Circulating Interleukin-18 as a Biomarker of Total-Body Radiation Exposure in Mice,Minipigs, and Nonhuman Primates (NHP)

We aim to develop a rapid, easy-to-use, inexpensive and accurate radiation dose-assessment assay that tests easily obtained samples (e.g., blood) to triage and track radiological casualties, and to evaluate the radioprotective and therapeutic effects of radiation countermeasures. In the present study, we evaluated the interleukin (IL)-1 family of cytokines, IL-1β, IL-18 and IL-33, as well as their secondary cytokines’ expression and secretion in CD2F1 mouse bone marrow (BM), spleen, thymus and serum in response to γ-radiation from sublethal to lethal doses (5, 7, 8, 9, 10, or 12 Gy) at different time points using the enzyme-linked immune sorbent assay (ELISA), immunoblotting, and cytokine antibody array. Our data identified increases of IL-1β, IL-18, and/or IL-33 in mouse thymus, spleen and BM cells after total-body irradiation (TBI). However, levels of these cytokines varied in different tissues. Interestingly, IL-18 but not IL-1β or IL-33 increased significantly (2.5–24 fold) and stably in mouse serum from day 1 after TBI up to 13 days in a radiation dose-dependent manner. We further confirmed our finding in total-body γ-irradiated nonhuman primates (NHPs) and minipigs, and demonstrated that radiation significantly enhanced IL-18 in serum from NHPs 2–4 days post-irradiation and in minipig plasma 1–3 days post-irradiation. Finally, we compared circulating IL-18 with the well known hematological radiation biomarkers lymphocyte and neutrophil counts in blood of mouse, minipigs and NHPs and demonstrated close correlations between these biomarkers in response to radiation. Our results suggest that the elevated levels of circulating IL-18 after radiation proportionally reflect radiation dose and severity of radiation injury and may be used both as a potential biomarker for triage and also to track casualties after radiological accidents as well as for therapeutic radiation exposure.     

Mesenchymal stromal cells inhibit graft-versus-host disease of mice in a dose-dependent manner

Background aimsGraft-versus-host disease (GvHD) remains a major complication after allogeneic hematopoietic cell transplantation (HCT). Recent literature demonstrates a potential benefit of human mesenchymal stromal cells (MSC) for the treatment of refractory GvHD; however, the optimal dose remains uncertain. We set out to develop an animal model that can be used to study the effect of MSC on GvHD.MethodsA GvHD mouse model was established by transplanting C3H/he donor bone marrow (BM) cells and spleen cells into lethally irradiated BALB/c recipient mice. MSC were obtained from C3H/he mice and the C3H/10T1/2 murine MSC line.ResultsThe mRNA expression of Foxp3 in regional lymph nodes (LN) localized with T cells was markedly increased by the addition of C3H10T1/2 cells in a real-time polymerase chain reaction (PCR). Using a mixed lymphocyte reaction, we determined the optimal splenocyte proliferation inhibition dose (MSC:splenocyte ratios 1:2 and 1:1). Three different C3H10T1/2 cell doses (low, 0.5 × 10⁶, intermediate, 1 × 10⁶, and high, 2 × 10⁶) with a consistent splenocyte dose (1 × 10⁶) were evaluated for their therapeutic potential in an in vivo GvHD model. The clinical and histologic GvHD score and Kaplan–Meier survival rate were improved after MSC transplantation, and these results demonstrated a dose-dependent inhibition.ConclusionsWe conclude that MSC inhibit GvHD in a dose-dependent manner in this mouse model and this model can be used to study the effects of MSC on GvHD.     

Correction to: Computational analysis of viable parameter regions in models of synthetic biological systems

Background

Myocardial infarction (MI) is a common cause of mortality in people. Mesenchymal stem cell (MSC) has been shown to exert therapeutic potential to treat myocardial infarction (MI). However, in patients with diabetes, the diabetic environment affected MSCs activity and could impair the efficacy of treatment. Interleukin-10 (IL-10) has been shown to attenuate MI by suppressing inflammation. In current study, the combination of MSC transplantation with IL-10 was evaluated in a diabetic mice model with MI.

Methods

We engineered bone marrow derived MSCs (BM-MSCs) to overexpress IL-10 by using CRISPR activation. We established the diabetic mice model with MI and monitored the IL-10 expression after BM-MSCs transplantation. We also evaluated the effects of BM-MSCs transplantation on inflammatory response, cell apoptosis, cardiac function and angiogenesis.

Results

CRISPR activation system enabled overexpression of IL-10 in BM-MSCs. Transplantation of BM-MSCs overexpressing IL-10 resulted in IL-10 expression in heart after transplantation. Transplantation of BM-MSCs overexpressing IL-10 inhibited inflammatory cell infiltration and pro-inflammatory cytokines production, improved cardiac functional recovery, alleviated cardiac injury, decreased apoptosis of cardiac cells and increased angiogenesis.

Conclusion

In summary, we have demonstrated the therapeutic potential of IL-10 overexpressed BM-MSCs in the treatment of MI in diabetic mice.

     

Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role of interleukin-6

Background aimsMesenchymal stromal cells (MSC) have been shown to possess immunomodulatory functions and proposed as a tool for managing or preventing graft-versus-host disease (GvHD) as well as promoting clinical transplantation tolerance. We investigated the capacity of human bone marrow (BM) MSC to modulate the proliferation of T cells obtained from peripheral blood (PB) and umbilical cord blood (CB). We addressed the importance of the MSC:T-cell ratio, requirement for cell contact and impact of soluble factors on the MSC-mediated effects. We also analyzed whether regulatory T cells could be modulated by MSC in co-cultures.MethodsThe effect of different MSC concentrations on T-cell proliferation induced by allogeneic, mitogenic or CD3/CD28 stimulation was analyzed using bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate–succinimidyl ester (CFDA-SE) labeling. The level of regulatory T cells was assessed using quantitative real-time polymerase chain reaction (PCR) and flow cytometry analysis.ResultsMSC induced a dose- and contact-dependent inhibition of T-cell proliferation but lymphocytes from CB and PB were differentially affected. At low concentrations, MSC supported both CB and PB T-cell proliferation, rather than inhibiting their proliferation. This supportive effect was contact independent and soluble factors such interleukin-6 (IL-6) appeared to be involved. Interestingly, among the expanded T-cell population in both CB and PB, regulatory T cells were increased and were a part of the new cells promoted by MSC at low doses.ConclusionsMSC represent an attractive tool for reducing the lymphocyte response by inhibiting T-cell activation and proliferation as well as promoting tolerance by maintaining and promoting the expansion of regulatory cells. Nevertheless, the dual ability of MSC to either sustain or suppress T-cell proliferation according to conditions should be considered in the context of clinical applications.     

Effect of fractionation and rate of radiation dose on human leukemic cells, HL-60

The capacity of HL-60 cells, human acute promyelocytic leukemic cells established in culture, to repair sublethal radiation damage was estimated from the response of the cells to fractionated irradiation or to a single irradiation at different dose rates. The HL-60 cells grown as a suspension culture in RPMI 1640 medium supplemented with 10% calf serum and antibiotics showed a cloning efficiency of about 0.46 in an agar culture bed. After exposure of cells to a single dose of X rays at a dose rate of 78 rad/min, the survival curve was characterized by n = 2.5, Dq = 80 rad, and D0 = 83.2 rad. Split-dose studies demonstrated that the cells were able to repair a substantial portion of sublethal radiation damage in 2 hr. The response of the cells to irradiation at different dose rates decreased with a decrease in the dose rates, which could be attributed to repair of sublethal radiation damage. The radiation response of leukemic cells is only one of the many factors which affect the clinical outcome of total-body irradiation (TBI) followed by bone marrow transplantation. Nevertheless, the possibility that some of the malignant hemopoietic cells, if not all, may possess a substantial capacity to repair sublethal radiation damage should not be underestimated in planning total-body irradiation followed by bone marrow transplantation.     

Lysis and phenotypic modulation of mesenchymal stromal cells upon blood contact triggers anti-inflammatory skewing of the peripheral innate immune repertoire

Background aimsMesenchymal stromal cells (MSCs) are used to treat immune-related disorders, including graft-versus-host disease. Upon intravenous infusion, MSCs trigger the instant blood-mediated inflammatory response, resulting in activation of both complement and coagulation cascades, and are rapidly cleared from circulation. Despite no/minimal engraftment, long-term immunoregulatory properties are evident. The aim of this study was to establish the effects of blood exposure on MSC viability and immunomodulatory functions.MethodsHuman, bone marrow derived MSCs were exposed to human plasma +/– heat inactivation or whole blood. MSC number, viability and cellular damage was assessed using the JC-1 mitochondrial depolarization assay and annexin V staining. C3c binding and expression of the inhibitory receptors CD46, CD55 and CD59 and complement receptors C3aR and C5aR were evaluated by flow cytometry. MSCs pre-exposed to plasma were cultured with peripheral blood mononuclear cells (PBMCs) and monocyte subsets characterized by flow cytometry. The PBMC and MSC secretome was assessed using enzyme-linked immunosorbent assays against tumor necrosis factor alpha, interleukin (IL)-6 and IL-10. Monocyte recruitment towards the MSC secretome was evaluated using Boyden chambers and screened for chemotactic factors including monocyte chemoattractant protein (MCP)-1. MSC effects on the peripheral immune repertoire was also evaluated in whole blood by flow cytometry.ResultsPlasma induced rapid lysis of 57% of MSCs, which reduced to 1% lysis with heat inactivation plasma. Of those cells that were not lysed, C3c could be seen bound to the surface of the cells, with a significant swelling of the MSCs and induction of cell death. The MSC secretome reduced monocyte recruitment, in part due to a reduction in MCP-1, and downregulated PBMC tumor necrosis factor alpha secretion while increasing IL-6 levels in the co-culture supernatant. A significant decrease in CD14⁺ monocytes was evident after MSC addition to whole blood alongside a significant increase in IL-6 levels, with those remaining monocytes demonstrating an increase in classical and decrease in non-classical subsets. This was accompanied by a significant increase in both mononuclear and polymorphonuclear myeloid-derived suppressor cells.ConclusionsThis study demonstrates that a significant number of MSCs are rapidly lysed upon contact with blood, with those surviving demonstrating a shift in their phenotype, including a reduction in the secretion of monocyte recruitment factors and an enhanced ability to skew the phenotype of monocytes. Shifts in the innate immune repertoire, towards an immunosuppressive profile, were also evident within whole blood after MSC addition. These findings suggest that exposure to blood components can promote peripheral immunomodulation via multiple mechanisms that persists within the system long after the infused MSCs have been cleared.     

Is the subarachnoid administration of mesenchymal stromal cells a useful strategy to treat chronic brain damage?

Background aimsTraumatic brain injury (TBI) is a leading cause of mortality and morbidity worldwide. Developing effective protocols for the administration of mesenchymal stromal cells (MSCs) is a promising therapeutic strategy to treat TBI. It is important to develop alternatives to direct parenchymal injection at the injury site because direct injection is an expensive and invasive technique. Subarachnoid transplantation, a minimally invasive and low-risk procedure, may be an important and clinically applicable strategy. The aim of this study was to test the therapeutic effect of subarachnoid administration of MSCs on functional outcome 2 months after an experimental TBI in rats.MethodsTwo months after TBI, 30 female Wistar rats were divided into 3 groups (n = 10 in each group): sham, MSC (received 2 × 10⁶ MSCs) and saline (received only saline) groups. Neurological function, brain and spinal cords samples and cerebrospinal fluid were studied.ResultsNo significant differences were found in neurological evaluation and after histological analysis; differences in the expression of neurotrophins were present but were not statistically significant. MSCs survived in the host tissue, and some expressed neural markers.ConclusionsSimilar to direct parenchymal injections, transplanted MSCs survive, migrate to the injury cavity and differentiate into mature neural cell types for at least 6 months after engraftment. These results open the possibility that MSC administration through subarachnoid administration may be a treatment for the consequences of TBI. The transplantation technique and cell number should be adjusted to obtain functional outcome and neurotrophin production differences.     

Predominance of NK1.1+TCR alpha beta+ or DX5+TCR alpha beta+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: "natural suppressor" cells

We developed a nonmyeloablative host conditioning regimen in a mouse model of MHC-mismatched bone marrow transplantation that not only reduces radiation toxicity, but also protects against graft-vs-host disease. The regimen of fractionated irradiation directed to the lymphoid tissues and depletive anti-T cell Abs results in a marked change in the residual host T cells, such that NK1.1+ or DX5+asialo-GM1+ T cells become the predominant T cell subset in the lymphoid tissues of C57BL/6 and BALB/c mice, respectively. The latter "natural suppressor" T cells protect hosts from graft-vs-host disease after the infusion of allogeneic bone marrow and peripheral blood cells that ordinarily kill hosts conditioned with sublethal or lethal total body irradiation. Protected hosts become stable mixed chimeras, but fail to show the early expansion and infiltration of donor T cells in the gut, liver, and blood associated with host tissue injury. Cytokine secretion and adoptive transfer studies using wild-type and IL-4(-/-) mice showed that protection afforded by NK1.1+ and DX5+asialo-GM1+ T cells derived from either donors or hosts conditioned with lymphoid irradiation is dependent on their secretion of high levels of IL-4.     

In vitro study of matrix metalloproteinase/tissue inhibitor of metalloproteinase production by mesenchymal stromal cells in response to inflammatory cytokines: the role of their migration in injured tissues

Background aimsThe transmigratory capacity of bone marrow (BM) mesenchymal stromal cells (MSC) through the endothelial cell barrier into various tissues and their differentiation potential makes them ideal candidates for cell therapy. Nevertheless, the mechanisms and agents promoting their migration are not fully understood. We evaluated the effects of several inflammatory cytokines on the migration of BM MSC and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) production.MethodsThe migratory potential of BM MSC was evaluated using a Boyden chamber coated with Matrigel^® in the presence and absence of stromal cell-derived (SDF)-1α, platelet-derived growth factor (PDGF)bb, insulin-like growth factor (IGF)-I and interleukin (IL)-6. The ability of inflammatory cytokines to induce MSC migration was tested in presence of their respective Ab or blocking peptide. We used immunofluorescence to check the expression of cytokine receptors, and MMP/TIMP production was analyzed at the protein (human cytokine array, enzyme-linked immunosorbent assay (ELISA), gelatine zymography and Western blot) and mRNA quantitative real-time polymerase chain reaction (qRT-PCR) levels.ResultsWe have demonstrated that inflammatory cytokines promote the migratory capacity of BM MSC according to the expression of their respective receptors. Higher migration through Matrigel was observed in response to IL-6 and PDGFbb. qRT-PCR and cytokine array revealed that migration was the result of the variable level of MMP/TIMP in response to inflammatory stimuli.ConclusionsOur observations suggest that chemokines and cytokines involved in the regulation of the immunity or inflammatory process promote the migration of MSC into BM or damaged tissues. One of the mechanisms used by MSC to promote their migration though the extracellular matrix is modulation of the production of MMP-1, MMP-2, MMP-13, TIMP-1 and TIMP-2.     

Local administration of allogeneic or autologous bone marrow-derived mesenchymal stromal cells enhances bone formation similarly in distraction osteogenesis

Background aimsDistraction osteogenesis (DO) is a surgical technique to promote bone regeneration that requires a long time for bone healing. Bone marrow-derived mesenchymal stromal cells (MSCs) have been applied to accelerate bone formation in DO. Allogeneic MSCs are attractive, as they could be ready to use in clinics. Whether allogeneic MSCs would have an effect similar to autologous MSCs with regard to promoting bone formation in DO is still unknown. This study compares the effect of autologous MSCs versus allogeneic MSCs on bone formation in a rat DO model.MethodsRat bone marrow-derived MSCs were isolated, characterized and expanded in vitro. Adult rats were subjected to right tibia transverse osteotomy. On the third day of distraction, each rat received one injection of phosphate-buffered saline (PBS), autologous MSCs or allogeneic MSCs at the distraction site. Tibiae were harvested after 28 days of consolidation for micro-computed tomography examination, mechanical test and histological analysis.ResultsResults showed that treatment with both allogeneic and autologous MSCs promoted bone formation, with significantly higher bone mass, mechanical properties and mineral apposition rate as well as expression of angiogenic and bone formation markers at the regeneration sites compared with the PBS-treated group. No statistical difference in bone formation was found between the allogeneic and autologous MSC treatment groups.ConclusionsThis study indicates that allogeneic and autologous MSCs have a similar effect on promoting bone consolidation in DO. MSCs from an allogeneic source could be used off-the-shelf with DO to achieve early bone healing.     

A simple method for identifying bone marrow mesenchymal stromal cells with a high immunosuppressive potential

Background aimsThe beneficial activity of mesenchymal stromal cells (MSC) in allogeneic hematopietic stem cell transplantation requires correct use in terms of cell dose and timing of infusion and the identification of biomarkers for selection. The immunosuppressive bone marrow (BM)-derived MSC (BM-MSC) functions have been associated with the production of soluble HLA-G molecules (sHLA-G) via interleukin (IL)-10. We have established a reliable method for evaluating BM-MSC HLA-G expression without the influence of peripheral blood mononuclear cells (PBMC).MethodsThirteen BM-MSC from donors were activated with recombinant IL-10 or co-cultured with 10 different phytohemagglutinin (PHA)-treated PBMC (PHA-PBMC). Membrane-bound and sHLA-G expression was evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively; lymphoproliferation was measured by (methyl-³H)thymidine.ResultsThe results demonstrated the ability of IL-10 to stimulate both membrane-bound and sHLA-G production by BM-MSC. The levels of HLA-G expression induced by IL-10 in BM-MSC were associated with the inhibition of PHA-PBMC proliferation (sHLA-G, P = 0.0008, r = 0.9308; membrane HLA-G, P = 0.0005, r = 0.9502).ConclusionsWe propose the evaluation of sHLA-G production in IL-10-treated BM-MSC cultures as a possible marker of immunoregulatory function.     

Clinicopathologic findings following intra-articular injection of autologous and allogeneic placentally derived equine mesenchymal stem cells in horses

Background aimsThe development of an allogeneic mesenchymal stem cell (MSC) product to treat equine disorders would be useful; however, there are limited in vivo safety data for horses. We hypothesized that the injection of self (autologous) and non-self (related allogeneic or allogeneic) MSC would not elicit significant alterations in physical examination, gait or synovial fluid parameters when injected into the joints of healthy horsesMethodsSixteen healthy horses were used in this study. Group 1 consisted of foals (n = 6), group 2 consisted of their dams (n = 5) and group 3 consisted of half-siblings (n = 5) to group 1 foals. Prior to injection, MSC were phenotyped. Placentally derived MSC were injected into contralateral joints and MSC diluent was injected into a separate joint (control). An examination, including lameness evaluation and synovial fluid analysis, was performed at 0, 24, 48 and 72 h post-injectionResultsMSC were major histocompatibility complex (MHC) I positive, MHC II negative and CD86 negative. Injection of allogeneic MSC did not elicit a systemic response. Local responses such as joint swelling or lameness were minimal and variable. Intra-articular MSC injection elicited marked inflammation within the synovial fluid (as measured by nucleated cell count, neutrophil number and total protein concentration). However, there were no significant differences between the degree and type of inflammation elicited by self and non-self-MSCConclusionsThe healthy equine joint responds similarly to a single intra-articular injection of autologous and allogeneic MSC. This pre-clinical safety study is an important first step in the development of equine allogeneic stem cell therapies.     

Mesenchymal stromal cells augment CD4+ and CD8+ T-cell proliferation through a CCL2 pathway

Background aimsMesenchymal stromal cells (MSC) derived from bone marrow are immunosuppressive in vitro and in vivo. Recent evidence, however, has shown that in certain settings, MSC can also be immunostimulatory. The mechanisms involved in this process are largely unknown.MethodsMouse spleen T cells were stimulated with allogeneic mixed lymphocyte reaction (MLR) or anti-CD3/CD28 beads and treated with autologous bone marrow MSC or MSC-conditioned medium. CD4+ and CD8+ T-cell proliferation was analyzed after treatment.ResultsWe show that MSC have both suppressive and stimulatory functions toward T cells after stimulation with anti-CD3/CD28 beads or in an MLR. This depended on the ratio of MSC to responder T cells, with low numbers of MSC increasing and higher numbers inhibiting T-cell proliferation. Immunostimulatory function was mediated, in part, by soluble factors. MSC immunosuppression of the MLR was indirect and related to inhibition of antigen-presenting cell maturation. Direct effects of MSC-conditioned medium during anti-CD3/CD28 stimulated proliferation were entirely stimulatory and required the presence of the T-cell receptor. MSC supernatant contained both CCL2 and CCL5 at high levels, but only CCL2 level correlated with the ability to augment proliferation. An anti-CCL2 antibody blocked this proliferative activity.ConclusionsCCL2 plays an important role in the immunostimulatory function of MSC, and we further hypothesize that the immunomodulatory role of MSC is determined by a balance between inhibitory and stimulatory factors, suggesting the need for caution when these cells are investigated in clinical protocols.     

Protective effect of sesamol against 60Co γ-ray-induced hematopoietic and gastrointestinal injury in C57BL/6 male mice

Abstract

Protection of γ-ray-induced injury in hematopoietic and gastrointestinal (GI) systems is the rationale behind developing radioprotectors. The objective of this study, therefore, was to investigate the radioprotective efficacy and mechanisms underlying sesamol in amelioration of γ-ray-induced hematopoietic and GI injury in mice. C57BL/6 male mice were pre-treated with a single dose (100 or 50 mg/kg, 30 min prior) of sesamol through the intraperitoneal route and exposed to LD_50/30 (7.5 Gy) and sublethal (5 Gy) dose of γ-radiation. Thirty-day survival against 7.5 Gy was monitored. Sesamol (100 mg/kg) pre-treatment reduced radiation-induced mortality and resulted survival of about 100% against 7.5 Gy of γ-irradiation. Whole-body irradiation drastically depleted hematopoietic progenitor stem cells in bone marrow, B cells, T cell subpopulations, and splenocyte proliferation in the spleen on day 4, which were significantly protected in sesamol pre-treated mice. This was associated with a decrease of radiation-induced micronuclei (MN) and apoptosis in bone marrow and spleen, respectively. Sesamol pre-treatment inhibited lipid peroxidation, translocation of gut bacteria to spleen, liver, and kidney, and enhanced regeneration of crypt cells in the GI system. In addition, sesamol pre-treatment reduced the radiation-induced pattern of expression of p53 and Bax apoptotic proteins in the bone marrow, spleen, and GI. This reduction in apoptotic proteins was associated with the increased anti-apoptotic-Bcl-x and PCNA proteins. Further, assessment of antioxidant capacity using ABTS and DPPH assays revealed that sesamol treatment alleviated total antioxidant capacity in spleen and GI tissue. In conclusion, the results of the present study suggested that sesamol as a single prophylactic dose protects hematopoietic and GI systems against γ-radiation-induced injury in mice.     

Traumatic brain injury and bone healing: radiographic and biomechanical analyses of bone formation and stability in a combined murine trauma model

Introduction:The combination of traumatic brain injury (TBI) and long-bone fractures has previously been reported to lead to exuberant callus formation. The aim of this experimental study was to radiographically and biomechanically study the effect of TBI on bone healing in a mouse model.Materials and methods:138 female C57/Black6N mice were assigned to four groups (fracture (Fx) / TBI / combined trauma (Fx/TBI) / controls). Femoral osteotomy and TBI served as variables: osteotomies were stabilized with external fixators, TBI was induced with controlled cortical impact injury. During an observation period of four weeks, in vivo micro-CT scans of femora were performed on a weekly basis. Biomechanical testing of femora was performed ex vivo.Results:The combined-trauma group showed increased bone volume, higher mineral density, and a higher rate of gap bridging compared to the fracture group. The combined-trauma group showed increased torsional strength at four weeks.Discussion:TBI results in an increased formation of callus and mineral density compared to normal bone healing in mice. This fact combined with a tendency towards accelerated gap bridging leads to increased torsional strength. The present study underscores the empirical clinical evidence that TBI stimulates bone healing. Identification of underlying pathways could lead to new strategies for bone-stimulating approaches in fracture care.     

HemaMax?, a Recombinant Human Interleukin-12, Is a Potent Mitigator of Acute Radiation Injury in Mice and Non-Human Primates

HemaMax, a recombinant human interleukin-12 (IL-12), is under development to address an unmet medical need for effective treatments against acute radiation syndrome due to radiological terrorism or accident when administered at least 24 hours after radiation exposure. This study investigated pharmacokinetics, pharmacodynamics, and efficacy of m-HemaMax (recombinant murine IL-12), and HemaMax to increase survival after total body irradiation (TBI) in mice and rhesus monkeys, respectively, with no supportive care. In mice, m-HemaMax at an optimal 20 ng/mouse dose significantly increased percent survival and survival time when administered 24 hours after TBI between 8–9 Gy (p<0.05 Pearson''s chi-square test). This survival benefit was accompanied by increases in plasma interferon-γ (IFN-γ) and erythropoietin levels, recovery of femoral bone hematopoiesis characterized with the presence of IL-12 receptor β2 subunit–expressing myeloid progenitors, megakaryocytes, and osteoblasts. Mitigation of jejunal radiation damage was also examined. At allometrically equivalent doses, HemaMax showed similar pharmacokinetics in rhesus monkeys compared to m-HemaMax in mice, but more robustly increased plasma IFN-γ levels. HemaMax also increased plasma erythropoietin, IL-15, IL-18, and neopterin levels. At non-human primate doses pharmacologically equivalent to murine doses, HemaMax (100 ng/Kg and 250 ng/Kg) administered at 24 hours after TBI (6.7 Gy/LD_50/30) significantly increased percent survival of HemaMax groups compared to vehicle (p<0.05 Pearson''s chi-square test). This survival benefit was accompanied by a significantly higher leukocyte (neutrophils and lymphocytes), thrombocyte, and reticulocyte counts during nadir (days 12–14) and significantly less weight loss at day 12 compared to vehicle. These findings indicate successful interspecies dose conversion and provide proof of concept that HemaMax increases survival in irradiated rhesus monkeys by promoting hematopoiesis and recovery of immune functions and possibly gastrointestinal functions, likely through a network of interactions involving dendritic cells, osteoblasts, and soluble factors such as IL-12, IFN-γ, and cytoprotectant erythropoietin.     

Radioprotection by whole body hyperthermia: possible mechanism(s)

Studies were carried out to gain an insight into the mechanisms underlying WBH induced radioprotection. The plasma levels of IL-1α, IL-6, TNF-α and GM-CSF, were elevated in WBH treated mice between 2 and 6 h after treatment. The total nucleated cell count of hemopoietic tissues such as spleen, thymus, bone marrow and peripheral blood showed drastic reduction without recovery until death in mice treated with TBI. However, the nucleated cell count in the above tissues showed significant recovery after initial drop in WBH and WBH+TBI treated groups and reached to a normal level by day 7 and day 28, respectively. The total WBC and RBC count in peripheral blood recovered to a control level by day 28 after treatment. Significant number of endogenous spleen colonies were detected, 14 days after TBI in WBH pre-treated mice whereas no such spleen colonies could be detected in TBI treated group. The transplantation of bone marrow derived from control, WBH, TBI and WBH+TBI treated groups of mice to lethally irradiated mice (8 Gy) showed formation of spleen colonies only in mice which received bone marrow from control, WBH and WBH+TBI treated groups. Transplantation of the bone marrow from these groups of mice resulted in prolonged survival of lethally irradiated mice as compared to mice receiving bone marrow from TBI treated mice. These results seem to suggest that WBH induced radioprotection of mice could be due to immunomodulation manifested through induction of cytokines responsible for protection and proliferative response, leading to accelerated recovery from hemopoietic damage-a major cause of radiation induced death.     

Allogeneic bone marrow transplantation in conventional mice: I. Effect of antibiotic therapy on long term survival of allogeneic chimeras.

In the present communication the beneficial effect of long term antimicrobial treatment with poorly absorbable antiboitics on the survival of allogeneic bone marrow chimeras was investigated. The combination of C57Bl mice as bone marrow donors and CBA/CA mice as irradiated recipients (800 rad) was used because of their strong histoincompatibility on the H-2 loci. All allografted recipients received 10 X 10(6) bone marrow cells. The majority of the recipients, which were rendered gnotobiotic by an antimicrobial treatment, achieved stable long term chimerism. In contrast, the conventional chimeras died from secondary disease within 9 weeks after transplantation. As early as 14 days after allogeneic bone marrow grafting the gnotobiotic recipients tolerated the reassociation with a conventional microflora without a change in the rate of mortality. Bone marrow cells (8 X 10(6) i.v.) and spleen cells (2 X 10(6) i.v.) collected from allogeneic chimeras failed to induce graft-versus-host-reaction (GVH) in a second lethally irradiated host. The data indicate, that the high rate of mortality in murine allogeneic bone marrow chimeras results from delayed GVH-reaction and systemic infection. The marrow graft, once established seems to exert tolerance against the allogeneic host. The pathogenesis of the systemic infection has not yet been worked out. It is assumed that it originates from bacteremia, induced by radiation dependent lesions of the epithelial integrity and defected lymphatic tissue in the gut.