The light microscopic demonstration of hydroperoxidase-positive Phi bodies and rods in leukocytes in acute myeloid leukemia.

Unique fusiform or spindle-shaped particles (Phi bodies) and rods with hydroperoxidase (catalase and/or peroxidase) activity are present in human granulocyte precursors only in acute myelogenous leukemia (AML). These newly recognized particles are much more numerous and prominent than Auer rods. They may be rapidly and readily identified using the microscope in marrow or peripheral blood films when the procedures recommended in this paper for fixation, incubation for hydroperoxidase demonstration in 3,3'-diaminobenzidine (DAB)/H2O2 medium, copper salt treatment and counterstaining (optional) with the Papanicolaou method are employed. Films prepared in the same manner but treated with benzidine/H2O2 medium for myeloperoxidase did not reveal these particles. We believe that Phi bodies are pathognomonic of AML since they are almost invariably present in AML patients with active disease. Their presence serves to distinguish AML from acute lymphocytic leukemia and from chronic granulocytic leukemia in blast crisis. Since the particles disappear in disease remission and reappear upon relapse, the recommended procedure is not only useful in diagnosis but in guiding therapy. When a very rapid diagnosis is needed, it is not necessary to counterstain the preparations, but the nuclei, cytoplasm and plasmalemma can readily be observed in the granulocyte precursors when they are counterstained by the Papanicolaou method. This treatment does not diminish the clarity of the Phi bodies and rods which stain by virtue of their peroxidatic activity. This cytochemical diagnostic procedure should be considered for adoption by hematology laboratories.     

Immunofluorescent demonstration of myeloperoxidase of phi bodies and rods in leukaemic leucocytes

Summary Previous studies employing enzyme histocytochemical methods based on the catalysis of the peroxidation of 3,3-diaminobenzidine (DAB) demonstrated the presence of hydroperoxidase activity in phi bodies and rods of immature leucocytes of patients with active acute myelogenous leukaemia. It could not be determined from these studies whether the DAB oxidation product was demonstrating a single hydroperoxidase, catalase or myeloperoxidase, or both. In the present study, immunofluorescence techniques for the two hydroperoxidases were applied in an attempt to identify this activity specifically. The results obtained indicate that myeloperoxidase is present in the phi bodies and rods, and that this enzyme may be the major on the only hydroperoxidase present. Its activity could account for the peroxidation of DAB under conditions which are more favourable for the demonstration of catalase.     

Cellular immunity in an annelid (Nereis diversicolor,Polychaeta): production of melanin by a subpopulation of granulocytes

Summary We attempted to identify the nature and origin of the pigment produced by the marine worm Nereis diversicolor in order to isolate, in inert brown capsules, foreign objects introduced into its body cavity. This brown pigment, characterized by cytochemical techniques, could be a melanin. The activity of the enzyme phenoloxidase responsible for melanin biosynthesis was detected by enzyme cytochemistry techniques in vacuoles and the Golgi apparatus of coelomocytes activated by the presence of foreign bodies. Morphological techniques combined with a monoclonal immunological probe enabled us to establish that the G₂ granulocytes contain both the precursor of the pigment in dense bodies and the capacity for phenoloxidase synthesis when activated to encapsulate foreign bodies. The G₂ granulocyte may therefore be compared to a melanocyte in which melanin is not stored as in mammals, but immediately extruded following synthesis in the form of a thick fluid.Abbreviations ACTH adrenocorticotropic hormone - Dopa L-3,4 dihydroxyphenylalanine - FITC fluorescein isothiocyanate - G ₁, G ₂, G ₃ granulocyte of types 1, 2, 3 - MSH melanocyte-stimulating hormone - proPo prophenoloxidase     

Serum-free generation and quantification of functionally active Leukemia-derived DC is possible from malignant blasts in acute myeloid leukemia and myelodysplastic syndromes

Functional dendritic cells (DC) are professional antigen presenting cells (APC) and can be generated in vitro from leukemic cells from acute myeloid leukemia AML patients, giving rise to APC of leukemic origin presenting leukemic antigens (DC_leu). We have already shown that DC can be successfully generated from AML and myeloplastic syndromes (MDS) cells in serum-free standard medium (X-vivo + GM-CSF + IL-4 +TNF + FL) in 10–14 days. In this study, we present that DC counts generated from mononuclear cells (MNC) varied between 20% (from 55 MDS samples), 34% (from 100 AML samples) and 25% (from 38 healthy MNC samples) medium. Between 53% and 58% of DC are mature CD83⁺ DC. DC harvests were highest in monocytoid FAB types (AML-M4/M5, MDS-CMML) and independent from cytogenetic risk groups, demonstrating that DC-based strategies can be applied for patients with all cytogenetic risk groups. Proof of the clonal derivation of DC generated was obtained in five AML and four MDS cases with a combined FISH/immunophenotype analysis (FISH-IPA): The clonal numerical chromosome aberrations of the diseases were regularly codetectable with DC markers; however, not with all clonal cells being convertible to leukemia-derived DC_leu (on average, 53% of blasts in AML or MDS). To the contrary, not all DC generated carried the clonal aberration (on average, 51% of DC). In 41 AML and 13 MDS cases with a suitable antigen expression, we could confirm FISH-IPA data by Flow cytometry: although DC_leu are regularly detectable, on average only 57% of blasts in AML and 64% of blasts in MDS were converted to DC_leu. After coculture with DC in mixed lymphocyte reactions (MLR), autologous T cells from AML and MDS patients proliferate and upregulate costimulatory receptors. The specific lysis of leukemic cells by autologous T cells could be demonstrated in three cases with AML in a Fluorolysis assay. In six cases with only few DC_leu or few vital T cells available after the DC/MLR procedure, no lysis of allogeneic or autologous leukemic cells was seen, pointing to the crucial role of both partners in the lysis process. We conclude: (1) the generation of DC is regularly possible in AML and also in MDS under serum-free conditions. (2) Clonal/leukemia-derived DC_leu can be regularly generated from MDS and AML-MNC; however, not with all blasts being converted to DC_leu and not all DC generated carrying leukemic markers. We recommend to select DC_leu for vaccinations or ex vivo T-cell activations to avoid contaminations with non-converted blasts and non-leukemia-derived DC and to improve the harvest of specific, anti-leukemic T cells. DC and DC-primed T cells could provide a practical strategy for the immunotherapy of AML and MDS.     

Medusa cells: cytostructure and cytochemistry of amoeboid eosinophils with pseudopod-like processes

Summary Eosinophils having one or more pseudopod-like processes of various lengths are observed in bone marrow, peripheral blood, sputum, nasal smears, and in other exfoliative cytology and tissue specimens after fixation and staining for histochemical study; they are called medusa cells. Although the conformations and lengths of the processes vary, they resemble protozoal pseudopodia. The form which first called our attention to these cells is a conical determinate projection that tapers to a fine tip, much like a protozoal axopod. A basal specialization in the form of a vesicle or thickening may be frequently observed where the process protrudes from, or appears attached to, the cell body. The processes of these eosinophil variants appear morphologically specialized to interact with other cellular elements of the blood and are occasionally seen in contact with, or engulfing, erythrocytes, platelets or other leukocytes.Two hydroperoxidases have been elucidated in eosinophils and medusa cells by virtue of different substrate specificities, subcellular localizations and inhibitor sensitivities. One of these hydroperoxidases is shown by 3,3-diaminobenzidine, is cyanide resistant, and is never observed in granules or rods in the medusa cell processes; it is frequently polarized to the sites of contact of medusa cells with other cellular elements of the blood. The other hydroperoxidase is revealed byp-phenylenediamine-pyrocatechol, is sensitive to cyanide and is frequently observed in granules and rods in medusa cell processes as well as in a population of larger granules in the cell bodies; the granules in the processes appear to be precursors to the rods, which may be related to Charcot-Leyden crystals.The extrusion of medusa cell processes is facilitated by the divalent cations calcium and magnesium and is inhibited by anions which sequester them such as phosphate, EDTA, citrate and oxalate. Medusa cells have been observed in samples from both rodents and humans and can be very prominent when eosinophilia accompanies allergy, parasitosis and malignancy.     

TIGAR cooperated with glycolysis to inhibit the apoptosis of leukemia cells and associated with poor prognosis in patients with cytogenetically normal acute myeloid leukemia

Background

Cancer cells show increased glycolysis and take advantage of this metabolic pathway to generate ATP. The TP53-induced glycolysis and apoptosis regulator (TIGAR) inhibits aerobic glycolysis and protects tumor cells from intracellular reactive oxygen species (ROS)-associated apoptosis. However, the function of TIGAR in glycolysis and survival of acute myeloid leukemia cells remains unclear.

Methods

We analyzed TIGAR expression in cytogenetically normal (CN-) AML patients and the correlations with clinical and biological parameters. In vivo and in vitro, we tested whether glycolysis may induce TIGAR expression and evaluated the combination effect of glycolysis inhibitor and TIGAR knockdown on human leukemia cell proliferation.

Results

High TIGAR expression was an independent predictor of poor survival and high incidence of relapse in adult patients with CN-AML. TIGAR also showed high expression in multiple human leukemia cell lines and knockdown of TIGAR activated glycolysis through PFKFB3 upregulation in human leukemia cells. Knockdown of TIGAR inhibited the proliferation of human leukemia cells and sensitized leukemia cells to glycolysis inhibitor both in vitro and in vivo. Furthermore, TIGAR knockdown in combination with glycolysis inhibitor 2-DG led leukemia cells to apoptosis. In addition, the p53 activator Nutlin-3α showed a significant combinational effect with TIGAR knockdown in leukemia cells. However, TIGAR expression and its anti-apoptotic effects were uncoupled from overexpression of exogenous p53 in leukemia cells.

Conclusions

TIGAR might be a predictor of poor survival and high incidence of relapse in AML patients, and the combination of TIGAR inhibitors with anti-glycolytic agents may be novel therapies for the future clinical use in AML patients.

     

Developmental Responses to Drought and Abscisic Acid in Sunflower Roots: I. ROOT GROWTH, APICAL ANATOMY, OSMOTIC ADJUSTMENT

Aeroponically grown sunflower seedlings (Helianthus annuus L.cv. Russian Giant) were droughted or treated with abscisic acid(ABA) for 7 d. Drought stress prompted a three-phase growthresponse in sunflower roots: an initial phase of increased rootelongation was followed by a period of almost complete inhibitionbetween about 6 h and 72 h; this was followed, in turn, by aphase of partial recovery in the rate of root elongation. Droughtdecreased the size of the apical meristem as cells in the proximalregion of the meristem vacuolated and elongated. Root-to-shootbiomass ratios (R:S) increased initially but declined after72 h. Drought stress decreased water potential () and osmoticpotential ( and increased turgor pressure _p in the apical 30mm of the roots. These initial changes were transitory, lastingabout 3 h. Thereafter, and began to rise; _p fell back to controllevels. In the later stages of treatment, fell as the stressgrew more severe, but fp was maintained by osmotic adjustment.Desiccation for 1 h increased turgor pressures in excised 30mm apical segments. The transitory increase in root elongationwas contemporary with the initial rise in _p in the root apices,while the periods of greatest inhibition and partial recoveryin root elongation were contemporary with the periods of declineand partial recovery in the length of the apical meristem respectively.The inhibition of root elongation and the anatomical changesin the root apices were not determined by loss of turgor orlack of photosynthate, but rather appeared to be an active responseby the meristem to a drop in external . Treatment with ABA triggeredmany of the same changes as drought stress: ABA promoted a three-phasegrowth response, increased R:S, triggered the same initial changesin , , and _p, increased _p in excised 3.0 mm apical segments,and induced the same pattern of anatomical changes in the rootapices as drought stress. It is proposed that ABA mediates drought-inducedchanges in the primary development of sunflower roots. Key words: Abscisic acid, apical meristem, drought, osmotic adjustmen     

The Effects of Auxin on the Mechanical Properties in Vivo of Cell Wall in Hypocotyl Segments from Gibberellin-Deficient Cowpea Seedlings

Auxin-induced changes in the mechanical properties of cell wallwere examined by both positive and negative pressure jump methodsusing hypocotyl segments excised from the 3-day-old seedlingsof cowpea that has been treated with uniconazole, a potent inhibitorof the biosynthesis of gibberellins. In such segments (U-segments)that were deficient in endogenous gibberellin, auxin increasedonly the effective turgor (Pⁱ–Y) and did not change theextensibility () of cell wall. As a result, the extent of theauxin-induced promotion of growth was halved. However, auxinwas able to increase of U-segments that has been pretreatedfor two hours with GA₃ prior to the application of IAA. Measurementof intracellular pressure (P_i) with a pressure probe revealedthat auxin did not change Pⁱ in either U-segments or GA₃-pretreatedsegments. The results suggest that auxin can decrease the yieldthreshold of the cell wall (Y) independently of gibberellinbut can increase only in the presence of gibberellin. The differencebetween and Y in terms of their requirement for gibberellinto respond to auxin suggests that they are mutually separablemechanical properties that originate from different molecularprocesses that occur in the architecture of yielding cell walls. ³Present address: Ohishi, Enden, Mori-machi, Shuchi-gun, Shizuoka,437-02 Japan     

Microarray-based analysis and clinical validation identify ubiquitin-conjugating enzyme E2E1 (<Emphasis Type="Italic">UBE2E1</Emphasis>) as a prognostic factor in acute myeloid leukemia

Background

Previous research suggested that single gene expression might be correlated with acute myeloid leukemia (AML) survival. Therefore, we conducted a systematical analysis for AML prognostic gene expressions.

Methods

We performed a microarray-based analysis for correlations between gene expression and adult AML overall survival (OS) using datasets GSE12417 and GSE8970. Positive findings were validated in an independent cohort of 50 newly diagnosed, non-acute promyelocytic leukemia (APL) AML patients by quantitative RT-PCR and survival analysis.

Results

Microarray-based analysis suggested that expression of eight genes was each associated with 1-year and 3-year AML OS in both GSE12417 and GSE8970 datasets (p?<?0.05). Next, we validated our findings in an independent cohort of AML samples collected in our hospital. We found that ubiquitin-conjugating enzyme E2E1 (UBE2E1) expression was adversely correlated with AML survival (p?=?0.04). Multivariable analysis showed that UBE2E1 ^high patients had a significant shorter OS and shorter progression-free survival after adjusting other known prognostic factors (p?=?0.03). At last, we found that UBE2E1 expression was negatively correlated with patients’ response to induction chemotherapy (p?<?0.05).

Conclusions

In summary, we demonstrated that UBE2E1 expression was a novel prognostic factor in adult, non-APL AML patients.

     

Hypothesis: Tim-3/Galectin-9, A New Pathway for Leukemia Stem Cells Survival by Promoting Expansion of Myeloid-Derived Suppressor Cells and Differentiating into Tumor-Associated Macrophages

Despite the improvements in chemotherapy, about 60 % of acute myeloid leukemia (AML) remission patients still relapse. Leukemic stem cells (LSCs) are the main causes for the relapse and refractory. T cell immunoglobulin mucin-3 (TIM-3), a specific surface molecule expressed on LSCs in most types of AML, is a candidate for AML LSC-targeted therapies. It is important to know how this molecule functions in the maintenance of LSCs and suppression of anti-tumor immunity. Recent data have shown that Tim-3 which expresses on T cells can suppress immune responses indirectly by inducing expansion of myeloid-derived suppressor cells (MDSCs). MDSCs at the leukemia site can also differentiate into tumor-associated macrophages (TAMs). TAMs can promote proliferation and survival of LSCs by the diversion of adaptive immunity and the facilitation of extracellular matrix remodeling, angiogenesis, and lymphangiogenesis. Our previous study in AML patient bone marrow samples showed CD68⁺ macrophages around AML clone. Based on the known evidence and our experimental findings, we hypothesize that Tim-3, which specifically expresses on LSCs, is beneficial for LSCs survival and AML progression by promoting expansion of MDSCs and differentiating into TAMs at the leukemia site.     

p62/SQSTM1 upregulation constitutes a survival mechanism that occurs during granulocytic differentiation of acute myeloid leukemia cells

The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34⁺ progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.p62 or sequestosome 1 (p62/SQSTM1) is a scaffold protein, implicated in a variety of biological processes including those that control cell death, inflammation, and metabolism.^{1, 2} Through its multi-domain structure, p62/SQSTM1 interacts specifically with key signaling proteins, including atypical PKC family members, NF-κB, and mTOR to control cellular responses.^{3, 4, 5, 6, 7} p62/SQSTM1 functions also as a key mediator of autophagy. Through its interaction with LC3, an essential protein involved in autophagy, p62/SQSTM1 selectively directs ubiquitinated substrates to autophagosomes leading to their subsequent degradation in lysosomes.^{8, 9} At the molecular level, p62/SQSTM1 acts as a pro-tumoral molecule by ensuring efficient and selective activation of cell signaling axes involved in cell survival, proliferation, and metabolism (i.e., NF-κB, mTOR, and Nrf-2 pathways).^{3, 5, 6, 7, 10, 11, 12, 13} p62/SQSTM1 can also signal anti-tumoral responses either by inactivating the pro-oncogenic signaling through BCR-ABL¹⁴ and Wnt pathways^{15, 16} or by inducing the activation of caspase 8, a pro-death protein.^{17, 18} Interestingly, in response to stress, autophagy promotes the degradation of p62, thus limits the activation of p62-regulatory pathways that control tumorigenesis.¹⁰ In addition, p62/SQSTM1 controls pathways that modulate differentiation of normal and cancerous cells. For example, p62/SQSTM1 has been shown to antagonize basal ERK activity and adipocyte differentiation.¹⁹ In contrast, p62/SQSTM1 favors differentiation of osteoclasts,²⁰ osteoblasts,²¹ neurons,²² megakaryocytes²³ and macrophages.²⁴ The role and regulation of p62/SQSTM1 during leukemia cell differentiation has been poorly documented.Acute myeloid leukemia (AML) is a hematological disease characterized by multiple deregulated pathways resulting in a blockade of myeloid precursors at different stages of maturation.^{25, 26} Acute promyelocyte leukemia (APL) is the M3 type of AML characterized by an arrest of the terminal differentiation of promyelocytes into granulocytes and frequently associated with the expression of the oncogenic PML-RAR alpha fusion gene.^{27, 28} All-trans retinoic acid (ATRA), a potent activator of cellular growth arrest, differentiation, and death of APL cells, has been shown to effectively promote complete clinical remission of APL when combined with chemotherapy.^{29, 30, 31} Despite the success of this treatment, some APL patients are refractory to ATRA treatment or relapse owing to the development of resistance to ATRA in leukemia cells.^{32, 33, 34}Our previous results revealed that autophagy flux is activated during granulocyte differentiation of myeloid leukemia cell lines induced by ATRA.³⁵ In the present study, we observed that p62/SQSTM1, an autophagic substrate, is markedly upregulated at both mRNA and protein levels during the granulocytic differentiation process. Here, we investigated the regulation and the function of p62/SQSTM1 during AML cells differentiation into neutrophils/granulocytes.     

Ribosomal proteins: XLIII. In vivo assembly of Escherichia coli ribosomal proteins

Isolation of ribosomal precursors from Escherichia coli K12 is described. The RNA and protein content of the precursor particles was determined.One physiologically stable precursor was found for the 30 S subunit. The assembly scheme is as follows: p16 S RNA + 9 proteins → p30 S (“21 S” precursor) p30 S + 12 proteins → 30 S subunit where p is precursor.Each of the two precursors for the 50 S subunit, P₁50 S and p₂50 S (“32 S” and “43 S” precursors, respectively), contains p5 S + p23 S RNA's in a 1:1 molar ratio. The assembly scheme is as follows: p23 S RNA + p5 S RNA + 16 or 17 proteins → p₁50 S

In contrast to the p₂50 S precursor the p₁50 S precursor is not similar to any core particles, which were obtained by treating 50 S subunits with different concentrations of LiCl or CsCl.The precursors p30 S and p₂50 S can be converted into active 30 S and 50 S sub-units, respectively, by incubation at 42 °C in the presence of ribosomal proteins and under RNA methylating conditions.     

An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

Background

Xenotransplantation of patient-derived AML (acute myeloid leukemia) cells in NOD-scid Il2rγ ^null (NSG) mice is the method of choice for evaluating this human hematologic malignancy. However, existing models constructed using intravenous injection in adult or newborn NSG mice have inferior engraftment efficiency, poor peripheral blood engraftment, or are difficult to construct.

Methods

Here, we describe an improved AML xenograft model where primary human AML cells were injected into NSG newborn pups intrahepatically.

Results

Introduction of primary cells from AML patients resulted in high levels of engraftment in peripheral blood, spleen, and bone marrow (BM) of recipient mice. The phenotype of engrafted AML cells remained unaltered during serial transplantation. The mice developed features that are consistent with human AML including spleen enlargement and infiltration of AML cells into multiple organs. Importantly, we demonstrated that although leukemic stem cell activity is enriched and mediated by CD34⁺CD117⁺ subpopulation, CD34⁺CD117^? subpopulation can acquire CD34⁺CD117⁺ phenotype through de-differentiation. Lastly, we evaluated the therapeutic potential of Sorafenib and Regorafenib in this AML model and found that periphery and spleen AML cells are sensitive to these treatments, whereas BM provides a protective environment to AML.

Conclusions

Collectively, our improved model is robust, easy-to-construct, and reliable for pre-clinical AML studies.

     

Atg5-dependent autophagy contributes to the development of acute myeloid leukemia in an MLL-AF9-driven mouse model

Acute myeloid leukemia (AML) is a hierarchical hematopoietic malignancy originating from leukemic stem cells (LSCs). Autophagy is a lysosomal degradation pathway that is hypothesized to be important for the maintenance of AML as well as contribute to chemotherapy response. Here we employ a mouse model of AML expressing the fusion oncogene MLL-AF9 and explore the effects of Atg5 deletion, a key autophagy protein, on the malignant transformation and progression of AML. Consistent with a transient decrease in colony-forming potential in vitro, the in vivo deletion of Atg5 in MLL-AF9-transduced bone marrow cells during primary transplantation prolonged the survival of recipient mice, suggesting that autophagy has a role in MLL-AF9-driven leukemia initiation. In contrast, deletion of Atg5 in malignant AML cells during secondary transplantation did not influence the survival or chemotherapeutic response of leukemic mice. Interestingly, autophagy was found to be involved in the survival of differentiated myeloid cells originating from MLL-AF9-driven LSCs. Taken together, our data suggest that Atg5-dependent autophagy may contribute to the development but not chemotherapy sensitivity of murine AML induced by MLL-AF9.Acute myeloid leukemia (AML) is a clonal hematopoietic malignancy characterized by the uncontrolled proliferation of immature myeloid cells within the bone marrow (BM), eventually suppressing normal hematopoiesis.¹ Recurrent chromosomal translocations frequently occur in AML, one of which involves the fusions of the KMT2A gene on chromosome 11 to a number of potential partners that are diagnosed as prognostically intermediate to poor.¹ Among these fusions, the MLL-AF9 fusion oncogene, resulting from the t(9;11)(p22;q23) translocation, is well studied owing to its robust phenotype in various mouse models of AML.^{2, 3, 4} It has been previously reported that BM transplantation of hematopoietic progenitors expressing exogenous MLL-AF9 leads to rapid in vivo transformation and progression of AML in a syngeneic, immunocompetent mouse model and recapitulates the poor chemotherapy response of t(9;11)(p22;q23) fusion human AML.^{2, 5}Autophagy is an evolutionarily conserved catabolic pathway by which cellular components are engulfed by double-membraned vesicles, called autophagosomes, and delivered to the lysosome for degradation and recycling. Autophagy is best characterized to be induced under stressful conditions, such as organelle damage or nutrient deprivation, and is followed by the elongation of the autophagosome membrane around its cargo. In Atg5-dependent autophagy, the conversion of LC3-I to LC3-II by lipidation is crucial for autophagosome membrane expansion, which is mediated by a series of ubiquitin-like conjugation systems.⁶ Within this pathway, the Atg5-Atg12-Atg16 complex acts as an E3-ubiquitin-ligase-like enzyme that specifically mediates the conjugation of LC3-I to phosphatidylethanolamine to form LC3-II, which inserts to the autophagosomal membrane. Autophagosome maturation is followed by fusion to lysosomes, at which time the inner compartment is degraded. The genetic ablation of Atg5 leads to a complete and highly selective inhibition of LC3-dependent autophagosome formation.^{6, 7}Autophagy is known to be implicated in cancer as both a tumor promoter and a tumor suppressor.⁸ The genetic ablation of autophagy in mouse hematopoietic stem cells (HSCs) has been shown to result in severe impairments to HSC maintenance.^{9, 10, 11, 12, 13} Autophagy dysregulation has also been implicated in AML,^{12, 13, 14} suggesting that targeting autophagy could be promising for AML treatment. As an expanding arsenal of pharmacological autophagy modulators are being developed,^{15, 16} it has become increasingly important to specifically determine whether autophagy has an important role in AML using a genetic mouse model. Therefore, we sought to dissect the role of autophagy through the in vivo homozygous deletion of Atg5 in MLL-AF9-driven murine AML. We discover in this study that Atg5 deletion during primary transplantation prolongs the survival of animals, whereas Atg5 deletion after secondary transplantation has no effect on animal survival, suggesting a role for autophagy in the initiation, but not maintenance, of AML in our model. We additionally assessed the effect of autophagy in chemotherapeutic response and found that Atg5 deletion in our MLL-AF9 model had no effect on the in vivo response to cytarabine and doxorubicin combination therapy, suggesting that autophagy does not significantly contribute to chemotherapy response in this model.     

A Mathematical Model for the Macrophage Response to Respiratory Viral Infection in Normal and Asthmatic Conditions

Respiratory viral infections are common in the general population and one of the most important causes of asthma aggravation and exacerbation. Despite many studies, it is not well understood how viral infections cause more severe symptoms and exacerbations in asthmatics. We develop a mathematical model of two types of macrophages that play complementary roles in fighting viral infection: classically \((\hbox {CA}\)-\(\hbox {M}\Phi )\) and alternatively activated macrophages \((\hbox {AA}\)-\(\hbox {M}\Phi )\). \(\hbox {CA}\)-\(\hbox {M}\Phi \) destroy infected cells and tissues to remove viruses, while \(\hbox {AA}\)-\(\hbox {M}\Phi \) repair damaged tissues. We show that a higher viral load or longer duration of infection provokes a stronger immune response from the macrophage system. By adjusting the parameters, we model the differences in response to respiratory viral infection in normal and asthmatic subjects and show how this skews the system toward a response that generates more severe symptoms in asthmatic patients.