantly reduced ratios of main organ weight to body weight at the postnatal ages of 30, 60 and 90 days were also observed within this dose range. The results indicate that irradiation with 0.5 to 2 Gy on day 15 of gestation caused permanent alterations in offspring that were dependent on dose. The alterations include permanent growth retardation, morphological malformations in main organs, including microcephaly, diminished reflex attainment, delayed appearance of physiological markers, and changes in adult behavior. Exposure to 1 to 2 Gy of radiation resulted in growth retardation and behavioral alterations that persisted throughout life. Accelerated carbon ions generally induced more detrimental effects than X rays.

Activated caspase expression and apoptosis increase in keloids: cytochrome c release and caspase-9 activation during the apoptosis of keloid fibroblast lines.

To characterize apoptosis in keloids and the mechanisms responsible for this process, the expression of activated caspase-9 and -3 in fibroblasts obtained from keloids was analyzed. Immunohistochemistry revealed that the number of fibroblasts positive for terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) or activated caspase-9 or -3 was low but was significantly higher in keloid tissues than in normal scar tissues. Significant relationships between the number of caspase-positive fibroblasts and TUNEL-positive fibroblasts suggested that the activation of caspase-9 and -3 induces apoptosis in a subpopulation of keloid fibroblasts. All keloid fibroblast cell lines established in this study showed activation of caspase-9 and -3 after serum deprivation for 3 or 4 hours, as shown using Western blotting. Furthermore, serum deprivation-induced apoptosis in a keloid fibroblast line was blocked by a caspase-9 inhibitor (acetyl-Leu-Glu-His-Asp-al), indicating that activation of caspase-9 was necessary for the process of apoptosis in keloid fibroblasts. Although serum deprivation did not significantly change the level of apoptosis protease activating factor-1 in any of the lines, cytochrome c release was detected in cytosolic fractions of the lines after serum deprivation for 3 or 4 hours. These results strongly suggest that keloid fibroblasts are predisposed to apoptosis and cytochrome c release and that caspase-9 activation may underlie regulation of apoptosis in keloid fibroblasts in vivo.

Adaptive response in embryogenesis: IV. Protective and detrimental bystander effects induced by X radiation in cultured limb bud cells of fetal mice.

The radioadaptive response and the bystander effect represent important phenomena in radiobiology that have an impact on novel biological response mechanisms and risk estimates. Micromass cultures of limb bud cells provide an in vitro cellular maturation system in which the progression of cell proliferation and differentiation parallels that in vivo. This paper presents for the first time evidence for the correlation and interaction in a micromass culture system between the radioadaptive response and the bystander effect. A radioadaptive response was induced in limb bud cells of embryonic day 11 ICR mice. Conditioning irradiation of the embryonic day 11 cells with 0.3 Gy resulted in a significant protective effect against the occurrence of apoptosis, inhibition of cell proliferation, and differentiation induced by a challenging dose of 5 Gy given the next day. Both protective and detrimental bystander effects were observed; namely, irradiating 50% of the embryonic day 11 cells with 0.3 Gy led to a successful induction of the protective effect, and irradiating 70% of the embryonic day 12 cells with 5 Gy produced a detrimental effect comparable to that seen when all the cells were irradiated. Further, the bystander effect was markedly decreased by pretreatment of the cells with an inhibitor to block the gap junction-mediated intercellular communication. These results indicate that the bystander effect plays an important role in both the induction of a protective effect by the conditioning dose and the detrimental effect of the challenge irradiation. Gap junction-mediated intercellular communication was suggested to be involved in the induction of the bystander effect.