myeloid progenitors mitigate radiation injury and …...sto-mp-hfm-223 30 - 1 myeloid progenitors...

STO-MP-HFM-223 30 - 1

Myeloid Progenitors Mitigate Radiation Injury and Improve Intestinal

Integrity after Whole-Body Irradiation

Vijay K. Singh,1 Thomas B. Elliott,

1 Ram Mandalam,

2 Holger Karsunky,

2 Anna K. Sedello

2

1Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, 8901

Wisconsin Ave, Bethesda MD 20889-5603, USA 2Cellerant Therapeutics, 1531 Industrial Road, San Carlos, CA 94070, USA

[email protected] , [email protected], [email protected]

[email protected], [email protected]

ABSTRACT

We have demonstrated the efficacy of mouse myeloid progenitor cells (mMPC) as a promising radiation

countermeasure with the potential to mitigate radiation injury across a broad range of lethal radiation doses

in unmatched recipients even when transfused days after radiation exposure. Here we investigated how

transfusion of mMPC mitigates death from supralethal doses of radiation known to cause death by

gastrointestinal injury. CD2F1 mice were exposed to different doses of radiation and then transfused with

mMPC intravenously after irradiation. Intestinal tissues were harvested at different times after irradiation

and analyzed for tissue architecture, surviving crypts, villus height and number. We also monitored the effect

of infused mMPC on bacterial translocation from gut to heart, spleen, and liver in irradiated mice by

bacterial tissue cultures, and estimated endotoxin in serum samples. We observed that the infusion of mMPC

significantly improved survival of mice receiving high doses of radiation, decreased the number of bacterial

infection, and lowered endotoxin levels in serum. Histopathology of jejunum from irradiated and mMPC-

transfused mice revealed significant mitigation of gastrointestinal tissue injury. In brief, results of this study

further supports our contention that the transfusion of mMPC acts as a bridging therapy for gastrointestinal

system recovery by improving intestinal structural integrity and inhibiting bacterial translocation in the

gastrointestinal tract of lethally irradiated mice. This novel cell therapeutic approach consisting of the

infusion of mMPC following acute radiation injury appears to be one of the most promising radiation

countermeasures for acute radiation syndrome.

1.0 INTRODUCTION

The risk of exposure to ionizing radiation due to terrorist activities is widely thought to be increasing [1].

Although efforts to find suitable radiation countermeasures were initiated more than half a century ago, no

safe and effective radiation countermeasure for acute radiation syndrome (ARS) has been approved by the

United States Food and Drug Administration (FDA). Thus, there is a pressing need for both radioprotectant

and radiomitigator therapeutics to address ARS, as recognized by civilian and military government agencies

[2]. Major themes of countermeasure development have been free radical scavengers and stimulation of

hematopoietic progenitors. Other therapeutic avenues are also being explored, such as enhancing DNA repair

or blocking cell death pathways. Therapies utilizing cytokine treatment and supportive care including

antibiotics and blood component transfusion have shown moderate success in animal models [3], prompting

intensified research to identify a new generation of countermeasures.

The biological effects of radiation are strongly dependent upon the dose of radiation received [4, 5]. ARS

developing from whole-body or partial-body irradiation can involve hematopoietic, gastrointestinal, and

cerebrovascular components [6]. Cerebrovascular damage invariably leads to death within several days. In

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Myeloid Progenitors Mitigate Radiation Injury and Improve Intestinal Integrity after Whole-Body Irradiation

30 - 2 STO-MP-HFM-223

contrast, mortality from hematopoietic and gastrointestinal syndromes occurs with lower frequency and more

slowly (over weeks rather than days) and is more likely to be amenable to radiation countermeasures. There

are a number of potential radiation countermeasures that are currently in different stages of development,

falling broadly into two categories depending upon their primary mechanism of action:

immunomodulators/cytokines/growth factors [7-12] and antioxidants/free-radical scavengers [13-15]. In large

part, the focus on cytokines and growth factors has been based on their potential ability to act as

radiomitigators enhancing recovery of the hematopoietic system from radiation damage, as demonstrated in

multiple in vitro and in vivo studies [11, 12]. Some cytokines have received FDA approval for treatment of

neutropenia and thrombocytopenia caused by anti-cancer radiotherapy and chemotherapy, and several others

are under development [16-18]. The anti-radiation potential of antioxidants and free-radical scavengers

derives from their ability to reduce levels of reactive oxygen species induced by radiation, thus decreasing

DNA damage, lipid peroxidation and other types of chemical modification [14]. Efficacy of this class of

agents is limited to prophylaxis of radiation injury.

Cellerant Therapeutics has developed culture conditions to produce large numbers of mouse myeloid

progenitors (mMPC) from hematopoietic stem cells as a radiomitigator. mMPC primarily consist of lineage-

/lowc-Kit

+ myeloid progenitors expressing varying levels of CD11b

+ and Gr1

+ indicating a commitment to the

myeloid lineage [12, 19]. In contrast to whole bone-marrow grafts, T and B cells are not present in detectable

levels among mMPC and the risk of graft-versus-host disease (GVHD) is negligible. Earlier published in vivo

studies suggest that allogeneic myeloid progenitors give rise to myeloid and erythroid cells that transiently

engraft and functionally protect mice from death due to invasive fungal infections in major histocompatibility

complex disparate recipient mouse strains [20, 21].

We demonstrate that mMPC are a promising radiation countermeasure with the potential to mitigate radiation

injury in unmatched recipients across a broad range of lethal radiation doses even when transfusion is delayed

seven days after radiation exposure [19]. With respect to efficacy, timing and practicality of administration,

mMPC appear to be one of the most promising radiation countermeasures for ARS among all candidate

therapeutics currently under development [12]. To understand the mechanism of radiomitigation by mMPC

transfused into lethally irradiated mice, we investigated the effects of mMPC transfusion on the extent of gut

injury by histopathology analysis. We also studied gut bacterial translocation in irradiated mice. Our results

demonstrated that mMPC transfusion significantly mitigated radiation injury in vital gastrointestinal tissue,

and inhibited bacterial translocation in mice exposed to high doses of 60Co γ-radiation when administered 2-

24 h after radiation exposure.

2. 0 MATERIALS AND METHODS

2.1 Mice

Male 6–8 week-old specific-pathogen-free (SPF) CD2F1 mice were purchased (Harlan, Indianapolis, IN,

USA) and housed (8 per cage) in an air-conditioned facility accredited by the Association for Assessment and

Accreditation of Laboratory Animal Care International at the Armed Forces Radiobiology Research Institute.

Upon arrival, mice were held in quarantine for 1 week. A microbiological examination of representative

samples ensured the absence of Pseudomonas aeruginosa. Mice were provided certified rodent rations

(Harlan Teklad Rodent Diet, Harlan Teklad, Madison, WI, USA), acidified water (HCl, pH = 2.5 - 2.8) ad

libitum, sterilized bedding and housed in rooms with a 12-h light/dark cycle. The mouse holding room was

maintained at 21 ± 2 C with 10–15 hourly cycles of fresh air and a relative humidity of 50 ± 10%. Research

was conducted according to the Guide for the Care and Use of Laboratory Animals prepared by the Institute

of Laboratory Animal Resources, National Research Council, U.S. National Academy of Sciences [22]. At the


STO-MP-HFM-223 30 - 3

time of experiment, mice were 8-9 wk old. Experimental mice were observed daily in the morning and any

mice found sick/morbid, but not moribund, were allowed to continue in the experiment.

All mice used at Cellerant Therapeutics for preparing mMPC were SPF males and were received at 6-8 weeks

of age, quarantined for at least one week and subjected to experimental procedures at 8-12 weeks of age.

AKR/J and B6.Pl-Thy1.1 mice were procured from Jackson Laboratories, Bar Harbor, ME. FVB mice were

procured from Charles River Laboratories, Hollister, CA, USA. Mice were housed in individually ventilated

cages of up to five mice per cage on a 12 h light/dark cycle at 21 ± 2 C with 10–15 hourly cycles of fresh air

and given sterilized bedding and acidified water. All animal procedures were performed according to a

protocol approved by the Institutional Animal Care and Use Committee at Cellerant Therapeutics.

2.2 Irradiation

Mice were placed in ventilated acrylic boxes compartmentalized to accommodate eight mice per box and

exposed to bilateral irradiation in the AFRRI cobalt-60 facility at a dose rate of 0.6 Gy/min to total midline

doses indicated in experiments as described recently [23]. After irradiation, mice were returned to their cages

and monitored. Sham-irradiated mice were treated in the same manner as irradiated animals except that the

cobalt-60 rods were not raised from the pool of shielding water. Radiation dosimetry was based on the

alanine/EPR (electron paramagnetic resonance) system [24, 25], currently accepted as one of the most

accurate methods and used for intercomparison between national metrology institutions. The calibration

curves used in dose measurements at AFRRI (spectrometer e-Scan, Burker Biospin, Inc., Germany) are based

on standard alanine calibration sets purchased from The United States National Institute of Standards and

Technology, Gaithersburg, MD, USA.

2.3 Derivation of mouse myeloid progenitor cells (mMPC)

mMPC were generated separately from three MHC disparate mouse strains: AKR/J, FVB, and B6.Pl-Thy1.1

mice. HSC for mMPC derivation were isolated from the bone marrow of donor mice as described previously

[26]. Monoclonal antibodies used for immunofluorescence staining for HSC isolation included PE-Cy7-

conjugated CD117 (2B8), APC-conjugated Sca-1 (Ly6A/E), FITC-conjugated Thy1.1 (HIS51), and a lineage

cocktail of PE-conjugated B220 (RA3-6B2), CD3 (145-2C11), CD4 (GK1.5), CD5 (53-7.3), CD8 (53-6.7),

CD11b (M1/70), Gr-1 (RB6-8C5), and Ter119 (TER-119) (eBioscience, San Diego, CA). HSC were double

sorted for purity as CD117+Thy-1.1

lowSca-1

+lin

-/low [27] using a 3-laser FACSAria (BD Biosciences, San Jose,

CA). Sorted HSC from B6.Pl-Thy1.1, AKR, or FVB mice were plated in X-VIVO 15 media (Lonza,

Walkersville, MD, USA) supplemented with recombinant mouse (rm) stem cell factor (rm SCF, Invitrogen,

Carlsbad, CA), rm thrombopoietin (rm TPO) (Invitrogen), rm Fms like tyrosine kinase 3 ligand (Flt3L) (R&D

Systems, Minneapolis, MN), Primocin (Invivogen, San Diego, CA), 2-mercaptoethanol (Sigma-Aldrich, St.

Louis, MO), and Glutamax (Invitrogen). On d 9 of culture, B6.Pl-Thy1.1 mMPC were harvested and

cryopreserved. AKR and FVB mMPC were harvested and cryopreserved on d 10. mMPC from each strain

were cryopreserved separately in X-VIVO 15 media containing 10% dimethyl sulfoxide (Protide

Pharmaceuticals, Lake Zurich, IL, USA) and 25% FCS at 20 million cells/ml.

2.4 Transfusion of mMPC to mice

Mice were anesthetized in a ComPac5 anesthesia system (VetEquip Inc., Pleasanton, CA) with isoflurane

(Abbott Laboratories, Chicago, IL, USA) aerosol used as the anesthetic agent. Anesthetized mice were

transfused intravenously (retro-orbital sinus) with a 0.5 ml insulin syringe and 28 G needle. Each mouse

received 100 l of cell suspension containing the desired number of mMPC pooled in equal parts from all


30 - 4 STO-MP-HFM-223

three donor strains or vehicle. Mice were continuously monitored until regaining consciousness before

transferring to cages.

2.5 Histopathology of jejunum

To evaluate the effect of mMPC transfusion on radiation-induced intestinal damage, mice were irradiated and

transfused with mMPC or vehicle as described above. Mice were euthanized by CO2 asphyxiation and

jejunum was collected for histopathology. Samples were gently perfused with formalin, placed in a cassette

and submerged in formalin for at least 12 h. Jejuna were immersion-fixed in a 20:1 volume of fixative (Z-

FIX®, Anatech Ltd., Battle Creek, MI, USA) to tissue for at least 24 h and up to seven days [28]. Paraffin

sections were used for immunohistochemistry examination. Cross sections of jejunum were cut with a manual

rotary microtome at 4 μm [29]. The crypt microcolony survival assay was performed on hematoxylin and

eosin (H&E) paraffin sections as described by Withers and Elkind [30]. The cross sections were observed

under a Nikon Eclipse TS100 microscope (Nikon Inc. Melville, NY)) equipped with the Retiga 2000R Q

imaging camera (Surrey, BC, Canada).The circumference of a transverse cross-section of the intestine was

used as a unit. The cross section tissue area was determined by subtracting the lumen area from total cross

section area of jejunum. Crypts of Lieberkühn were considered viable if they contained at least 10 epithelial

cells (either columnar enterocytes or goblet cells), a lumen and at least one Paneth cell. The number of

surviving crypts was counted in each circumference. The total number of villi was scored in each

circumference. Six circumferences were scored per mouse and 8 mice were used in each group. The six

longest villi were measured in each circumference.

2.6 Evaluation of gut bacterial translocation

Heart ventricular blood, liver, and spleen of mice were collected aseptically as described recently [31] and

cultured on sheep blood agar, colistin-nalidixic acid in sheep-blood agar, and xylose-lysine-desoxycholate

agar media. Single colonies of isolated microorganisms were observed for their characteristics including

morphology and color. Pertinent characteristics were recorded. A portion of an isolated colony was

subcultured to obtain a pure culture and the remaining portion of the same colony was used to prepare a

Gram-stain. The Gram-stain characteristics for each isolate were observed under oil immersion at 1000X

magnification and recorded (cell-wall structure, cellular shape, and cellular arrangement). The subcultures on

SBA were incubated in 5% CO2 at 35C for 18–24 h and then observed to assure a pure culture. Pure cultures

were identified by a Vitek-2 Compact automated system (bioMérieux, Inc., Durham, NC, USA) according to

the manufacturer’s validated procedure.

2.7 Determination of bacterial endotoxin in serum

Concentration of bacterial endotoxin was determined in serum obtained from mMPC-treated and vehicle-

treated mice by a kinetic turbidimetric method of the limulus amebocyte lysate assay (ENDOSAFE®

KTA2™, Charles River Laboratories, Inc., Charleston, SC) [32].

2.8 Statistical analysis

Means with standard error or percentage were reported if applicable. Analysis of variance (ANOVA) was

used to detect whether there was a significant difference among groups. If significant, then a pair-wise

comparison by Tukey-Kramer was used to identify which groups were different from the others. For survival

data, a log-rank test was used to compare survival curves. Fisher’s exact test was used to compare survival

rates at the end of 30 d, with Bonferroni correction used to control type-I error if multiple comparisons


STO-MP-HFM-223 30 - 5

were used. ANOVA was used to detect whether there were significant differences between groups. If

significant, a Tukey’s post-hoc test was used to determine significant differences between particular

groups. A significance level was set at 5% for each test. All statistical tests were two-sided with a 5%

significance level. Statistical software, SPSS version 19, was used for statistical analyses.

3. 0 RESULTS

3.1 mMPC mitigate death from a potentially lethal dose of 60

Co -radiation in CD2F1 mice

when administered days after irradiation

To investigate how long mMPC

administration can be delayed after

irradiation, CD2F1 mice (H-2d)

were irradiated using 60

Co -

radiation (LD90/30 dose, 9.2 Gy) and

transfused with mMPC pooled from

AKR (H-2k), B6.Pl-Thy1.1 (H-2

b),

and FVB (H-2q) mice at different

times after irradiation. For each

administration time point a control

group of mice was irradiated with 60

Co -radiation and received

vehicle. The irradiated mice were

monitored for survival over 30 d and

survival curves were plotted. mMPC

administration was delayed for 5, 6,

and 7 days after irradiation. When

administered 5 or 6 d after 60

Co -

radiation (LD90/30 dose, 9.2 Gy),

88% of mice treated with 4 million mMPC survived compared to 13% in the vehicle control treatment groups

(figure 1). Administration of 6 million mMPC 7 d after 60

Co -irradiation (LD90/30 dose) significantly mitigated

death in CD2F1 mice compared to vehicle control group (56 % survivors in mMPC treated mice compared

with 0% survival in vehicle control). When 12 million mMPC were administered, there was no additional

benefit.

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Su

rviv

al

(%)

Time post-irradiation (d)

4 million mMPC 5 day

Vehicle 5 day


Vehicle 6 day


Vehicle 7 day

**

*

Figure 1. Efficacy of delayed administration of mMPC in 60Co -irradiated CD2F1 mice.

Mice were transfused mMPC 5/6 d (4 million cells) or 7 d (6 million cells) (n = 16) after 60Co -irradiation (9.2 Gy). Death by radiation was significantly mitigated in mice treated

with mMPC compared with vehicle controls * Denotes statistically significant difference

from vehicle control.


30 - 6 STO-MP-HFM-223

3.2 mMPC mitigated death from supralethal doses of radiation

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Su

rviv

al (

%)


14.5 Gy + Vehicle

14.5 Gy + 5 million mMPC

15.0 Gy + Vehicle


15.5 Gy + Vehicle


**

Figure 2. Effect of mMPC transfusion on survival of CD2F1 mice when administered 24 h after

high doses of 60Co -irradiation. Five million mMPC or vehicle were transfused into mice 24 h

after irradiation with either 14.5, 15.0, or 15.5 Gy 60Co -radiation. Survival was monitored for

30 d. Death by radiation was mitigated in all mice transfused with mMPC (n = 16 each group)

given 14.5 and 15.0 Gy. * Denotes statistically significant difference between mMPC- and

vehicle-transfused mice exposed to similar radiation dose.

To investigate whether mMPC transfusion could be used to treat injuries due to supralethal doses of ionizing

radiation causing gastrointestinal injury, three sets of mice were irradiated with 14.5, 15.0, and 15.5 Gy,

respectively, and transfused with 5 million mMPC 24 h after irradiation. Three additional sets of mice

receiving the same radiation exposures were transfused with vehicle. All mice were observed for 30 d post-

irradiation. Results presented in figure 2 demonstrate that all mice transfused with mMPC receiving 14.5 and

15 Gy radiation survived and all mice given 15.5 Gy died by d 11 after irradiation. All mice in vehicle-

transfused groups (14.5, 15 and 15.5 Gy) also died by d 11 after irradiation. These results suggest that a single

transfusion of 5 million mMPC can mitigate radiation injury in mice exposed to 60

Co -radiation as high as 15

Gy.

3.3 Effect of mMPC transfusion on radiation-induced intestinal damage

To assess the effect of mMPC transfusion on radiation-induced intestinal damage, 6 million mMPC or vehicle

were transfused to recipient mice 2 h after irradiation with 13 Gy and jejunum (which is the most sensitive

organ to enumerate effects of high doses of radiation) was analyzed by histopathology 4 and 8 days post

irradiation (figure 3). Histological analyses indicated that the jejunal tissue from mMPC treated groups had

improved normal histological structure and integrity compared to vehicle treated animals. A variety of

histological assessments of intestinal damage were carried out, including evaluation of viable crypts of

Lieberkühn, cross section tissue area, villus height, and villus number were selected as parameters to evaluate

intestinal damage. The cross section tissue areas of jejunum in mMPC transfused mice were significantly

higher compared to vehicle control mice on days 4 and 8 (p < 0.001). Mice receiving mMPC had a greater

number of crypts compared to vehicle control on d 8 after irradiation (p < 0.001). Our results also demonstrate

that mean villus height and circumference were higher in mMPC-transfused mice compared to vehicle control

on days 4 and 8 (p < 0.001). These results demonstrate overall improved structural integrity of jejunum in

mMPC treated mice.


STO-MP-HFM-223 30 - 7

Figure 3. The effect of mMPC and vehicle transfusion on jejunum tissue recovery in CD2F1 mice exposed to 60Co γ–irradiation.

CD2F1 male mice were irradiated with 13 Gy and transfused 2 h after irradiation with 6 million pooled, allogeneic mMPC or vehicle.

Jejunum samples were collected 4 and 8 days after irradiation, and scored for mean tissue area, mean crypt number per circumference,

mean villus height, and mean villus number per circumference. Upper two panels show representative photomicrographs of jejunal

cross sections stained with hematoxylin and eosin (circumference at 40x, villus height at 100x). Lower panel shows the quantification

of representative cross sections (*p<0.05).

3.4 Effect of mMPC transfusion on the translocation of intestinal bacteria in irradiated

mice

The effects of mMPC transfusion on intestinal mucosal integrity were analyzed by evaluating the

translocation of gut bacteria to various organs. Mice were irradiated with 13 Gy and transfused with 6 million

mMPC or vehicle 24 h later. Heart blood, liver, and spleen samples of mMPC- or vehicle-transfused mice

were collected aseptically on d 9, 11, and 14 post-irradiation, and cultured on appropriate media. Table 1

shows all bacterial species identified in samples from mMPC-transfused and vehicle-control groups at each

time point after irradiation. No bacteria were isolated from mMPC-transfused mice at any time point (total of

18 mice). In contrast, at least two bacterial species were isolated from each of six vehicle-control mice on d 9,

4 d 8 d

Vehicle mMPC Vehicle mMPC

Average crypt per

circumference Mean tissue area per

circumference Mean villus height

Mean villus number per circumference

0

10

20

30

40

50

60

70

80

4 8

Avera

ge c

ryp

t p

er

cir

cu

mfe

ren

ce


Vehicle

mMPC

*

0.0

0.2

0.4

0.6

0.8

1.0

1.2

4 8

Mean

ti

ssu

e a

rea (

mm

2)

per

cir

cu

mfe

ren

ce


*

*

0

50

100

150

200

250

300

350

400

4 8

Mean

vil

li h

eig

ht (μ

m)


*

*

0

5

10

15

20

25

30

35

40

4 8

Mean

vil

li n

um

ber

per

cir

cu

mfe

ren

ce


**


30 - 8 STO-MP-HFM-223

which demonstrate polymicrobial sepsis in all six vehicle-control mice. Both Gram-positive and Gram-

negative bacterial species were isolated from tissues in five of the six control mice and two Gram-positive

species were isolated from one mouse. A total of eight species of bacteria were isolated from the six mice. Six

species were Gram-positive, Staphylococcus aureus (2 mice), Streptococcus uberis (1 mouse), Streptococcus

mitis/Streptococcus oralis (1 mouse), Streptococcus sanguinis (1 mouse), Staphylococcus haemolyticus (1

mouse), and Enterococcus durans/Enterococcus hirae (1 mouse); and two species were Gram-negative,

Escherichia coli (3 mice) and Sphingobacterium thalpophilum (2 mice). It is noteworthy that E. coli and either

S. aureus or S. haemolyticus appeared in combination in three mice and S. thalpophilum and either S.

sanguinis or E. durans/E. hirae appeared together in two mice. These cases demonstrate incidence of

polymicrobial infection of both Gram-positive and Gram-negative bacteria. Isolated bacterial species were

detected in either heart blood, liver, or spleen from each mouse. No vehicle-control mice survived for

sampling on days 11 and 14. On day 9, the concentration of bacterial endotoxin in the serum of eight mMPC-

treated mice was <0.5 endotoxin unit (EU)/ml, whereas concentrations in serum of five vehicle-treated mice

ranged between 131.8 and 13100.0 EU/ml. These findings correlate with the isolation of bacterial species in

vehicle-treated and mMPC-treated mice. That is, bacteria and endotoxin were detected in vehicle-treated but

not in mMPC-treated mice. As stated above, no vehicle-treated mice survived for collection of serum samples

on days 11 and 14.

Table 1. Effect of mMPC transfusion on the translocation of gut bacteria to heart, spleen, and liver in mice

exposed to 60

Co –radiation.

Treatment

Mice with

bacterial growth

on d 9 post-

irradiation

Days after irradiation

9 11 14

Vehicle 6/6

Escherichia coli (3)

Staphylococcus aureus (2)

Streptococcus uberis (1)

Streptococcus mitis/Streptococcus oralis (1)

Sphingobacterium thalpophilum (2)

Streptococcus sanguinis (1)

Staphylococcus haemolyticus (1)

Enterococcus durans/Enterococcus hirae (1)

No

survivors

No

survivors

mMPC 0/6 No bacteria isolated No bacteria

isolated

No

bacteria

isolated

4. 0 DISCUSSION

The potential for nuclear incidents is expected to grow in coming years, and the need to develop

countermeasures for radiation victims is pressing. The radiation-induced gastrointestinal syndrome is a major

lethal radiation injury that might occur after a nuclear accident or radiation terrorism (dirty bomb). The


STO-MP-HFM-223 30 - 9

gastrointestinal syndrome involves destruction of crypt/villus units, loss of mucosal integrity, and infection by

resident enterobacterial flora, characterized clinically as anorexia, vomiting, diarrhea, dehydration, systemic

infection, and in extreme cases, septic shock and death [33-35]. There are no radiation countermeasures

(protectors, mitigators or therapeutics) against radiation-induced gastrointestinal syndrome lethality for first

responders, military personnel, or other workers entering a contaminated area. Pathophysiology of this

syndrome requires depletion of stem cell clonogens within the crypts of Lieberkühn, necessary for post-injury

regeneration of gut epithelium. Infusion of cryopreservable, culture-derived mMPC pooled from MHC-

disparate donors are effective in preventing death across a wide range of high radiation doses in unmatched

recipient mice. We were able to significantly mitigate radiation injury in mice and delay administration of

mMPC as late as 7 d after irradiation to rescue mice exposed to hematopoietic dose of radiation (9.2 Gy,

LD90/30) [19]. The ability to delay the administration of any radiation countermeasure is critical to allow for

dosing of radiation victims in an emergency situation. We have previously used tocopherol succinate to

mobilize progenitors and used mobilized progenitors as a radiomitigator to protect 60

Co -irradiated mice

when administered as late as 48 h after irradiation [36, 37] and were able to rescue mice against 11 Gy

radiation exposure. With mMPC, we demonstrate that mice can be rescued from radiation exposure as high as

15 Gy (LD90/10). The present study confirms our previous report that mMPC transfusion after irradiation

ameliorates radiation injury and reduces lethality across a broad range of radiation doses. Further, the ability

to cryopreserve mMPC prior to use and administration without matching donor and recipient makes them

logistically more feasible compared to bone marrow transplant. mMPC can be stockpiled and readily made

available on demand in case of an emergency. Currently there is no report of any radiation countermeasure

under development, which can be administered as late as this mMPC product to a wide range of people and

still confer significant mitigation of ARS [11, 12].

Our histopathology data demonstrate that transfusion of mMPC into irradiated mice mitigates radiation-

induced gastrointestinal injury. There was significant recovery in mMPC recipients compared to vehicle

control. We have observed similar recovery of radiation-induced gastrointestinal injury when either

tocopherol- or tocopherol succinate-mobilized progenitors were administered to mice [31, 37]. Tocopherol

succinate was used as a radioprotector (24 h prior to irradiation) and tocopherol succinate-mobilized

progenitors were used 24 or 48 h after irradiation as a mitigator. Gamma-tocotrienol has also been shown to

improve post-irradiation survival and intestinal radiation injury [38, 39]. Gamma-tocotrienol was used as a

radioprotector (24 h before irradiation). In this study with mMPC, we have used a much higher dose of

radiation (13 Gy, LD75/10) and observed significant protection of gastrointestinal tissue. Our results show

significantly increased tissue area, numbers of crypt, villus number and height in jejunal samples of mMPC

transfused mice compared to vehicle control mice.

Radiation is known to cause bacterial translocation from the gastrointestinal tract to different organs [40, 41],

which is considered to be a clinically important event during radiation-induced gastrointestinal injury [42, 43].

Our results presented in table 1 suggest the prevention of bacterial translocation and polymicrobial infections

in mMPC-transfused mice compared to recipients of vehicle. We have earlier reported that tocopherol

succinate treatment inhibits bacterial translocation in 60

Co -irradiated CD2F1 mice [31]. The protective role

of α-tocopherol on intestinal mucosa in mice and rats has also been reported by others [44, 45]. These results

support our current findings.

Our results demonstrate that pooled, allogeneic mMPC prevent death from lethal radiation known to cause

death through hematopoietic and gastrointestinal injury. Administration of mMPC can be delayed as late as 7

d after irradiation to mitigate radiation injury. The effect of mMPC on the gastrointestinal tract presented here

point to a function of mMPC and their progeny outside the hematopoietic system at supra-lethal radiation


30 - 10 STO-MP-HFM-223

doses. With respect to efficacy across a broad range of radiation doses, as well as timing and practicality of

administration, mMPC appear to be one of the most promising radiation countermeasures for ARS among all

candidate therapeutics currently under development. Further studies will be needed to understand the

mechanism by which this benefit is mediated.

5. 0 CONCLUSION

There are a number of major advantages that make mMPC ideal for the treatment of patients/casualities with

ARS: a) myeloid progenitor cells can be cryopreserved and stored without compromise in function, b)

treatment can be delayed to provide survival benefit, and c) myeloid progenitors provide radiomitigation in

unmatched irradiated recipients without signs of rejection or graft-versus-host disease. These characteristics

make myeloid progenitors a prime candidate as a bridging therapy for acute radiation victims that can be

administered in the field with minimal infrastructure requirements. With further preclinical development in

large animals (nonhuman primate, canine or minipig), we may be able to provide an appropriate protocol in

the near future for the clinical management of individuals suffering from exposure to high doses of ionizing

radiation. Cellerant Therapeutics has developed a culture system to expand CD34+ enriched human HSC in

vitro and to direct their differentiation into human myeloid progenitor cells. These hMPC (human myelod

progenitor cells, CLT-008) are currently in two Phase 1 clinical trials in patients undergoing cord blood

transplants and in patients receiving high dose chemotherapy for the treatment of hematological malignancies.

6.0 ACKNOWLEDGEMENTS

The authors are thankful to Dr. Christopher R. Lissner, Scientific Director of AFRRI, for helpful discussions,

and to Dr. Mark H. Whitnall, AFRRI Radiation Countermeasures Program Advisor, and Dr. Robert Tressler,

Vice President of Research and Development at Cellerant Therapeutics, for critical review of this manuscript.

We gratefully acknowledge Miss Elizabeth Ducey, Mr. Oluseyi Fatanmi, David L. Bolduc for technical help,

Dr. Cara Olsen and Miss Anna Posarac for statistical analysis, and Dr. Pankaj Singh for photomicrography.

This study was supported by a U.S. Department of Defense Threat Reduction Agency grant

(CBM.RAD.01.10.AR.012) to VKS.

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