Page 259 - Read Online

Mondal et al. Neuroimmunol Neuroinflammation 2018;5:34 I http://dx.doi.org/10.20517/2347-8659.2018.13 Page 11 of 19

bearing group both in the LDC and HDC. Further, in situ immunofluorescence imaging studies [Figure 4E] of
isolated BMHSCs showed a sharp (P < 0.001) increase in mean fluorescence intensity of cytoplasmic Ang-1
in HSCs of ENU induced glioma-bearing group compared to that in normal BMHSCs in both the immature
and mature compartments which showed moderate fluorescence staining and hence the moderate mean
intensity of fluorescence. However, T11TS treatment was able to bring down the fluorescence intensity of
cytoplasmic Ang-1 in dose dependent manner (P < 0.001) in glioma-associated HSCs of ET1, ET2, and ET3
groups compared to that in HSCs of the glioma-bearing ENU group.

T11TS therapy inhibits Tie-2 expression
Flowcytometric studies showed [Figure 5A and B] that there was simultaneous remarkable (P < 0.001) elevated
expression of Tie-2 in BMHSCs following glioma induction of the ENU group in both the compartments LDC
and HDC compared to low basal expression in normal BMHSCs in both the LDC and HDC. T11TS therapy
in glioma model, which significantly (P < 0.001) decreased Tie-2 expression in glioma associated BMHSCs of
both LDC and HDC compartment in all the three ET1, ET2 and ET3 dose levels in dose dependent manner
as compared to the remarkably high expression in ENU group both in the LDC and HDC.

In situ immunofluorescence staining of isolated BMHSCs of rats from different experimental groups showed
that glioma induction in the ENU group caused a remarkable increase in Tie-2 expression [Figure 5C] in
HSCs within bone marrow both in the LDC and HDC when compared to that in isolated HSCs from
a normal control group in both the compartments. T11TS therapy caused a significant (P < 0.001) dose-
dependent down regulation of Tie-2 expression in glioma-bearing HSCs within the bone marrow of T11TS
treated groups both in the LDC and HDC compartments.

Tie-2 expression pattern in glioma-associated BMHSCs as observed from flowcytometric studies concurred
with the in-situ immunofluorescence imaging results.

DISCUSSION
There is global immune suppression during glioma growth. Intraperitoneal administration of ENU in 5-6
days old neonatal rats produce grade IV glioma after 5-6 months of age. The salient features of ENU induced
full-blown grade IV glioma, have been elaborately elucidated histologically , by Glial fibrillary acidic
[56]
protein (GFAP) staining , by cell proliferation index studies , and by in situ immunofluorescences . The
[59]
[57]
[58]
published literature has also conclusively documented the gliomagenic global immune suppression on glioma-
bearing animals and the beneficial immunomodulatory effect of T11TS immunotherapy [60-63] . Following
T11TS administration, there is profound immune stimulation which in turn results in the proliferation of
the immunocytes and rejuvenates immune suppressive state of the malignant glioma [61,62,64-67] . Therefore,
the novelty of our work is that this is the first reporting of the status of HSCs during glioma occurrence
and thereafter following immunotherapy, which has not been explored so far. Both, in HDC comprised
of mature cells and LDC composed of relatively immature cells, the suppressive effect on HSCs during
a chemical carcinogen-induced glioma condition has been documented and reversal of this suppressive
situation after T11TS administration has also been noted.

The Sialomucin CD34 is a critical marker for primitive HSCs, the expression pattern of which appears to be
responsible for most of the early phases of hematopoietic activity [50,68] . Our study demonstrated that T11TS
therapy significantly stimulates the level of expressions of CD34 both at the LDC and HDC in BMHSCs
+
of glioma-bearing rats. The diminished level of CD34 on HSCs of glioma-bearing animals indicates a
diminution of primitive HSCs and suppression of normal hematopoiesis during glioma progression, which
might be associated with the accelerated pre-mature apoptosis of the hematopoietic machinery due to the
invading glioma . From our experiments, it was delineated that LDC in all the experimental groups contains
[41]