Improved nonclinical safety profile of a novel, highly selective inhibitor of the immunoproteasome subunit LMP7 (M3258).

M3258 is the first selective inhibitor of the immunoproteasome subunit LMP7 (Large multifunctional protease 7) in early clinical development with the potential to improve therapeutic utility in patients of multiple myeloma (MM) or other hematological malignancies. Safety pharmacology studies with M3258 did not reveal any functional impairments of the cardiovascular system in several in vitro tests employing human cardiomyocytes and cardiac ion channels (including hERG), guinea pig heart refractory period and force contraction, and rat aortic contraction as well as in cardiovascular function tests in dogs. Following single dose M3258 administration to rats, no changes were observed on respiratory function by using whole body plethysmography, nor did it change (neuro)behavioral parameters in a battery of tests. Based on pivotal 4-week toxicity studies with daily oral dosing of M3258, the identified key target organs of toxicity were limited to the lympho-hematopoietic system in rats and dogs, and to the intestine with its local lymphoid tissues in dogs only. Importantly, the stomach, nervous system, heart, lungs, and kidneys, that may be part of clinically relevant toxicities as reported for pan-proteasome inhibitors, were spared with M3258. Therefore, it is anticipated that by targeting highly selective and potent inhibition of LMP7, the resulting favorable safety profile of M3258 together with the maintained potent anti-tumor activity as previously reported in mouse MM xenograft models, may translate into an improved benefit-risk profile in MM patients.

Immunopathogenesis of hematopoietic tissues in response to Vibrio parahaemolyticus (VPAHPND) infection in Macrobrachium rosenbergii.

In crustacean, hemocytes are known as crucial components of crustaceans' innate immunity against pathogens. Drastic hemocytes reduction during infectious disease is apparently related to disease severity and calls for a health status evaluation and aquaculture management. The molecular pathogenesis of hemocytes loss during bacterial infection was elucidated with VPAHPND challenged in M. rosenbergii. We report herein a correlation between hemocyte loss and the pathogenicity and aggressive immune response in hematopoietic tissues of moribund M. rosenbergii. In this study, adult freshwater prawn was administered an LC50 dose of VPAHPND; bacterial clearance ensued, and success was reached within 24 h. Hemocytes increased in survival, yet drastically decreased in moribund prawn. Pathological analysis of hematopoietic tissue of moribund prawn showed apparent abnormal signs, including the presence of bacteria, a small number of mitotic cells, cellular swelling, loosening of connective tissue, and karyorrhectic nuclei cells. A significant upregulation of a core apoptotic machinery gene, caspase-3, was detected in hematopoietic tissue of moribund shrimp, but not in those of Escherichia coli DH5α (non-pathogenic bacteria) and VPAHPND survival prawn. The highest level was found in the moribund group, which confirms the occurrence of apoptosis in this hematopoietic tissue. Further, our results suggest that hematopoietic tissue damage may arise from inflammation triggered by an aggressive immune response. Immune activation was indicated by the comparison of immune-related gene expression between controls, E. coli (DH5α)-infected (non-pathogenic), and VPAHPND-infected survival groups with moribund prawn. RT-PCR revealed a significant upregulation of all genes in hematopoietic tissues and hemocytes within 6-12 h and declined by 24 h. This evident related to the almost VPAHPND are clearance in survival and E. coli (DH5α) challenged group in contrast with drastic high expression was determined in moribund group. We conclude that a reduction of renewing circulating hemocytes in fatally VPAHPND-infected prawn was caused by an acute self-destructive immune response by hematopoietic cells.

Dysregulated Cardiac IGF-1 Signaling and Antioxidant Response Are Associated with Radiation Sensitivity.

Acute exposure to ionizing radiation leads to Hematopoietic Acute Radiation Syndrome (H-ARS). To understand the inter-strain cellular and molecular mechanisms of radiation sensitivity, adult males of two strains of minipig, one with higher radiosensitivity, the Gottingen minipig (GMP), and another strain with comparatively lower radiosensitivity, the Sinclair minipig (SMP), were exposed to total body irradiation (TBI). Since Insulin-like Growth Factor-1 (IGF-1) signaling is associated with radiation sensitivity and regulation of cardiovascular homeostasis, we investigated the link between dysregulation of cardiac IGF-1 signaling and radiosensitivity. The adult male GMP; n = 48, and SMP; n = 24, were irradiated using gamma photons at 1.7-2.3 Gy doses. The animals that survived to day 45 after irradiation were euthanized and termed the survivors. Those animals that were euthanized prior to day 45 post-irradiation due to severe illness or health deterioration were termed the decedents. Cardiac tissue analysis of unirradiated and irradiated animals showed that inter-strain radiosensitivity and survival outcomes in H-ARS are associated with activation status of the cardiac IGF-1 signaling and nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated induction of antioxidant gene expression. Our data link H-ARS with dysregulation of cardiac IGF-1 signaling, and highlight the role of oxidative stress and cardiac antioxidant response in radiation sensitivity.

A multicenter study of patients with multisystem Langerhans cell histiocytosis who develop secondary hemophagocytic lymphohistiocytosis.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare myeloid neoplasm characterized by the presence of abnormal CD1a-positive (CD1a+ )/CD207+ histiocytes. Hemophagocytic lymphohistiocytosis (HLH) represents a spectrum of hyperinflammatory syndromes typified by the dysregulated activation of the innate and adaptive immune systems. Patients with LCH, particularly those with multisystem (MS) involvement, can develop severe hyperinflammation mimicking that observed in HLH. Nevertheless, to the authors' knowledge, little is known regarding the prevalence, timing, risk factors for development, and outcomes of children and young adults who develop HLH within the context of MS-LCH (hereafter referred to LCH-associated HLH). METHODS: To gain further insights, the authors conducted a retrospective, multicenter study and collected data regarding all patients diagnosed with MS-LCH between 2000 and 2015. RESULTS: Of 384 patients with MS-LCH, 32 were reported by their primary providers to have met the diagnostic criteria for HLH, yielding an estimated 2-year cumulative incidence of 9.3% ± 1.6%. The majority of patients developed HLH at or after the diagnosis of MS-LCH, and nearly one-third (31%) had evidence of an intercurrent infection. Patient age <2 years at the time of diagnosis of LCH; female sex; LCH involvement of the liver, spleen, and hematopoietic system; and a lack of bone involvement each were found to be independently associated with an increased risk of LCH-associated HLH. Patients with MS-LCH who met the criteria for HLH had significantly poorer 5-year survival compared with patients with MS-LCH who did not meet the criteria for HLH (69% vs 97%; P < .0001). CONCLUSIONS: Given its inferior prognosis, further efforts are warranted to enhance the recognition and optimize the treatment of patients with LCH-associated HLH.

Treatment Outcome of Children With Multisystem Langerhans Cell Histiocytosis: The Experience of a Single Children's Hospital in Shanghai, China.

We reported the outcome of 150 children newly diagnosed with multisystem langerhans cell histiocytosis following a langerhans cell histiocytosis-II-based protocol (arm B). However, the continuation treatment was extended to 56 weeks and etoposide was omitted from the continuation treatment. Risk organ (RO) involvement was defined as: liver (≥3 cm with or without functional impairment); spleen (≥2 cm below the costal margin in the midclavicular line); hematopoietic system (hemoglobin <100 g/L, and/or white blood cell count <4.0×10/L, and/or platelets <100×10/L). The lungs are not considered a RO in the current study. For the 59 patients with RO involvement (RO+), the rapid response rate (week 6) was 61.0% and the 3-year overall survival 73.4%±5.9%. Rapid responders had a better 3-year survival rate than poor responders (90.9%±5.0% vs. 45.7%±11.0%, P<0.001). Ninety-one patients without RO involvement (RO-) had a relatively low 3-year cumulative reactivation rate (10.7%). No deaths occurred in this subgroup and the 3-year overall survival of RO- patients was 100%. Poor responders of RO+ patients had an extremely poor prognosis. An effective salvage therapy is essential for this high-risk group. The initial treatment intensity and duration of continuation therapy both impact disease reactivation in RO- patients.

Low hazard of silver nanoparticles and silver nitrate to the haematopoietic system of rainbow trout.

Silver nanoparticles (Ag NPs) are known for their antibacterial properties and are used in a growing number of nano-enabled products, with inevitable concerns for releases to the environment. Nanoparticles may also be antigenic and toxic to the haematopoietic system, but the immunotoxic effect of Ag NPs on non-target species such as fishes is poorly understood. This study aimed to assess the effect of Ag NP exposure via the water on the haematopoietic system of rainbow trout, Oncorhynchus mykiss, and to determine whether or not the hazard from Ag NPs was different from that of AgNO3. Fish were exposed for 7 days to a control (dechlorinated Plymouth freshwater), dispersant control, 1µgl-1 Ag as AgNO3 or 100µgl-1 Ag NPs. Animals were sampled on days 0, 4 and 7 for haematology, tissue trace metal concentration, biochemistry for evidence of oxidative stress/inflammation in the spleen and histopathology of the blood cells and spleen. The Ag NP treatment significantly increased the haematocrit, but the haematological changes were within the normal physiological range of the animal. Thrombocytes in spleen prints at day 4, and melanomacrophage deposits at day 7 in the spleen, of Ag NP exposed-fish displayed significant increases compared to all the other treatments within the time point. A dialysis experiment confirmed that dissolution rates were very low and any pathology observed is likely from the NP form rather than dissolved metal released from it. Overall, the data showed subtle differences in the effects of Ag NPs compared to AgNO3 on the haematopoietic system. The lack of pathology in the circulating blood cells and melanomacrophage deposits in the spleen suggests a compensatory physiological effort by the spleen to maintain normal circulating haematology during Ag NP exposure.

Zymosan-a Protects the Hematopoietic System from Radiation-Induced Damage by Targeting TLR2 Signaling Pathway.

BACKGROUND/AIMS: The hematopoietic system is vulnerable to ionizing radiation and is often severely damaged by radiation. Molecules affecting radioresistance include Toll-like receptor 2. We investigated whether Zymosan-A, a novel TLR2 agonist, can protect the hematopoietic system from radiation-induced damage after total body irradiation. METHODS: Mice were exposed to total body radiation after treatment with Zymosan-A or normal saline, and their survival was recorded. Tissue damage was evaluated by hematoxylin-eosin staining. The number of nucleated cells in bone marrow was determined by flow cytometry. Cell viability and apoptosis assay were determined by CCK-8 assay and flow cytometry assay. Enzyme-linked immunosorbent assay was used to detect the level of cytokines. RESULTS: Zymosan-A protected mice from radiation-induced death and prevented radiation-induced hematopoietic system damage. Zymosan-A also promoted cell viability and inhibited cell apoptosis caused by radiation, induced radioprotective effects via TLR2, upregulated IL-6, IL-11, IL-12, and TNF-α in vivo. CONCLUSION: Zymosan-A can provide protection against radiation-induced hematopoietic system damage by targeting the TLR2 signaling pathway. Thus, Zymosan-A can be potentially effective radioprotectant.

Linking functional decline of telomeres, mitochondria and stem cells during ageing.

The study of human genetic disorders and mutant mouse models has provided evidence that genome maintenance mechanisms, DNA damage signalling and metabolic regulation cooperate to drive the ageing process. In particular, age-associated telomere damage, diminution of telomere 'capping' function and associated p53 activation have emerged as prime instigators of a functional decline of tissue stem cells and of mitochondrial dysfunction that adversely affect renewal and bioenergetic support in diverse tissues. Constructing a model of how telomeres, stem cells and mitochondria interact with key molecules governing genome integrity, 'stemness' and metabolism provides a framework for how diverse factors contribute to ageing and age-related disorders.

Loss of beta-catenin impairs the renewal of normal and CML stem cells in vivo.

A key characteristic of stem cells and cancer cells is their ability to self-renew. To test if Wnt signaling can regulate the self-renewal of both stem cells and cancer cells in the hematopoietic system, we developed mice that lack beta-catenin in their hematopoietic cells. Here we show that beta-catenin-deficient mice can form HSCs, but that these cells are deficient in long-term growth and maintenance. Moreover, beta-catenin deletion causes a profound reduction in the ability of mice to develop BCR-ABL-induced chronic myelogenous leukemia (CML), while allowing progression of acute lymphocytic leukemia (ALL). These studies demonstrate that Wnt signaling is required for the self-renewal of normal and neoplastic stem cells in the hematopoietic system.

Conditional TRF1 knockout in the hematopoietic compartment leads to bone marrow failure and recapitulates clinical features of dyskeratosis congenita.

TRF1 is part of the shelterin complex, which binds telomeres and it is essential for their protection. Ablation of TRF1 induces sister telomere fusions and aberrant numbers of telomeric signals associated with telomere fragility. Dyskeratosis congenita is characterized by a mucocutaneous triad, bone marrow failure (BMF), and presence of short telomeres because of mutations in telomerase. A subset of patients, however, show mutations in the shelterin component TIN2, a TRF1-interacting protein, presenting a more severe phenotype and presence of very short telomeres despite normal telomerase activity. Allelic variations in TRF1 have been found associated with BMF. To address a possible role for TRF1 dysfunction in BMF, here we generated a mouse model with conditional TRF1 deletion in the hematopoietic system. Chronic TRF1 deletion results in increased DNA damage and cellular senescence, but not increased apoptosis, in BM progenitor cells, leading to severe aplasia. Importantly, increased compensatory proliferation of BM stem cells is associated with rapid telomere shortening and further increase in senescent cells in vivo, providing a mechanism for the very short telomeres of human patients with mutations in the shelterin TIN2. Together, these results represent proof of principle that mutations in TRF1 lead to the main clinical features of BMF.

HIF-1α is a negative regulator of plasmacytoid DC development in vitro and in vivo.

Hypoxia-inducible factors (HIFs) regulate hematopoiesis in the embryo and maintain hematopoietic stem cell function in the adult. How hypoxia and HIFs contribute to hematopoietic lineage differentiation in the adult is ill defined. Here we provide evidence that HIF-1 limits differentiation of precursors into plasmacytoid dendritic cells (pDCs). Low oxygen up-regulated inhibitor of DNA binding 2 (ID2) and suppressed Flt3-L-induced differentiation of bone marrow cells to pDCs in wild-type but not HIF-1α(fl/fl) LysM-Cre bone marrow cells. Moreover, pDC differentiated normally in hypoxic ID2(-/-) bone marrow cultures. Finally, we observed elevated pDC frequencies in bone marrow, blood, and spleen of HIF-1α(fl/fl) LysM-Cre and ID2(-/-), but not HIF-2α(fl/fl) LysM-Cre mice. Our data indicate that the low oxygen content in the bone marrow might limit pDC development. This might be an environmental mechanism to restrict the numbers of these potentially autoreactive cells.

Forced homo-oligomerization of RARalpha leads to transformation of primary hematopoietic cells.

Almost 100% of APL patients carry chimeric transcripts encoding truncated RARalpha fused to homo-oligomerization domains from partner proteins. To gain further insights into the cellular transformation mechanisms mediated by RARalpha fusion proteins, thorough structure/function analyses have been performed and identified the POZ homo-oligomerization domain as the minimal transformation domain that is necessary and sufficient for PLZF-RARalpha-mediated in vitro transformation of primary hematopoietic cells. A transformation-incompetent PLZF-RARalpha mutant defective in homo-oligomerization but not corepressor interaction could be rescued by synthetic FKBP-oligomerization domains. Furthermore, an artificial FKBP-RARalpha construct not only mimicked various biochemical properties of bona fide RARalpha fusion proteins but also mediated an ATRA-dependent transformation. Taken together, these findings endorse an oligomerization-dependent mechanism for RARalpha-mediated transformation and suggest a potential avenue for molecular therapy.

Role of major histocompatibility complex class II expression by non-hematopoietic cells in autoimmune and inflammatory disorders: facts and fiction.

It is well established that interactions between CD4(+) T cells and major histocompatibility complex class II (MHCII) positive antigen-presenting cells (APCs) of hematopoietic origin play key roles in both the maintenance of tolerance and the initiation and development of autoimmune and inflammatory disorders. In sharp contrast, despite nearly three decades of intensive research, the functional relevance of MHCII expression by non-hematopoietic tissue-resident cells has remained obscure. The widespread assumption that MHCII expression by non-hematopoietic APCs has an impact on autoimmune and inflammatory diseases has in most instances neither been confirmed nor excluded by indisputable in vivo data. Here we review and put into perspective conflicting in vitro and in vivo results on the putative impact of MHCII expression by non-hematopoietic APCs--in both target organs and secondary lymphoid tissues--on the initiation and development of representative autoimmune and inflammatory disorders. Emphasis will be placed on the lacunar status of our knowledge in this field. We also discuss new mouse models--developed on the basis of our understanding of the molecular mechanisms that regulate MHCII expression--that constitute valuable tools for filling the severe gaps in our knowledge on the functions of non-hematopoietic APCs in inflammatory conditions.

The structure of the hematopoietic system can explain chronic myeloid leukemia progression.

Almost all cancer types share the hallmarks of cancer and a similar tumor formation: fueled by stochastic mutations in somatic cells. In case of chronic myeloid leukemia (CML), this evolutionary process can be tracked from an asymptomatic long-lasting chronic phase to a final rapidly evolving blast phase. Somatic evolution in CML occurs in the context of healthy blood production, a hierarchical process of cell division; initiated by stem cells that self-renew and differentiate to produce mature blood cells. Here we introduce a general model of hierarchical cell division explaining the particular progression of CML as resulting from the structure of the hematopoietic system. Driver mutations confer a growth advantage to the cells carrying them, for instance, the BCR::ABL1 gene, which also acts as a marker for CML. We investigated the relation of the BCR::ABL1 mutation strength to the hematopoietic stem cell division rate by employing computer simulations and fitting the model parameters to the reported median duration for the chronic and accelerated phases. Our results demonstrate that driver mutations (additional to the BCR::ABL1 mutation) are necessary to explain CML progression if stem cells divide sufficiently slowly. We observed that the number of mutations accumulated by cells at the more differentiated levels of the hierarchy is not affected by driver mutations present in the stem cells. Our results shed light on somatic evolution in a hierarchical tissue and show that the clinical hallmarks of CML progression result from the structural characteristics of blood production.

Hematopoietic Fas deficiency does not affect experimental atherosclerotic lesion formation despite inducing a proatherogenic state.

The Fas death receptor (CD95) is expressed on macrophages, smooth muscle cells, and T cells within atherosclerotic lesions. Given the dual roles of Fas in both apoptotic and nonapoptotic signaling, the aim of the present study was to test the effect of hematopoietic Fas deficiency on experimental atherosclerosis in low-density lipoprotein receptor-null mice (Ldlr(-/-)). Bone marrow from Fas(-/-) mice was used to reconstitute irradiated Ldlr(-/-) mice as a model for atherosclerosis. After 16 weeks on an 0.5% cholesterol diet, no differences were noted in brachiocephalic artery lesion size, cellularity, or vessel wall apoptosis. However, Ldlr(-/-) mice reconstituted with Fas(-/-) hematopoietic cells had elevated hyperlipidemia [80% increase, relative to wild-type (WT) controls; P < 0.001] and showed marked elevation of plasma levels of CXCL1/KC, CCL2/MCP-1, IL-6, IL-10, IL-12 subunit p70, and soluble Fas ligand (P < 0.01), as well as systemic microvascular inflammation. It was not possible to assess later stages of atherosclerosis because of increased mortality in Fas(-/-) bone marrow recipients. Our data indicate that hematopoietic Fas deficiency does not affect early atherosclerotic lesion development in Ldlr(-/-) mice.

Differentiation of rodent immune and hematopoietic system reactive lesions from neoplasias.

The immune and hematopoietic systems play an important role in the normal homeostasis of blood and blood cells and for immune responses to endogenous and exogenous processes and insults. In order to interpret histopathologic changes in the immune and hematopoietic systems, it is important to understand the normal anatomy and histology of the thymus, spleen, lymph nodes, bone marrow, and other tissues. The thymus, spleen, and lymph nodes can be categorized by anatomical compartments, each of which contributes to specific immune functions. Lesions may be diagnosed by interpretive or descriptive (semiquantitative) methods. The interpretation of these tissues by lesion in anatomical compartments should allow for better understanding of these reactions and more definitive pathologic findings. Proliferative lesions may be difficult to differentiate from lymphomas and leukemias. The use of immunohistochemistry, compartmental pathology, and methods for the evaluation of clonality will make interpretation easier.

Dimerization of MLL fusion proteins immortalizes hematopoietic cells.

MLL fusion proteins are leukemogenic, but their mechanism is unclear. Induced dimerization of a truncated MLL immortalizes bone marrow and imposes a reversible block on myeloid differentiation associated with upregulation of Hox a7, a9, and Meis1. Both dimerized MLL and exon-duplicated MLL are potent transcriptional activators, suggesting a link between dimerization and partial tandem duplication of DNA binding domains of MLL. Dimerized MLL binds with higher affinity than undimerized MLL to a CpG island within the Hox a9 locus. However, MLL-AF9 is not dimerized in vivo. The data support a model in which either MLL dimerization/exon duplication or fusion to a transcriptional activator results in Hox gene upregulation and ultimately transformation.

[The role of radiation and non-radiation factors in the development and progression of acute leukemia in children].

At 278 ALL and AML patients the kind of hereditary pathology in the family; clinikal-laboratory features and a course of acute leukemia and terms of longevity of children were studied. Degree of integrated pollution of territory (air, water, soil) by heavy metals and radionuclides and also the irradiation doses have been considered. Results which have been received testify about the certain contribution of genetic components and adverse factors of environment in of development of acute leukemia and their course at children. The definition of different leukemogenic factors action on the condition of hemopoietic system gives the chance to generate the abnormally high risk groups of oncological hematological pathologies among the children's population and to improve the diagnostic and treatment-and-prophylactic actions.

[The analysis of relationship between numbers of hemopoietic hematoblasts and laboratory indicators of state of hepatobiliary system in children with congenital and hereditary diseases of liver].

The content of CD34/CD45dim-positive cells in peripheral blood of children with congenital and hereditary diseases of hepatobiliary system is studied. The analysis of relationship between numbers of studied cells and level of C-reactive protein, sCD40L, sCD30 and laboratory parameters specific to liver functions is applied The number of CD34-positive hemopoietic hematoblasts in children with hepatocirrhosis correlated with the level of C-reactive protein, albumin, hemoglobin concentration and quantity of blood erythrocytes. No relationship was established with the levels of sDC40L and sDC30. The number ofstudied cells in children with liver diseases was higher than in healthy adult donors.