Clostridium haemolyticum Infection: A Cause of Hemolytic Anemia in a Patient with Bone Marrow Necrosis

Clostridium haemolyticum is a sporulating Gram-positive anaerobic rod that is considered to be one of the most fastidious and oxygen-sensitive anaerobes. It is a well-known animal pathogen and the cause of bacillary hemoglobinuria primarily in cattle. To date, human infections caused by C. haemolyticum have been reported in three patients with malignant underlying diseases. We present herein the case of a 30-year-old obese woman with no significant past medical history who developed bacteremia caused by C. haemolyticum with massive intravascular hemolysis associated with bone marrow necrosis and acute renal failure. Because of subculture failure, the diagnosis was made on the basis of 16S rDNA sequencing and next-generation sequencing. The patient, who had been afebrile for 20 days after a 17-day-course of antibiotics, experienced a second bacteremic episode caused by C. haemolyticum. After having been successfully treated for 42 days with clindamycin and amoxicillin-clavulanic acid, the patient developed acute myeloid leukemia as a result of bone marrow regeneration. Although uncommon in humans, infections caused by C. haemolyticum are severe and should be considered in a febrile patient who has severe hemolytic anemia. This case also highlights the importance of using molecular techniques for the identification of this fastidious anaerobic organism.

RRx-001 Radioprotection: Enhancement of Survival and Hematopoietic Recovery in Gamma-Irradiated Mice

The present studies evaluate the in vivo prophylactic radioprotective effects of 1-bromoacetyl-3, 3-dinitroazetidine (RRx-001), a phase III anticancer agent that inhibits c-myc and downregulates CD-47, after total body irradiation (TBI), in lethally and sublethally irradiated CD2F1 male mice. A single dose of RRx-001 was administered by intraperitoneal (IP) injection 24 h prior to a lethal or sublethal radiation dose. When irradiated with 9.35 Gy, the dose lethal to 70% of untreated mice at 30 days (LD70/30), only 33% of mice receiving RRx-001 (10 mg/kg) 24 h prior to total body irradiation (TBI) died by day 30, compared to 67% in vehicle-treated mice. The same pretreatment dose of RRx-001 resulted in a significant dose reduction factor of 1.07. In sublethally TBI mice, bone marrow cellularity was increased at day 14 in the RRx-001-treated mice compared to irradiated vehicle-treated animals. In addition, significantly higher numbers of lymphocytes, platelets, percent hematocrit and percent reticulocytes were observed on days 7 and/or 14 in RRx-001-treated mice. These experiments provide proof of principle that systemic administration of RRx-001 prior to TBI significantly improves overall survival and bone marrow regeneration.

Bone marrow regeneration requires mitochondrial transfer from donor Cx43-expressing hematopoietic progenitors to stroma

The fate of hematopoietic stem and progenitor cells (HSPC) is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation preconditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers after irradiation is unknown. We report that BM mesenchymal stromal cells (MSC) undergo massive damage to their mitochondrial function after irradiation. Donor healthy HSPC transfer functional mitochondria to the stromal ME, thus improving mitochondria activity in recipient MSC. Mitochondrial transfer to MSC is cell-contact dependent and mediated by HSPC connexin-43 (Cx43). Hematopoietic Cx43-deficient chimeric mice show reduced mitochondria transfer, which was rescued upon re-expression of Cx43 in HSPC or culture with isolated mitochondria from Cx43 deficient HSPCs. Increased intracellular adenosine triphosphate levels activate the purinergic receptor P2RX7 and lead to reduced activity of adenosine 5′-monophosphate–activated protein kinase (AMPK) in HSPC, dramatically increasing mitochondria transfer to BM MSC. Host stromal ME recovery and donor HSPC engraftment were augmented after mitochondria transfer. Deficiency of Cx43 delayed mesenchymal and osteogenic regeneration while in vivo AMPK inhibition increased stromal recovery. As a consequence, the hematopoietic compartment reconstitution was improved because of the recovery of the supportive stromal ME. Our findings demonstrate that healthy donor HSPC not only reconstitute the hematopoietic system after transplantation, but also support and induce the metabolic recovery of their irradiated, damaged ME via mitochondria transfer. Understanding the mechanisms regulating stromal recovery after myeloablative stress are of high clinical interest to optimize BMT procedures and underscore the importance of accessory, non-HSC to accelerate hematopoietic engraftment.

Polysaccharide-based Scaffolds for Bone Marrow Regeneration: Recent Work and Commercial Utility (Patent)

Biochemical applications of polysaccharide are often demonstrated in various drug delivery systems and bone tissue engineering. Perhaps they are similar in biochemical properties with human components of the extracellular matrix, the body recognized them easily. In this manuscript, the polysaccharides, such as chitosan chitin, carrageenan, chondroitin sulfate,and cellulose used as scaffolds for bone regeneration, are discussed. Scaffolds have a porous structure which is extremely interconnected & permits cell penetration. They provide a 3-dimensional environment for bone regeneration. Polysaccharides such as chitosan have great mechanical characteristics and biocompatibility. Present manuscript deals with the polysaccharides based scaffolds that should possess osteoconductivity, biocompatibility,and mechanical strength like property during the tissue repairing process.They also show decreased degradation rate which means that they are present for tissue regeneration for an extended period of time. So it can be concluded that the polysaccharide-based scaffolds have good mechanical strength and stimulate the natural extracellular of bone for the regeneration process. In this manuscript, various patentsbased on applications of polysaccharide in bone marrow and tissue regeneration is also included.

Lin-Sca-1+c-KitlowCD48+CD71+ Cells Are the Engine of Bone Marrow Regeneration

The hematopoietic tissue is the most suitable tissue for studies into the biology of regeneration. We examined bone marrow regeneration starting from a very low number of repopulating cells. Mice were irradiated by a sublethal dose of 6 Gy and recovery of hematopoiesis was examined in its three basic compartments: stem and progenitor cells, precursors of blood cells, and peripheral blood between days 8 – 90 following irradiation. The irradiation reduced existing pools of progenitors and stem cells to ̴0.1%. We focused on the pools of stem cells and progenitors that were evaluated by several assays. A novel assay based on engraftment of a defined number of transplanted competitors revealed delayed filling of the niches for long-term repopulating cells (LTRCs; stem cells) as compared to filling of the niches for short-term repopulating cells (STRCs; progenitors) by endogenous STRCs and LTRCs generated by cells surviving irradiation. This indicated a very slow recovery of the pool of stem cells, lagging after recovery of the pool of progenitors. This was confirmed by a traditional competitive transplantation assay, as well as by another novel assay that measured the capacity of bone marrow to establish pools of stem cells and progenitors in lethally irradiated mice. Direct assays of progenitors by the spleen colony (CFU-S) and in vitro CFC-GM assays indicated a low frequency of the clonogenic cells in regenerating bone marrow. Productive hematopoiesis was re-established about 10 days after irradiation in presence of very low pools of progenitors and stem cells. Next we examined numbers of immature hematopoietic cells by flow cytometry focused at lineage negative (Lin-), Sca-1 positive (Sca-1+), c-Kit+ cells (LSK cells), characterized further by means of the CD150, CD48 and CD71 antigenic markers. In absolute terms, only the population of LSK CD150+CD48+ recovered and overshot normal values after day 12 following irradiation. In relative terms, the LSK CD48+ cells (CD150+ and CD150-) represented more than 99% of all LSK cells two weeks after irradiation. Also in relative terms, the LSK CD150+CD48- cells, which are highly enriched for hematopoietic stem cells in normal bone marrow, recovered by day 30 and became relatively abundant later on. Another striking change consisted in initial lack and later severe scarcity of cells with the phenotype of LSK CD150-CD48-. These cells, serving as multipotent progenitors in normal bone marrow thus remained severely depleted in regenerating bone marrow during the whole follow-up period. LSK cells were mostly negative for CD71 (transferrin receptor) in normal bone marrow but became highly positive for this marker between days 10 – 20 after irradiation. In conclusion, our semi-quantitative evaluation of regenerating hematopoiesis reveals its highly peculiar features. Those include an early recovery of blood cell production, followed by significant expansion of the Lin-Sca-1+c-KitlowCD48+CD71+ cells in presence of a highly reduced pool of hematopoietic stem cells. Disclosures No relevant conflicts of interest to declare.

Association of Hematological Nadirs and Survival in a Non-Human Primate Model of Hematopoietic Syndrome of Acute Radiation Syndrome

Recombinant human interleukin-12 (rHuIL-12, HemaMaxTM) is being developed for mitigation of HSARS under the FDA Animal Rule using a NHP model of HSARS for proof of efficacy and clinical studies in healthy human subjects to demonstrate safety. We have shown previously that a single injection of rHuIL-12 administered 24-25 hours after lethal total body irradiaton (TBI), in the absence of antibiotics, fluids or blood products, resulted in improved survival while filgrastim (G-CSF) did not provide any survival benefit in our NHP HSARS model (Basile et al 2012, Gluzman-Poltorak et al 2014, Gluzman-Poltorak et al 2014). The mechanism by which IL-12 mitigates HSARS following TBI appears to involve multiple effects of IL-12 on hematopoieses. In our previous studies, animals treated with rHuIL-12 showed statistically significant reductions in the occurrence of severe neutropenia and severe thrombocytopenia, as well as attenuated nadirs for lymphocytes, neutrophils, platelets, and reticulocytes. To characterize further relationship between survival and hematological nadirs (lymphocytes, neutrophils, platelets, RBCs and reticulocytes) in the rhesus model of HSARS and to better understand the effects of rHuIL-12 versus G-CSF on blood cell nadirs, we undertook a meta-analysis analysis across three studies in irradiated rhesus monkeys. Animals used in this analysis were irradiated (700 cGy) and treated with a single subcutaneous injection of vehicle (n=64) or rHuIL-12 50-500ng/kg (n=108) 24-25 hours after irradiation, or daily subcutaneous injections of G-CSF at 10μg/kg/d for 18 days starting 24-25 hours after irradiation (n=26). Males and females were equal in each group. Lymphocytes, neutrophils and platelets were significantly lower in decedents versus monkeys that survived to day 60 overall and this was true in each treatment group (p<0.001, Wilcoxon rank-sum test). The mean RBC nadir was significantly higher in survivors compared to non-survivors in the rHuIL-12 group (p = 0.008), but not in the control group or the G-CSF group. Lymphocytes nadir appears to be the strongest and most consistent predictor of death followed by neutrophils and platelets (Spearman’s rank correlation). RBCs and reticulocytes are less informative in terms of predicting survival status. In addition, the receiver operating characteristic (ROC) curve area under the curve (AUC) by nadir value was assessed. The operating characteristics for lymphocytes, neutrophils and platelets allow for relatively large positive predictive values (PPV) of death with relatively high sensitivity. A cutoff value for lymphocytes nadir of 0.08x109/L (values less than or equal predicting death and higher values predicting life) allows for largest PPVs (97.2% and 92.5%) with 76.1% and 62.7% sensitivities for control and rHuIL-12 treatments, respectively. A cutoff value for neutrophils nadir of 0.03x109/L permits for 84% and 71.1% PPVs with sensitivities of 91.3% and 91.5 % for control and rHuIL-12 treatments, respectively. A cutoff value for platelets nadir of 9x109/L permits for 84.1% and 76.8% PPVs with sensitivities of 80.4% and 72.9 % for control and rHuIL-12 treatments, respectively. RBCs and reticulocytes were found less informative. To conclude, in the rhesus model of HSARS we have observed that an augmented hematological nadirs generally predicts an increased potential for survival as this effect reflects early bone marrow regeneration. The nadir for lymphocytes appears to be the strongest and most consistent predictor of death. Decrease of lymphocyte counts has been established as a best marker of bone marrow damage in a large database of human victims of acute radiation (METREPOL, Fliedner et al 2001). Thus, the correlation of our results with the human data supports the validity of our animal model as an accurate representation of human HSARS and its ability to predict effectiveness in humans exposed to lethal radiation. These data also suggest that the significant increase in early bone marrow regeneration seen in our studies, resulting in increases in nadir values for all major blood cell types, may be the main mechanism of action by which rHuIL-12 mitigates the lethality of HSARS. This project has been entirely funded with Federal funds from BARDA/ASPR/DHHS under Contract No. HHSO100201100037C. Disclosures Gluzman Poltorak: Neumedicines Inc.: Employment, Equity Ownership. Vladimir:Neumedicines Inc.: Consultancy, Employment, Equity Ownership. Basile:Neumedicines Inc.: Employment, Equity Ownership, Neumedicines Inc. Patents & Royalties.

Industry Watch

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