mFISH analysis of chromosomal damage in bone marrow cells collected from CBA/CaJ mice following whole body exposure to heavy ions (<Superscript>56</Superscript>Fe ions)

To date, there is scant information on in vivo induction of chromosomal damage by heavy ions found in space (i.e. ⁵⁶Fe ions). For radiation-induced response to be useful for risk assessment, it must be established in in vivo systems especially in cells that are known to be at risk for health problems associated with radiation exposure (such as hematopoietic cells, the known target tissue for radiation-induced leukemia). In this study, the whole genome multicolor fluorescence in situ hybridization (mFISH) technique was used to examine the in vivo induction of chromosomal damage in hematopoietic tissues, i.e. bone marrow cells. These cells were collected from CBA/CaJ mice at day 7 following whole-body exposure to different doses of 1 GeV/amu ⁵⁶Fe ions (0, 0.1, 0.5 and 1.0 Gy) or ¹³⁷Cs γ rays as the reference radiation (0, 0.5, 1.0 and 3.0 Gy, at the dose rate of 0.72 Gy/min using a GammaCell40). These radiation doses were the average total-body doses. For each radiation type, there were four mice per dose. Several types of aberrations in bone marrow cells collected from mice exposed to either type of radiation were found. These were exchanges and breaks (both chromatid- and chromosome-types). Chromosomal exchanges included translocations (Robertsonian or centric fusion, reciprocal and incomplete types), and dicentrics. No evidence of a non-random involvement of specific chromosomes in any type of aberrations observed in mice exposed to ⁵⁶Fe ions or ¹³⁷Cs γ rays was found. At the radiation dose range used in our in vivo study, the majority of exchanges were simple. Complex exchanges were detected in bone marrow cells collected from mice exposed to 1 Gy of ⁵⁶Fe ions or 3 Gy of ¹³⁷Cs γ rays only, but their frequencies were low. Overall, our in vivo data indicate that the frequency of complex chromosome exchanges was not significantly different between bone marrow cells collected from mice exposed to ⁵⁶Fe ions or ¹³⁷Cs γ rays. Each type of radiation induced significant dose-dependent increases (ANOVA, P < 0.01) in the frequencies of chromosomal damage, including the numbers of abnormal cells. Based upon the linear-terms of dose-response curves, ⁵⁶Fe ions were 1.6 (all types of exchanges), 4.3 (abnormal cells) and 4.2 (breaks, both chromatid- and chromosome-types) times more effective than ¹³⁷Cs γ rays in inducing chromosomal damage.     

Comparison of Cesium-137 and X-ray Irradiators by Using Bone Marrow Transplant Reconstitution in C57BL/6J Mice

Mice are used extensively in transplantation studies involving bone marrow ablation. Due to the increasing security issues and expenses involved with γ irradiators, self-contained X-ray irradiators have been increasing in popularity. We hypothesized that bone marrow ablation by irradiation of mice with a ¹³⁷Cs irradiator would be comparable to that from an X-ray source irradiator. A lethal-dose curve was obtained by irradiating C57BL/6J mice with 500, 700, 900, and 1100 cGy from either source. These data were used to determine the lethal radiation exposure range for a noncompetitive bone marrow engraftment curve for each source. At 90 d after reconstitution, the bone marrow engraftment curves revealed significant differences between the 2 sources in the establishment of B cell, myeloid, and T cell lineages. Murine B cell reconstitution after exposure to a ¹³⁷Cs source was greater than that after X-ray exposure at each dose level, whereas the converse was true for myeloid cell reconstitution. At the 1050- and 1100-cGy doses, mice irradiated by using the X-ray source demonstrated higher levels of T cell reconstitution but decreased survival compared with mice irradiated with the ¹³⁷Cs source. We concluded that although both sources ablated endogenous bone marrow sufficiently to enable stem cell engraftment, there are distinct physiologic responses that should be considered when choosing the optimal source for use in a study and that irradiation from the ¹³⁷Cs source was associated with lower overall morbidity due to opportunistic infection.Abbreviations: B3, barrier level; B4, barrier level 4; BMT, bone marrow transplantationStudies of hematopoietic stem cell transplantation have evolved over the past 60 y.^1,9 Many preclinical investigations involving cell and gene therapy and hematopoietic stem cell function are performed in mouse models, and techniques such as adoptive cell transfer and bone marrow transplant are commonly used in these studies. Such techniques often require a supralethal dose of irradiation to ensure adequate engraftment with donor cells and subsequent survival. Conventional γ-emitting irradiators (¹³⁷Cs and ⁶⁰Co sources) have been used to deliver myeloablative doses of radiation prior to bone marrow transplantation (BMT). After the terrorist attack of 11 September 11 2001, security measures regarding active radioactive source irradiators have been heightened. In 2005, the US Congress passed the Energy Policy Act, in which the US Nuclear Regulatory Commission was assigned to evaluate and prevent malicious misuse of radioactive materials. As a result, increased security controls were imposed on radioactive material sources and quantities of concern, including shielded active source irradiators.¹⁴ Mandated security measures now include fingerprinting and a criminal-history record check to allow persons unescorted access to various radioactive materials.¹⁵ Background checks and fingerprinting procedures can be time-consuming and present an additional expense that usually is passed on to individual investigators. These enhanced security measures have significantly increased the expense associated with use of these irradiators, and federal regulations as proposed in 10 CFR Part 37 are likely to become more stringent in coming years.¹⁶ This situation has correspondingly led to an increased interest in the use of X-ray irradiators as a substitute for γ-ray sources such as ¹³⁷Cs, and many animal facilities across the country have begun to purchase these units, even though there is no unbiased comparative information regarding the effectiveness of the instruments.In addition to decreased security requirements, X-ray irradiators are substantially less expensive to purchase than are active-source irradiators. After reviewing quotes, we estimate that the initial purchase price of an X-ray irradiator is about one sixth that of a cesium source. These figures do not include the costs of shipping, installation, or disposal of old active-source machines, and thus actual starts up costs are much higher. Annual maintenance as well as annual or semiannual dosimetry assessment costs are relatively comparable between the 2 sources. X-ray irradiators offer an additional financial advantage in that they do not require the strict security measures required for active γ-source irradiators. Given the number of disadvantages for the possession and use of γ-emitting irradiators, the use of X-ray irradiators in research likely will increase in the future.Extensive review of the literature did not reveal any studies in which bone marrow transplantation (BMT) efficiencies, kinetics, or overall responses in mice were compared between ¹³⁷Cs and X-ray irradiators. We hypothesized that both the ¹³⁷Cs and X-ray sources would ablate the bone marrow effectively and allow for comparable donor bone marrow reconstitution, and we sought to compare any differences in cell population engraftment after the use of each source. Recipient hematologic recovery after irradiation and reconstitution with bone marrow was assessed by determining the percentages of B and T lymphocytes and myeloid cells in the peripheral blood at 90 d after engraftment. In light of previously published work, we hypothesized that using the X-ray source before BMT would require a reduced dosage of radiation compared with that for the ¹³⁷Cs source.^4,6,9Historically, lethal-dose curves have been generated to calculate the dose which is lethal for 50% of the irradiated animals (mice, in this case) over a 30-d period (that is, the LD_50:30); this method allows approximation of the radiation sensitivity of a cohort of experimental mice.¹¹ A mouse in which 100% of the bone marrow has been ablated will be unable to recover hematopoietic function and will die. A priori, if the animal dies, one can assume that the minimal lethal dose has been reached or exceeded; conversely, if the mouse survives, the minimal lethal dose was not achieved. Because of the number of mice needed to calculate an accurate LD_50:30, we elected to perform a broad lethal-dose curve (1100 to 500 cGy in 200-cGy increments) to determine the point at which 100% death was reached for both sources. We then used this information as the lower radiation exposure limit for a bone marrow reconstitution curve (refined into 50-cGy increments), thereby allowing us to examine the bone marrow reconstitution response after differing radiation exposures.⁹ There is ample support in the literature for the broad lethal-dose test range chosen in this study.^4,9 Previous work with thymocyte reconstitution after bone marrow ablation has demonstrated that irradiation exposures of approximately 400 cGy are required to establish a population of donor-derived thymocytes in the recipient.⁵ Therefore, we used a dosage test range above 400cGy in the current study. The dose range for this study was 500 to 1100 cGy, and we expected to see morbidity and mortality primarily due to bone marrow failure between 8 and 20 d in the lethal-dose curve.¹²     

Investigation of unmatured hematopoietic cell migration in a mouse model

The objective of this work was to examine the migration of transplanted bone marrow hematopoietic lin⁻ cell population using the BALB/c mouse contact hypersensitivity model in vivo and to determine the time point at which they reach the site of injury (paw edema) as well as other undamaged organs, such as liver and spleen. Female BALB/c mice with induced contact hypersensitivity reaction were intravenously injected with 1×10⁶ cells/mouse lin⁻ cells, labeled with PKH67. The presence of lin⁻ stained cells in mouse tissue sections was evaluated by fluorescent microscopy. After one hour, the labeled cells were found in mice paw edema and liver, and after 4 hours in spleen tissue. Migrated hematopoietic lin⁻ cells remained in liver tissue for 48 h, and in spleen and paw edema at least for 72 h. Migrated stained cells in untreated paw were not found. The results prove that bone marrow unmatured hematopoietic cells are first found in paw edema, where they participate in the inhibition of tissue inflammation; these cells subsequently migrate to the liver and are found in the spleen shortly afterwards.     

Doses from external irradiation and ingestion of 134Cs, 137Cs and 90Sr of the population of Belarus accumulated over 35 years after the Chernobyl accident

This study considers the exposure of the population of the most contaminated Gomel and Mogilev Oblasts in Belarus to prolonged sources of irradiation resulting from the Chernobyl accident. Dose reconstruction methods were developed and applied in this study to estimate the red bone-marrow doses (RBMs) from (i) external irradiation from gamma-emitting radionuclides deposited on the ground and (ii) ¹³⁴Cs, ¹³⁷Cs and ⁹⁰Sr ingestion with locally produced foodstuffs. The mean population-weighted RBM doses accumulated during 35 years after the Chernobyl accident were 12 and 5.7 mGy for adult residents in Gomel and Mogilev Oblasts, respectively, while doses for youngest age groups were 20–40% lower. The highest mean area-specific RBM doses for adults accumulated in 1986–2021 were 63, 56 and 46 mGy in Narovlya, Vetka and Korma raions in Gomel Oblast, respectively. For most areas, external irradiation was the predominant pathway of exposure (60–70% from the total dose), except for areas with an extremely high aggregated ¹³⁷Cs soil to cow’s milk transfer coefficient (≥?5.0 Bq L^?1 per kBq m^?2), where the contribution of ¹³⁴Cs and ¹³⁷Cs ingestion to the total RBM dose was more than 70%. The contribution of ⁹⁰Sr intake to the total RBM dose did not exceed 4% for adults and 10% for newborns in most raion in Gomel and Mogilev Oblasts. The validity of the doses estimated in this study was assessed by comparison with doses obtained from measurements by thermoluminescence dosimeters and whole-body counters done in 1987–2015. The methodology developed in this study can be used to calculate doses to target organs other than RBM such as thyroid and breast doses. The age-dependent and population-weighted doses estimated in this study are useful for ecological epidemiological studies, for projection of radiation risk, and for justification of analytical epidemiological studies in populations exposed to Chernobyl fallout.

     

Administration of recombinant human IL11 after supralethal radiation exposure promotes survival in mice: interactive effect with thrombopoietin

In the present study, we evaluated the therapeutic potential of recombinant human IL11 in lethally irradiated C57BL6/J mice exposed to gamma rays. IL11 administered for 5 consecutive days beginning 2 h after total-body irradiation with 8 or 9 Gy 60Co gamma rays resulted in a significant increase in 30-day survival. When IL11 was administered, only a slight improvement in the hematopoietic status (both blood cell counts and progenitor cells) was observed after an 8-Gy exposure, and no improvement in hematopoietic reconstitution was observed after 9 Gy total-body irradiation. The enhancement of fibrinogen in the plasma of irradiated animals suggested the importance of infections in the death of animals. IL11 was able to limit the increase in fibrinogen levels. However, prevention of bacterial infections by antibiotic treatment, although it delayed death, was ineffective in promoting survival either in placebo-treated and IL11-treated mice. IL11 was administered along with thrombopoietin (TPO) or bone marrow transplantation to limit the hematopoietic syndrome, in addition to antibiotic treatment. When IL11 was combined with TPO, a potent stimulator of hematopoiesis, the survival of animals which had been irradiated with 10 Gy 137Cs gamma rays was increased significantly compared to those treated with IL11 or TPO alone. Furthermore, an interactive effect of TPO and IL11 on hematopoietic reconstitution was observed. Similarly, IL11 in combination with bone marrow transplantation enhanced survival after 15 Gy 137Cs gamma rays. These data suggest that the effect of IL11 on the hematopoietic system is only moderate when it is used alone in supralethally irradiated mice but that the effect is improved in the presence of a hematopoietic growth factor or bone marrow transplantation.     

The protocol for the isolation and cryopreservation of osteoclast precursors from mouse bone marrow and spleen

Osteoclasts are responsible for physiological bone remodeling as well as pathological bone destruction in osteoporosis, periodontitis and rheumatoid arthritis, and thus represent a pharmacological target for drug development. We aimed to characterize and compare the cytokine-induced osteoclastogenesis of bone marrow and spleen precursors. Established protocols used to generate osteoclasts from bone marrow were modified to examine osteoclastogenesis of the spleen cells of healthy mice. Osteoclast formation was successfully induced from spleen precursors using receptor activator of nuclear factor κB ligand (50 ng/ml) and macrophage colony stimulating factor (50 ng/ml). Compared to bone marrow cultures, differentiation from spleen required a longer cultivation time (9 days for spleen, as compared to 5 days for marrow cultures) and a higher plating density of non-adherent cells (75,000/cm² for spleen, as compared to 50,000/cm² for bone marrow). Osteoclasts generated from spleen precursors expressed osteoclast marker genes calcitonin receptor, cathepsin K and matrix metalloproteinase 9 and were capable of resorbing hydroxyapatite. The differentiation capacity of spleen and bone marrow precursors was comparable for BALB/c, C57BL/6 and FVB mice. We also developed and tested a cryopreservation protocol for the osteoclast precursors. While 70–80 % of cells were lost during the first week of freezing, during the subsequent 5 weeks the losses were within 2–5 % per week. Osteoclastogenesis from the recovered bone marrow precursors was successful up to 5 weeks after freezing. Spleen precursors retained their osteoclastogenic capacity for 1 week after freezing, but not thereafter. The described protocol is useful for the studies of genetically modified animals as well as for screening new osteoclast-targeting therapeutics.     

Chronic Internal Exposure to Low Dose 137Cs Induces Positive Impact on the Stability of Atherosclerotic Plaques by Reducing Inflammation in ApoE-/- Mice

After Chernobyl and Fukushima Daï Chi, two major nuclear accidents, large amounts of radionuclides were released in the environment, mostly caesium 137 (¹³⁷Cs). Populations living in contaminated territories are chronically exposed to radionuclides by ingestion of contaminated food. However, questions still remain regarding the effects of low dose ionizing radiation exposure on the development and progression of cardiovascular diseases. We therefore investigated the effects of a chronic internal exposure to ¹³⁷Cs on atherosclerosis in predisposed ApoE^-/- mice. Mice were exposed daily to 0, 4, 20 or 100 kBq/l ¹³⁷Cs in drinking water, corresponding to range of concentrations found in contaminated territories, for 6 or 9 months. We evaluated plaque size and phenotype, inflammatory profile, and oxidative stress status in different experimental groups. Results did not show any differences in atherosclerosis progression between mice exposed to ¹³⁷Cs and unexposed controls. However, ¹³⁷Cs exposed mice developed more stable plaques with decreased macrophage content, associated with reduced aortic expression of pro-inflammatory factors (CRP, TNFα, MCP-1, IFNγ) and adhesion molecules (ICAM-1, VCAM-1 and E-selectin). Lesions of mice exposed to ¹³⁷Cs were also characterized by enhanced collagen and smooth muscle cell content, concurrent with reduced matrix metalloproteinase MMP8 and MMP13 expression. These results suggest that low dose chronic exposure of ¹³⁷Cs in ApoE^-/- mice enhances atherosclerotic lesion stability by inhibiting pro-inflammatory cytokine and MMP production, resulting in collagen-rich plaques with greater smooth muscle cell and less macrophage content.     

A peptide from Testudo horsfieldii tortoise spleen as a potential helper for reducing acute radiation syndrome

The Middle Asian tortoise Testudo horsfieldii is one of the most radioresistant animals, with Lethal Dose (LD) 50/30 around 500 Gy. Extracts were prepared from different organs of the tortoise, and their biological activity was evaluated. Crude extract from the spleen was found to significantly increase survival of mice treated with lethal doses of radiation. In an iterative process, the active extract was purified by chromatography, and the fractions were screened for biological activity. Various vital parameters were monitored: peripheral blood leukocytes, spleen colonies, mitosis in the bone marrow, and survival after 30 days. The process concluded with the isolation, characterization, and synthesis of the tetrapeptide FTGN, which accelerated repopulation of the irradiated bone marrow at very low concentrations both in vivo and ex vivo. A fluorescently labeled derivative of the peptide was found to selectively associate to CD34⁺ stem cells, suggesting that the peptide mediates their proliferation and allows fast repopulation of hematopoietic lineages. Interestingly, the peptide protected animals from alopecia. The studies in experimental animals suggest that treatment with FTGN can potentially benefit patients who suffer bone marrow damage due to radiotherapy or chemotherapy and patients undergoing autologous or allogenic bone marrow transplantation.     

The GIY-YIG Type Endonuclease Ankyrin Repeat and LEM Domain-Containing Protein 1 (ANKLE1) Is Dispensable for Mouse Hematopoiesis

Ankyrin repeat and LEM-domain containing protein 1 (ANKLE1) is a GIY-YIG endonuclease with unknown functions, mainly expressed in mouse hematopoietic tissues. To test its potential role in hematopoiesis we generated Ankle1-deficient mice. Ankle1^Δ/Δ mice are viable without any detectable phenotype in hematopoiesis. Neither hematopoietic progenitor cells, myeloid and lymphoid progenitors, nor B and T cell development in bone marrow, spleen and thymus, are affected in Ankle1^Δ/Δ-mice. Similarly embryonic stress erythropoiesis in liver and adult erythropoiesis in bone marrow and spleen appear normal. To test whether ANKLE1, like the only other known GIY-YIG endonuclease in mammals, SLX1, may contribute to Holliday junction resolution during DNA repair, Ankle1-deficient cells were exposed to various DNA-damage inducing agents. However, lack of Ankle1 did not affect cell viability and, unlike depletion of Slx1, Ankle1-deficiency did not increase sister chromatid exchange in Bloom helicase-depleted cells. Altogether, we show that lack of Ankle1 does neither affect mouse hematopoiesis nor DNA damage repair in mouse embryonic fibroblasts, indicating a redundant or non-essential function of ANKLE1 in mouse.     

Epithelial entry rather than the ensuing systemic immune response determines the pathogenicity of two Salmonella enterica serovar Typhimurium strains in a mouse model

Most studies of Salmonella enterica serovar Typhimurium infection focus only on the pathogenicity of one strain. We investigated whether differences in pathogenicity of two wild-type S. Typhimurium strains; DT120 and SL1344, were related to gut invasion or the resulting immune response.Oral administration of a ten-fold lower number of SL1344 (10⁶ CFU) as compared to DT120 (10⁷ CFU) resulted in higher bacterial counts in liver and lymph nodes, and led to massive neutrophil infiltration of the spleen, while DT120 administration did not. In contrast, administration of the same dose (10³ CFU) of the two strains intravenously resulted in the same levels of bacteria and neutrophils in spleen and bone marrow. Oral administration of SL1344 led to an increase in neutrophil apoptosis in both spleen and the bone marrow and four out of five mice died before Day 8, while in DT120 mice, no increase in neutrophil apoptosis was observed and all mice survived until Day 8. This study reveals that two wild-type S. Typhimurium strains, despite evoking highly comparable immune responses upon intravenous injection, exhibit diverse pathogenicity in mice and thus suggests that differences in their invasiveness and survival during gut passage determines the success of the ensuing immune response.     

B lymphocyte differentiation in lethally irradiated and reconstituted mice. I. The effect of Strontium-89 induced bone marrow aplasia on the recovery of the B cell compartment in the spleen.

The influence of ⁸⁹Sr-treatment on the recovery of the B cell compartment in lethally irradiated, fetal liver reconstituted mice was studied by means of membrane fluorescence. ⁸⁹Sr is a bone-seeking radio-isotope which causes in a dose of 3 μCi ⁸⁹Sr/g body weight a depletion of all nucleated cells, including immunoglobulin-bearing (B) cells, of the bone marrow.Treatment of irradiated and fetal liver reconstituted mice with 3 μCi ⁸⁹Sr/g body weight immediately and at 17 days after irradiation and reconstitution prevented recovery of the nucleated cell population, including B cells, in the bone marrow. In the spleen of such mice both nucleated cells and B cells reappeared at day 7 and 14 respectively. The B cell population in the spleen did not recover up to normal values during the experimental period of 45 days. It is concluded that B cell differentiation in lethally irradiated, fetal liver reconstituted mice can take place outside the bone marrow. The efficiency of this extra-medullary differentiation is discussed. The conclusion was drawn that mice with a ⁸⁹Sr-induced bone marrow aplasia are able to generate B lymphocytes. Consequently the bone marrow microenvironment seems not to be obligate to the differentiation of B lymphocytes. The peripheral lymphoid organs of such mice were found to be unable to compensate completely for the absence of B lymphocyte production in the bone marrow.     

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34⁺ cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34⁺ cord blood stem cell preparations.     

137Cs and 40K isotopes in forest and wasteland soils in a selected region of eastern Poland 20 years after the Chernobyl accident

The vertical ¹³⁷Cs profile of forest and wasteland soils was analyzed in the south of the Podlasie Lowland area (Eastern Poland) about 20 years after the Chernobyl accident. In addition, the concentration of ⁴⁰K in soils of the investigated area was measured. Below the litter layer (mean thickness 3 cm), the soil samples were collected up to a depth of 12 cm and then divided into three layers: 0–3, 3–7, 7–12 cm. The behavior of ¹³⁷Cs and ⁴⁰K isotopes in soils was analyzed depending on the depth from which the soil samples were collected, as well as on the content of organic carbon, pH of soil and its granulometric composition. It was established that the density of ¹³⁷Cs in the litter layer equals 2.17 kBq m⁻²; it is the highest in layer 0–3 cm where it equals 3.44 kBq m⁻², and it decreases with the depth to the value of 0.76 kBq m⁻² in layer 7–12 cm. No similar pattern was observed in wasteland soils. The concentrations of ⁴⁰K in forest and wasteland soils did not change significantly with depth.     

The Soluble Interleukin-6 Receptor Is a Mediator of Hematopoietic and Skeletal Actions of Parathyroid Hormone

Both PTH and IL-6 signaling play pivotal roles in hematopoiesis and skeletal biology, but their interdependence is unclear. The purpose of this study was to evaluate the effect of IL-6 and soluble IL-6 receptor (sIL-6R) on hematopoietic and skeletal actions of PTH. In the bone microenvironment, PTH stimulated sIL-6R protein levels in primary osteoblast cultures in vitro and bone marrow in vivo in both IL-6^+/+ and IL-6^−/− mice. PTH-mediated hematopoietic cell expansion was attenuated in IL-6^−/− compared with IL-6^+/+ bone marrow, whereas sIL-6R treatment amplified PTH actions in IL-6^−/− earlier than IL-6^+/+ marrow cultures. Blocking sIL-6R signaling with sgp130 (soluble glycoprotein 130 receptor) inhibited PTH-dependent hematopoietic cell expansion in IL-6^−/− marrow. In the skeletal system, although intermittent PTH administration to IL-6^+/+ and IL-6^−/− mice resulted in similar anabolic actions, blocking sIL-6R significantly attenuated PTH anabolic actions. sIL-6R showed no direct effects on osteoblast proliferation or differentiation in vitro; however, it up-regulated myeloid cell expansion and production of the mesenchymal stem cell recruiting agent, TGF-β1 in the bone marrow microenvironment. Collectively, sIL-6R demonstrated orphan function and mediated PTH anabolic actions in bone in association with support of myeloid lineage cells in the hematopoietic system.     

Localization of hematopoietic cells in the bullfrog (<Emphasis Type="Italic">Lithobates catesbeianus</Emphasis>)

Amphibians represent the first phylogenetic group to possess hematopoietic bone marrow. However, adult amphibian hematopoiesis has only been described in a few species and with conflicting data. Bone marrow, kidney, spleen, liver, gut, stomach, lung, tegument, and heart were therefore collected from adult Lithobates catesbeianus and investigated by light microscopy and immunohistochemical methods under confocal laser microscopy. Our study demonstrated active hematopoiesis in the bone marrow of vertebrae, femur, and fingers and in the kidney, but no hematopoietic activity inside other organs including the spleen and liver. Blood cells were identified as a heterogeneous cell population constituted by heterophils, basophils, eosinophils, monocytes, erythrocytic cells, lymphocytes, and their precursors. Cellular islets of the thrombocytic lineage occurred near sinusoids of the bone marrow. Antibodies against CD34, CD117, stem cell antigen, erythropoietin receptor, and the receptor for granulocyte colony-stimulating factor identified some cell populations, and some circulating immature cells were seen in the bloodstream. Thus, on the basis of these phylogenetic features, we propose that L. catesbeianus can be used as an important model for hematopoietic studies, since this anuran exhibits hematopoiesis characteristics both of lower vertebrates (renal hematopoiesis) and of higher vertebrates (bone marrow hematopoiesis).     

Tissue-dependent preventive effect of metallothionein against DNA damage in dyslipidemic mice under repeated stresses of fasting or restraint

AimsTo investigate the effect of repeated stress on DNA damage in seven organs of dyslipidemic mice, and the preventive role of metallothionein (MT).Main methodsFemale adult 129/Sv wild-type and MT-null mice fed high-fat diet (HFD) were repeatedly subjected to mild stress of fasting or restraint in weeks 2 to 4 of 4-week study period. Serum cholesterol level, DNA damage in the liver, pancreas, spleen, bone marrow, kidney, lung and gastric mucosa, and other parameters were determined.Key findingsBody weights were increased in both types of mice fed HFD compared to those fed standard diet (STD), and further increased by 12 h-fasting, while they were markedly decreased by 1–3 h-restraint. Fasting accelerated accumulation of fat in the liver, and increase in serum cholesterol of both types of mice fed HFD. Feeding of HFD increased DNA damage in the pancreas, spleen and bone marrow of both types of mice, compared with those fed STD. In the wild-type mice fed HFD, 24 h-fasting increased DNA damage in the liver and spleen, while restraint increased the damage in the liver, pancreas, spleen and bone marrow. DNA damage in the cells of organs was markedly increased in the MT-null mice. Specifically, damage in the liver, pancreas, spleen and bone marrow was greatly increased with the intensity of stress increased, and the damage was much greater in the restraint mice than in the fasting mice.SignificanceMT plays a tissue-dependent preventive role against DNA damage in various murine organs induced by repeated stress.     

Age-related changes in hematopoietic stem cell populations of a long-lived hybrid mouse

Age-related changes in the number and concentration of pluripotential and unipotential hematopoietic stem cells in the femoral bone marrow and spleen of BC3F₁ mice were investigated. Pluripotential stem cells were assayed by the spleen colony technique, and unipotential stem cells were determined by an agar cloning method and by erythropoietin responsiveness in polycythemic mice. Changes with senescence were observed in the concentration of both uni- and pluripotential stem cells in the bone marrow; the size of the stem cell compartment in the marrow did not change significantly with age. Also, a reduction in the seeding of transplanted spleen colony-forming units into the spleens of aged recipients was demonstrated. The implications of these findings for the kinetics of hematopoietic stem cell proliferation in aged animals are discussed.     

Isolation of osteoprogenitors from murine bone marrow by selection of CD11b negative cells

Selection of cells having the most osteogenic potential is a strategy used in bone tissue engineering. Preclinical studies using murine bone marrow cells must consider the large amount of hematopoietic cells in the adherent fraction. The aim of this study was to enrich a murine bone marrow cell population with osteoprogenitors by using a simple and reliable method. Bone marrow from C57Bl/6 mice was extracted and cells which adhered onto plastic were expanded in primary culture for 14 days. Immunolabeling of the CD11b surface antigen was performed and the CD11b⁻ cell fraction was isolated by FACS. Sorted and unsorted populations were analyzed for gene expression of osteoblast differentiation, alkaline phosphatase (AlkP) activity and matrix mineralization capacities. Selection of CD11b⁻ cells increased the number of AlkP⁺ cells from the plastic adherent fraction from 6.3% ± 0.8 to 56% ± 3.3 with a sevenfold increase in AlkP activity. mRNA analysis revealed a significant increase in the CD11b⁻ fraction for Osterix (41-fold), RANKL (17-fold), M-CSF (8-fold) and Runx-2 (8-fold). An osteogenic population was obtained with improved capacities to produce a mineralized extracellular matrix in vitro, independently of the presence of glucocorticoids in the culture medium.     

Isoform-dependent effects of apoE on doublecortin-positive cells and microtubule-associated protein 2 immunoreactivity following 137Cs irradiation

Previously we found apoE isoform-dependent effects of ¹³⁷Cs irradiation on cognitive function of female mice 3 months following irradiation. Alterations in the number of immature neurons and in the levels of the dendritic marker microtubule-associated protein 2 (MAP-2) might contribute to the cognitive changes following irradiation. Therefore, in the present study we determined if, following ¹³⁷Cs irradiation, there are apoE isoform-dependent effects on loss of doublecortin-positive neuroprogenitor cells or MAP-2 immumonoreactivity. In the dentate gyrus, CA1 and CA3 regions of the hippocampus, enthorhinal and sensorimotor cortex, and central and basolateral nuclei of the amygdala of apoE3 female mice, MAP-2 immunoreactivity increased 3 months following ¹³⁷Cs irradiation. In addition, at 8 h following irradiation, the number of doublecortin-positive cells was higher in apoE3 than apoE2 or apoE4 mice. Together, these data indicate that brains of apoE3 mice respond differently to ¹³⁷Cs irradiation than those of apoE2 or apoE4 mice.