`
`ACTA BIOMED 2013; 84: 102-109
`
`© Mattioli 1885
`
`O R I G I N A L
`
`A R T I C L E
`
`Modulation of expression of Programmed Death-1 by
`administration of probiotic Dahi in DMH-induced
`colorectal carcinogenesis in rats
`Dheeraj Mohania1, 2, Vinod K. Kansal2, Manoj Kumar3, Ravinder Nagpal4, Yuichiro Yamashiro4,
`Francesco Marotta5
`1 Department of Research, Sir Ganga Ram Hospital, New Delhi, India; 2 Animal Biochemistry Division, National Dairy Re-
`search Institute, Karnal, Haryana, India; 3 Department of Microbiology & Immunology, National Institute of Nutrition, Hy-
`derabad, India; 4 Probiotic Research Laboratory, Juntendo University Graduate School of Medicine, Tokyo, Japan; 5 ReGenera
`Research Group for Aging Intervention, Milan, Italy
`
`Abstract. Background and Aim: Interaction of probiotic bacteria with the host immune system elicits benefi-
`cial immune modulating effects. Although, there are many published studies on interaction of probiotics with
`immune system focusing on activation of immune system by bacterial cell wall through the engagement of
`Toll-like receptor family, very few studies have focused on molecules involved in the T-cell activation, and
`not much work has been executed to study the correlation of probiotics and programmed death-1 in col-
`orectal carcinogenesis in animal models. Hence, the present study was carried out to assess the effect of pro-
`biotic Dahi on expression of programmed death (PD-1) in colorectum of 1, 2-dimethylhydrazine treated
`Wistar rats. Methods: DMH was injected subcutaneously at the rate of 40 mg/kg body weight per animal
`twice a week for 2 weeks. A total of 168 male Wistar rats were randomly allocated to seven groups, each
`group having twenty-four animals. The rats were euthanized at the 8th, 16th and 32nd week of the experi-
`ment and examined for the expression of PD-1 in colorectal tissues by immunohistochemical staining. Re-
`sults: PD-1 expression was observed in colorectal tissues of normal and DMH-treated rats. An increase in
`PD-1 expression upon DMH treatment was observed and its reversion by two different preparations of the
`probiotic Dahi, an effect also observed with the NSAID piroxicam. The effects of probiotic Dahi and pirox-
`icam were synergistic. Feeding rats with probiotic Dahi or piroxicam treatment decreased the expression of
`PD-1 in DMH-induced colorectal mucosa. Combined treatment with probiotic Dahi and piroxicam was
`significantly more effective in reducing the expression of PD-1. Conclusion: PD-1 is expressed independent-
`ly of carcinogen administration in normal colonic mucosa and may play a role in immune response modu-
`lation in DMH-induced colorectal carcinogenesis. The present study suggests that probiotic Dahi can be
`used as an effective chemopreventive agent in the management of colorectal cancer (www.actabiomedica.it)
`
`Key words: colorectal cancer, 1,2-dimethylhydrazine, piroxicam, programmed death-1, probiotics
`
`Introduction
`
`Gastrointestinal tract of humans and mammals
`are the biggest reservoir of large number of commen-
`sal microbial flora that contributes to the beneficial
`
`health of the host via immunomodulation. The reser-
`voir of these symbiotic bacteria participates in nutrient
`assimilation, production of amino acids, vitamins for
`their host and plays a key role in the development of
`healthy immune system. Immunomodulatory compo-
`
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`Probiotics reduce programmed death-1 expression in rats
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`103
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`nents such as cell surface components and peptidogly-
`can components of these bacteria may play an impor-
`tant role in activating immune-proficient cells in the
`gastrointestinal (GI) tract (1). Additionally, function-
`al association of the intestinal microflora in im-
`munomodulatory responses and maintenance of
`homeostasis accentuate the important role of the mi-
`crobiota in the GI tract (2, 3). Furthermore, intestinal
`microbiota, which includes various species of Lacto-
`bacillus, interacts regularly with colonic epithelial cells
`(4-6). A growing body of evidence suggests that mod-
`ulations of the immune system could be mediated by
`ingestion of probiotics which confer a health benefit
`upon the host by promoting humoral and cell mediat-
`ed immunity (7). However,
`little is known about the
`relationship between the probiotics and proteins in-
`volved in the regulation of T-cell activation in 1, 2-di-
`methylhydrazine (DMH)-induced colorectal carcino-
`genesis. Programmed cell death 1 (PD-1) is one
`among these proteins, a homolog of CD28 and CT-
`LA-4, involved in the regulation of T-cell activation
`(8). PD-1 is expressed on T cells, B cells and myeloid
`cells. It, plays an important role in lymphocyte acti-
`vation at tissue level based upon the expression of
`PDL-1 in non-lymphoid organs (9-13). Furthermore,
`PD-1 mediates immune regulation via not only on ac-
`tivated T cells, but also on B cells and monocytes (11,
`14). Up-regulation of PD-1 might be associated with
`immune evasion and inhibition in tumor-bearing
`hosts (14).
`Probiotic Dahi, an Indian fermented milk prod-
`uct, has been studied in tumor-bearing animals; how-
`ever, the relationship between probiotics and expres-
`sion of PD-1 in colorectal cancer is not known. Con-
`sequently, in the present investigation, we prepared
`the buffalo milk-based probiotic Dahi by co-culturing
`two combinations of selected strains of lactobacillus
`with Dahi: (A) Lactobacillus acidophilus (LaVK2)
`along with Lactobacillus plantarum (Lp9) and Dahi
`culture (B) Lactobacillus acidophilus (LaVK2) along
`with Bifidobacterium bifidum BbVK3 and Dahi cul-
`ture. Consumption of the fermented product was
`then evaluated for its effects on the expression of pro-
`gramme death-1 (PD-1) in DMH-induced colorectal
`carcinogenesis in rats.
`
`Materials and methods
`
`Bacterial strains
`
`Lactococcus lactis ssp. cremoris NCDC-86 and Lac-
`tococcus lactis ssp. lactis biovar diacetylactis NCDC-60
`were obtained from National Collection of Dairy Cul-
`tures, National Dairy Research Institute , Karnal, In-
`dia. Lactobacillus acidophilus LaVK2 and Bifidobacteri-
`um bifidum BbVK3 are our laboratory isolates with
`probiotic attributes tested through in vitro tests as per
`FAO/WHO guidelines. Lactobacillus plantarum (Lp9)
`was a generous gift from Dr. V. K. Batish, Scientist
`Emeritus, Dairy Microbiology Division, NDRI, Kar-
`nal, India. Lactobacilli and lactococci were propagat-
`ed and maintained in MRS-broth and M17 broth
`(Himedia Laboratories Pvt. Ltd., Mumbai, India) at
`37° and 30°C, respectively, and were stored at 4-8°C
`between transfers. B. bifidum BbVK3 was cultured and
`propagated under anaerobic conditions at 37°C.
`
`Probiotic Dahi and Dahi preparation
`
`Bacterial cultures were revitalized three times in
`reconstituted and autoclaved skim milk prior to use for
`preparation of fermented milk. Buffalo milk obtained
`from the cattle yard of the institute and standardized to
`3.0% fat was heated to 90°C for 15 min and then
`cooled to 37°C. Dahi was prepared by culturing stan-
`dardized buffalo milk with Dahi starter (Lactococcus
`lactis ssp. cremoris and Lactococcus lactis ssp. lactis biovar
`diacetylactis, 1% each) at 30°C for 8 h. Probiotic LaLp-
`Dahi was prepared by co-culturing standardized buffa-
`lo milk with L. acidophilus LaVK2, L. plantarum Lp9
`and Dahi starter under aseptic condition. The final
`product contained lactococci, 20x108 cfu/g, L. aci-
`dophilus LaVK2, 2x108 cfu/g and L. plantarum Lp9,
`2x108 cfu/g. Probiotic LaBb-Dahi was prepared by cul-
`turing standardized buffalo milk with L. acidophilus
`LaVK2, B. bifidum BbVK3 and Dahi starter. The final
`product contained lactococci, 1-2x109 cfu/g, L. aci-
`dophilus, 2-20x108 cfu/g and B. bifidus, 2- 20x108cfu/g.
`
`Chemicals
`
`1, 2-dimethylhydrazine dihydrochloride (DMH),
`piroxicam (PXC), Harris’ haematoxylin and 3,3-di-
`
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`104
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`D. Mohania, V.K. Kansal, M. Kumar, R. Nagpal, Y. Yamashiro, F. Marotta
`
`aminobenzidine were purchased from Sigma-Aldrich
`Chemical Co. (St. Louis, MO, USA). The source of
`primary antibody and Ultravision Kit was Abcam
`(Cambridge, UK). All other chemicals were obtained
`from S.D. Fine Chemicals Ltd., Mumbai, India, or
`Hi-Media Lab. Ltd., Mumbai, India.
`
`Animals and Diet
`
`Male Wistar rats (21 d old) were obtained from
`Small Animal House of National Dairy Research In-
`stitute, Karnal, India and maintained in a small ani-
`mal house. Animals were housed in stainless steel
`cages (three animals per cage) throughout the study
`and room temperature was maintained at 25 ± 2ºC
`with 55 ± 5% humidity and at a 12-h light/12-h dark
`cycle and allowed water ad libitum. The animals were
`used and cared for in accordance with the principles
`and guidelines for humane use and protocols were ap-
`proved the Institutional Ethics Committee. Basal di-
`et composition is given in Table 1.
`
`Experimental design
`
`Animals randomly distributed into 7 groups (24
`in each group) were given the following dietary treat-
`
`Table 1. Composition of hypercholesterolaemic basal diet.a
`
`Ingredients
`
`Casein
`Hydrogenated vegetable oil
`Cellulose
`Choline chloride
`Starch
`Sucrose
`D, L-methionine
`Salt mixture
`Vitamin mixture
`
`Amount
`
`20.0%
`20.0%
`5.0%
`0.2%
`19.25%
`30.0%
`0.3%
`5.3%
`1.0%
`
`a Salt mixture (AOAC, 2005) required for 10 kg diet (500 g)
`contained CaCO3, 190.7 g; CoCl2.6H2O, 0.0115 g; Cu-
`SO4.5H2O, 0.238 g; FeSO4.7H2O, 13.5 g; KH2PO4, 194.5 g;
`KI, 0.4 g; MgSO4.7H2O, 58.62 g; MnSO4.H2O, 2.005 g; NaCl,
`69.65 g; and ZnSO4.7H2O, 0.274 g. Vitamin mixture (100 g)
`comprised of biotin, 4 mg; folic acid, 20 mg; vitamin B12, 0.3
`mg; menadione, 50 mg; para aminobezoic acid, 1 g; meso-inos-
`itol, 1 g; thiamine, 50 mg; riboflavin, 80 mg; pyridoxine, 50 mg;
`calcium pentothenate, 0.4g and starch, 76.946 g. Vitamin A (2
`x 105 IU), vitamin E (103 IU) and vitamin D (2 × 104 IU) were
`administered to the diet through oil / fat (for 10Kg diet)
`
`ment in addition to basal diet for 32 weeks: 1) Milk
`group, fed buffalo milk; 2) Milk-DMH group, fed
`buffalo milk (3% fat adjusted) and administered 1,2-
`dimethylhydrazine dihydrochloride (DMH); 3) Milk-
`DMH-PXC group, fed buffalo milk (3% fat adjusted)
`and administered DMH and piroxicam; 4) LaLp
`Dahi-DMH group, fed LaLp Dahi and administered
`DMH; 5) LaLp Dahi-DMH-PXC group, fed LaLp
`Dahi and administered DMH and piroxicam; 6)
`LaBb Dahi-DMH group, fed LaBb Dahi and admin-
`istered DMH; 7) LaBb Dahi-DMH-PXC group, fed
`LaBb Dahi and administered DMH and piroxi-
`cam.Each rat was fed 20 g supplements of buffalo
`milk (3% fat adjusted) or probiotic Dahi, followed by
`basal diet ad libitum. Following 28 day feeding, each
`animal was injected (s.c.) DMH (40 mg/kg body
`weight) twice a week for 2 weeks except for the Milk
`group. Piroxicam (4 mg/day/rat) was delivered
`through milk/probiotic Dahi, start one week after the
`last DMH dose till termination of the experiment.
`Eight rats from each group were sacrificed by cervical
`dislocation at 8, 16 and 32 weeks, and their colorectal
`tissues were
`examined for PD-1 expression.
`These periods correspond to 2, 10 and 26 weeks past
`DMH administration, and 1, 9 and 25 weeks from
`piroxicam introduction in the drug treatment groups.
`Colorectal tissue specimens were fixed in formalin and
`embedded in paraffin for routine histological diagno-
`sis and immunohistochemical analysis.
`
`Immunohistochemical staining
`
`Four microns sections of the specimens were cut
`from paraffin-embedded tissue, mounted on poly-L-
`lysine coated slides, air dried for 10 min and fixed at
`65ºC for 15 min. Endogenous peroxidase activity was
`blocked with 1.0% hydrogen peroxide in PBS for 30
`min at room temperature. Sections were then washed
`three times in PBS. Non-specific background staining
`was blocked with protein blocking agent (PBA) for 20
`min at room temperature. Sections were incubated
`with primary antibody (Anti-PD-1) at the final con-
`centration of 1:100 in a humidified chamber at 37ºC
`for 2 h. Bound primary antibodies were detected with
`Ultravision Kit according to the manufacturer’s in-
`structions. Sections were washed with deoinized wa-
`
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`Probiotics reduce programmed death-1 expression in rats
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`105
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`Table 2. Effect of feeding probiotic Dahi and piroxicam (PXC) treatment on feed intake and body weight of dimethylhydrazine
`(DMH) treated rats. Values are mean ± SD for n=8
`
`Groups
`
`Milk
`Milk-DMH
`Milk-DMH-PXC
`LaLp Dahi-DMH
`LaLp Dahi-DMH-PXC
`LaBb Dahi-DMH
`LaBb Dahi-DMH-PXC
`
`Average Feed Intake
`(g/d/rat)
`17.8a ± 1.9
`11.4b ± 2.0
`16.8a ± 2.0
`17.8a ± 1.5
`18.9a ± 1.8
`17.7a ± 1.3
`18.6a ± 2.0
`
`Body weight (g)
`Initial
`22.2a ± 0.3
`22.8a ± 0.4
`23.2a ± 0.4
`21.7a ± 0.3
`24.2a ± 0.3
`22.7a ± 0.3
`23.2a ± 0.3
`
`Body weight (g)
`Initial
`325.2a ± 1.0
`237.0b ± 3.5
`304.5c ± 3.1
`342.0d ± 3.8
`345.8d ± 3.3
`338.0d ± 4.8
`340.8d ± 2.9
`
`DMH, 1,2-dimethylhydrazine dihydrochloride; PXC, piroxicam.
`a,b,c,d Mean values within a column with unlike superscript letters were significantly different (P<0.05).
`
`ter for 5 min and counterstained with Harris’ haema-
`toxylin. The expression levels of PD-1 were calculat-
`ed according to Detre S’ method (15).
`
`Statistical analysis
`
`The results were expressed as mean ± SD for each
`group (n=8) and analyzed by 1-way analysis of variance
`(ANOVA) followed by the Tukey post hoc test (SYS-
`TAT version 6.0.1, SPSS Inc, Chicago, IL, USA). Dif-
`ferences were considered significant at P < 0.05.
`
`Results
`
`A significant decline in feed intake as well as in
`body weight gain was observed in rats treated with
`DMH (Table 2). Treatment of DMH induced rats
`with either piroxicam (PXC), or LaBb Dahi, or LaLp
`Dahi, or piroxicam plus probiotic Dahi combined
`(LaBb or LaLp), restored feed intake to normal levels
`and increased weight gain significantly (P< 0.05). The
`DMH induced animals treated with LaBb Dahi or
`piroxicam and LaBb Dahi grew at rates even faster
`than the control rats. Typically, lymphocyte infiltra-
`tion occurred in colorectal tissues of DMH-treated
`rats (Fig. 1). In these tissues, mononuclear infiltration
`of lymphocytes showed positive membrane staining
`with anti-PD-1 (Fig. 2). The pattern of staining was
`consistent with the fact that PD-1 molecules are asso-
`ciated with cell membranes. PD-1 positive cells
`showed a U-shaped pattern throughout colonic ep-
`ithelial membrane in colorectal mucosa. The expres-
`
`sion of PD-1 in colorectal tissues of DMH-treated
`rats varied from 8 wk to 32 wk of the experimental
`study (Table 3).
`In the present study, PD-1 expression in the col-
`orectal mucosa of normal and DMH-treated animals
`supplemented with buffalo milk and different probi-
`otic Dahi preparations was detected and measured
`(Fig. 2). PD-1 expression was significantly greater in
`milk fed and DMH-treated rats when compared to all
`other groups from 8 wk to 32 wk (Table 3). When an-
`imals milk-fed animals were injected with DMH, the
`expression of PD-1 was substantially enhanced at 32
`wk reaching 7-fold of that at 0 d level (Table 3).
`Treatment with piroxicam or probiotic Dahi signifi-
`cantly decreased DMH-induced expression of PD-1
`in epithelial cells of colorectum. Piroxicam and both
`probiotic Dahi (LaLp and LaBb Dahi) were almost
`equally effective in reducing DMH-induced accumu-
`lation of PD-1 in epithelial cells of colorectum. The
`reductions in DMH-induced accumulation of PD-1
`in epithelial cells of colorectum were more pro-
`nounced in animals treated with the combination of
`piroxicam and probiotic Dahi. In animals treated with
`LaBb Dahi along with piroxicam, PD-1 expression in
`epithelial cells of colorectum at 32 wk of experimental
`period reached levels similar to untreated normal an-
`imals.
`
`Discussion
`
`Probiotic microorganisms have a long history of
`consumption in the form of fermented foods and are
`
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`106
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`D. Mohania, V.K. Kansal, M. Kumar, R. Nagpal, Y. Yamashiro, F. Marotta
`
`Figure 1. Longitudinal section of colonic mucosa showing aberrant crypt focus stained with haematoxylin and eosin (200X) at 32
`week. (A) Normal looking colonic mucosa; (B, C, D) Colonic mucosa showing crypt disarray and inflammatory cell infiltration in
`lamina propria in DMH-treated rats. Crypts are smaller than normal, variable in shape and show branching. There is loss of nor-
`mal crypt architecture with widening of lamina propria. Submucosa reveals dense lymphomononuclear infiltrate
`
`known to interact with the immune system and elicit
`beneficial immune modulating effects (1, 16-18). In
`line of these evidences, we have prepared probiotic
`fermented milk namely probiotic Dahi (LaLp Dahi or
`LaBb Dahi) along with mixed Dahi cultures of lacto-
`cocci for the delivery of probiotic strains that can pro-
`vide protection against colorectal cancer in animal
`models. In this study, we examined the expression of
`PD-1 in normal colorectal mucosa and investigated
`the effects of probiotic Dahi (LaLp or LaBb Dahi) or
`their combination with piroxicam on the expression of
`PD-1 in colorectal tissues of DMH-treated rats. Both
`probiotic Dahi (LaBb Dahi or LaLp Dahi) were
`equally effective, and were even more effective than
`piroxicam in reducing PD-1 expression in the col-
`orectum of DMH-treated rats. Furthermore, the com-
`
`bination of piroxicam and probiotic Dahi treatment
`decreased DMH-induced initiation and progression
`of neoplastic lesions more effectively, suggesting that
`this treatment combination is effective in preventing
`the initiation and progression of carcinogenesis.
`Hence, the role of probiotic Dahi (LaBb Dahi or
`LaLp Dahi) as an alternate biotherapeutic agent in
`the treatment of colorectal cancer may also be ex-
`plored. Probiotic intervention may decrease exposure
`of the colonic epithelial cells to cytotoxic and geno-
`toxic agents or may modulate the balance of colonic
`cell proliferation and apoptosis, and/or enhance the
`production of butyrate acetate, thereby improving im-
`munomodulation of colorectal mucosa (19-21). In-
`testinal bacteria are capable of activating or deactivat-
`ing proximal carcinogens, behaving as promoters or
`
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`Probiotics reduce programmed death-1 expression in rats
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`107
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`Figure 2. Longitudinal section of colonic mucosa showing immunohistochemical staining of normal and DMH treated colorectal
`tissues for programmed death-1 (PD-1) at 32 week. A: PD-1 expression in normal colonic mucosa (200X); (B, 200X; C, 400X; D,
`600X): Immunohistochemical staining of colonic mucosa showing positive staining (arrow head) for PD-1 treated with DMH
`showing crypt disarray and inflammatory cell infiltration in lamina propria
`
`anti-promoters in colon carcinogenesis (22). Recently,
`the author’s laboratory has shown that probiotic LaBb
`Dahi down regulate carcinogen activating cytochrome
`P450 enzymes CYP1A1, CYP1A2 and CYP1B1 in
`liver, and up regulate carcinogen detoxifying γ-glu-
`tamyltranspeptidase, UDP-glucuronosyl
`transferase
`and quinone reductase activities in liver as well as in
`colon (23). The potential of LaBb Dahi to improve
`macrophage and lymphocyte functions (24) and an-
`tioxidative status (25) has been also established. The
`results presented herein could be due to the mainte-
`nance of gut homeostasis by balancing proinflamma-
`tory (secretion of IL-12, a critical factor in switching
`memory T cells to Th1 response) and anti-inflamma-
`tory (secretion of IL-10 and IL-14 which promote the
`
`generation of a Th2 response) mucosal responses with
`the help of intestinal bacteria and probiotics, includ-
`ing lactobacilli and bifidobacteria (26).
`
`Conclusion
`
`From the results obtained, it could be concluded
`that PD-1 is expressed independent of carcinogen ad-
`ministration and is upregulated by DMH administra-
`tion in colorectal carcinogenesis. The findings of this
`study indicate that consumption of probiotic Dahi
`(LaLp or LaBb Dahi) or piroxicam or probiotic Dahi
`combined results in the decreased expression of PD-1
`antigen. Moreover, the effects of probiotic Dahi and
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`D. Mohania, V.K. Kansal, M. Kumar, R. Nagpal, Y. Yamashiro, F. Marotta
`
`Table 3. Effect of feeding probiotic Dahi and piroxicam (PXC) treatment on programmed death 1 (PD-1) expression in colorectal
`tissue of dimethylhydrazine (DMH) treated rats
`
`Group
`
`Buffalo milk
`Buffalo milk-DMH
`Buffalo milk-DMH-PXC
`LaLp Dahi-DMH
`LaLp Dahi-DMH-PXC
`LaBb Dahi-DMH
`LaBb Dahi -DMH-PXC
`
`0 day
`
`1.75a ± 1.04
`1.75a ± 1.04
`1.75a ± 1.04
`1.75a ± 1.04
`1.75a ± 1.04
`1.75a ± 1.04
`1.75a ± 1.04
`
`8 week
`
`1.63a ± 0.52
`8.50bc ± 2.00
`9.75c ± 2.55
`6.38bde ± 1.85
`3.50a ± 1.69
`4.00ae ± 2.07
`1.63a ± 0.52
`
`16 week
`
`1.50a ± 0.54
`13.88b ± 2.23
`9.00cd ± 3.42
`7.00de ± 2.56
`3.63ae ± 1.92
`3.63ae ± 1.92
`3.38a ± 1.77
`
`32 week
`
`1.63a ± 0.52
`14.33b ± 3.29
`7.63cd ± 2.62
`6.50d ± 2.98
`3.00a ± 0.76
`3.13a ± 0.99
`2.00a ± 0.93
`
`Values are mean ± SD for n=8.
`a,b,c,d,e,f Values within column with different superscripts letters are significantly different (P<0.05).
`
`piroxicam were synergistic. Furthermore, the PD-1ex-
`pression is significantly higher in DMH-induced ade-
`nocarcinoma than in normal colonic mucosa and cor-
`related with the number of infiltrating lymphocytes,
`indicating the importance of PD-1 in tumor develop-
`ment. The study has demonstrated that traditionally
`used dairy based fermented foods could be used as a
`good medium or potential nutraceutical intervention
`for the delivery of probiotic strains of bacteria to
`achieve health-benefits to the consumers.
`
`Acknowledgements
`
`The authors wish to acknowledge the research fellowships
`and necessary facilities provided by UGC and ICAR, New
`Delhi, and NDRI, Karnal, India. The generous gift of L. plan-
`tarum Lp9 strain by Dr. V.K. Batish, Dairy Microbiology Di-
`vision, NDRI, Karnal, is also duly acknowledged.
`
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`
`Accepted: 27 May 2013
`Correspondence: Dr. Dheeraj Mohania,
`Associate Scientist Consultant,
`Department of Research, Sir Ganga Ram Hospital,
`Rajinder Nagar, New Delhi 110060, India.
`Tel. + 91-011-42251624
`Fax: +91-011-42251633
`E-mail: dmohania@gmail.com
`
`
`
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