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Mondal et al. Neuroimmunol Neuroinflammation 2018;5:34 I http://dx.doi.org/10.20517/2347-8659.2018.13 Page 3 of 19

and microglial cells . A recent finding by our lab also documented that T11TS favors T cells survival
[35]
not only by inhibiting glioma mediated apoptogenic death of T cells but also by repairing gliomagenic
[36]
impairment of phosphatidylinositol 3-kinase/AKT (PI3K-AKT) signaling cascades . Further study in
[37]
our lab also deciphered that T11TS immunotherapy, mediate functional activation of T cells by rectifying
gliomagenic anti-proliferative action on T-cell by correcting CD2-mediated nuclear factor of activated T-cell
calcineurin pathway . Some of our previous publications also delineated that T11TS reduces glioma mass
[38]
simultaneously by accelerating the apoptotic death of brain tumor cells and by decreasing the number of
dividing glioma-bearing cells [39,40] .

The above immune rejuvenation and induced increased potentiality during T11TS treatment might have
an important bearing on the concomitant production of activated immunocompetent cells through the
hematopoietic machinery in the bone marrow. The query for such regenerative immunocompetence
remains unanswered on the hematopoietic level. Interestingly, in our recent publication, we have elucidated
for the first time that T11TS also protects the BMHSCs by inhibiting the premature apoptogenic death by
counteracting gliomagenic stimulation of intrinsic, extrinsic apoptogenic pathway and also by inhibiting
Granzyme-B mediated apoptotic fate of HSCs within bone marrow milieu .
[41]

Hence, the present study emphasized on possible modulations of key elements of the early phases of hematopoiesis
on bone marrow HSCs during glioma growth and following T11TS therapy. Our finding shows modulatory
effects of T11TS therapy towards differentiation, proliferation, activation of HSCs against gliomagenic shock.
This entirely new finding not only illuminates the role of HSCs in glioma and also the relevance of T11TS therapy
against this dreadful disease, but it may also drive us towards an important new target for basic investigation
and, potentially, therapeutic intervention against many more hematological malignancies.

METHODS
Animals
Healthy Swiss albino rat pups of both sexes (4.5-6 g) were maintained in our Institutional animal facility
as per Institutional Ethical Committee guidelines monitored by Committee for the Purpose of Control
and Supervision of Experiments on Animals, Govt. of India regulations . Six animals in each group were
[41]
weaned at 1 month of age and housed in individual cages at 22 ˚C in a 12 h light/dark cycle. Animals were
fed with standard autoclaved food pellets along with water ad libitium.

The experimental animals were grouped into the following 5 groups: (1) age matched normal healthy control
[N]; (2) 3-5 days-old neonatal animals intraperitoneally (i.p.) injected with ENU and reared for 5 months
(optimal period for glioma development) [ENU]; (3) ENU animals (5 months of age) treated with single dose
of (i.p.) T11TS [ET1]; (4) ENU animals treated with 2 doses of (i.p.) T11TS [ET2] at an interval 6 days for each
dose; (5) ENU animals treated with 3 doses of (i.p.) T11TS [ET3] at an interval 6 days for each dose.

Brain tumor induction with ethyl nitrosourea
ENU the engineered alkylating compound is a strong mutagen and is observed to be the most intense of
neurocarcinogens engendering brain tumors with architectural and physiological likenesses to typically
ensuing neural neoplasms in mankind . ENU does not require any metabolic computation for its
[42]
enactment . ENU can enter into brain in spite of an intact blood brain barrier and the alkylated products
[43]
of the DNA are readily formed within the brain tissue .
[44]
ENU was freshly prepared by dissolving 10 mg/mL in sterile saline and adjusting the pH to 4.5 with
crystalline ascorbic acid. ENU was injected i.p. to newborn rats (3-5 days old) with a dose of 80 mg/kg body
weight . Maintenance of age-matched control was done by rearing healthy rat pups up to 5 months of age.
[36]