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Purpose: Immunomodulatory as well as in vivo antimicrobial properties of Lactobacillus casei were investigated. L. casei was administered orally in Wistar rats. Methods: Enteropathogenic Escherichia coli were used. Both live and dead culture of L. casei were used. 2x10 8 CFU/ml of each culture was given to respective group daily from day 1st upto 28th days. Results: Beneficial effects such as increased adhesive property shown by L. casei, increased hemoglobin content and RBC and decreased WBC counts were observed in LL ( live L. casei ) and LD (dead L. casei ) group when compared with EC group (Administerd E. coli). Serum biomarkers like Serum glutamic pyruvic transaminase (SGPT), Serum glutamic oxaloacteic transaminase (SGOT) and total proteins (TP) were also decreased in LL and LD group. Moreover, L. casei in both live and dead form has decreased the production of pro-inflammatory cytokine (IL-6 and α TNF) while it has increased anti-inflammatory cytokine (IL-10). Histopathological analysis also confirmed the protective effect of L. casei against enteropathogenic E. coli. Conclusion: Present study revealed that L. casei consumption may provide gastrointestinal tract immunity against enteropathogenic E. coli Key words: Lactobacillus casei, Enteropathogenic Escherichia coli, IL-6, IL-10, TNF α Running Title: Lactobacillus casei modulate the expression of anti-inflammatory cytokines in gastrointestinal tract.
 Enteropathogens are the sole source of agents responsible for diarrhoeal sickness. These are the common cause of morbidity and even mortality. An increasing wide variety of pathogens are recognized as cause of serious illness, such as Escherichia coli, Campylobacter jejuni, Clostridium difficile, Salmonella, Shigella and Giardia.  E. coli are responsible for 200 million cases of diarrhoea each year, and death of 380,000 people mainly children in developing countries [1].  E. coli is the normal microflora of human gastrointestinal tract [2]. Establishment of normal bacterial flora is important to prevent growth of potential pathogens as well as, contribute to the adequate maturation of the immune system, since imbalances in the normal microflora composition have been consistently associated with several disease consequences. Intestinal mucosa also provides protective host defense against the food antigens and microorganisms in the gut lumen [3]. Intestinal mucosa also provides protective host defense against the constant presence of food antigens and microorganisms in the gut lumen. Various therapeutic treatments have been used through out of the world. Administration of probiotics, prebiotics or their association (symbiotics) has as a new and interesting strategy to modulate intestinal microbiota [4]. Consumption of Lactobacillus, Bifidobacterium and Enterococcus etc improves intestinal health [5-7]. Various studies have been performed for the immunomodulating properties of Lactobacillus against various pathogens like E. coli, Salmonella, Listeria [8 -15].  Lactobacillus species has been used previously for its antagonistic activity in in vivo and in vitro. The present study aimed at understanding the protective properties L. casei both in live and dead form against  enteropathogenic E. coli used to infect Wistar rats.
Reagents All the chemicals used in this study were of analytical grade. These were purchased from Peekay Chemicals & Glasswares and Scientific Systems & Chemicals, Bhopal, Madhya Pradesh. Cytokines assay kits were purchased from Ray Biotech, Norcross G A and DNA bio, Hyderabad, Andhra Pradesh India. Kits of Liver functional test were purchased from Acurex Private Ltd. Thane; India and Aggappe Diagnostics, Kerela; India.  Cultures: Lyophilized culture L. casei ATCC 334 was obtained from Hi Media (Navi Mumbai, India).  Culture of Enteropathogenic E coli was obtained culture collection from Department of Microbiology, Barkatullah University, Bhopal. Culture of E. coli was streaked on Nutrient Agar and L. casei on MRS agar. Animals 60 female Wistar rats, two months old (190-200 gm) were purchased from Institute of Industrial Toxicological Research Centre, Luckhnow, India. Animals were housed in Institute of Biomedical Sciences, Bundelkhand University, Jhansi, Uttar Pradesh. Stainless steel cages (34 x 47 x 18 cm) with soft wood shavings were used as bedding. Wistar rats were fed with normal commercial pellet diet (Amrut feed Ltd. India) and water ad libitum and maintained under laboratory conditions (Temperature 20-22°C, Relative humidity 60-70%, and 12 hrs light-dark cycles). All procedures and techniques used in this study were in accordance CPCSEA guidelines and were approved by the Departmental Ethics committee. Animals were randomly divided into five groups (n=12). Groups were designated as: LL group   :  Live culture of L. casei  in 1 ml distilled water LD group   :  Dead culture L. casei in 1 ml distilled water EC group   :  Live culture of E. coli in 1 ml distilled wa     LE group   :  Live culture of both L. cei and E. coli in 1 ml distilled water CON group  : Control group fed on 1ml distilled water Experimental schedule Dose 2x108 CFU /ml were suspended in 1ml distilled water and orally administered with syringe cannula to the prescribed group according to the protocol from day 1st to 28th day. For dead culture of L. casei, the method given by Bhatia K and Rani U [16] was used with some modification. 2x 108 CFU/ml was taken and suspended in 1ml distilled water and kept in water bath for 60 min at 90 °C. Control group was administered with 1 ml distilled water. These treatments were given up to day 28 th. On 29 th day animals were euthanized with the help of Di ethyl ether. Sample collection and processing Blood samples were drawn. Blood was mixed with EDTA for the hematological parameters. Serum was obtained by centrifuging the blood sample at 3500 RPM for 30 minutes at 4°C. Serum were isolated from blood and stored at -80°C for further investigations. Spleen and thymus were also taken. Piece of small intestine (2 cm long) were taken for histopathological analysis. Body weight Weight of all the animals were checked on day 0, 7th, 14 th, 21 th and 28th day. Spleen and Thymus of all the wistar rats were isolated and weighed on an electronic balance. Hematological parameters At 29 th blood sample was collected. Hematological parameters were analyzed. Red blood cell count (RBC count), White blood cell count (WBC Count) and Hemoglobin content (Hb %) were observed. Cytokines assay For the estimation of cytokines, serum was separated from blood. Estimation of IL-6 and TNF- α (Pro-inflammatory cytokines) and IL-10 (Anti-inflammatory cytokines) was done with the ELISA Reader (Lisa Plus). IL-6 and TNF-α (Ray Bio ®) and IL-10 (DNA BIO) ELISA kits were used. Assays were performed according to the instructions of manufacturer. Bacterial count in feces of Wistar rats Freshly voided fecal materials were collected at day 0 and 4 from each rat (1 gm/rat) of LE group. This was done to check the growth of L. casei in presence of E. coli. Faeces were homogenized in normal saline and serially diluted. 0.1 ml fecal sample was spread on MRS agar for the enumeration of L. casei and on MacConkey agar for the enumeration of E. coli. Plates were incubated at 37 Û« C for 24 hours and colony forming units were on recorded [15]. Biochemical test Some of major serum biochemical markers were assayed. Enzyme assays for liver functioning i,e Serum glutamic pyruvic transaminase (SGPT), Serum glutamic oxaloacteic transaminase (SGOT) and Total protein (TP) were performed with the help of Spectrophotometer (UV-Vi Shimadzu, Japan, 1601). Enzyme assays were done according to the instruction of manufacturer. Histopatological analysis Small intestines of wistar rats were removed. It was preserved in 10% formalin, dehydrated in increasing percentages of ethanol. These tissues were than Cleared in xylene for 2 hours for embedding. The embedded organs were sectioned using microtome and stained with Haematoxylin-eosin [17]. Statistical analysis The results were presented as a Mean ± SEM of twelve rats per group. The significance of the difference was evaluated by one-way ANOVA followed by Dunnettâs multiple comparisons test. Data were considered statistically significant if P
Effect of cultures on the diarrhoeal symptoms Rats of CON, LL and LD group were not showing any symptoms of diaarhoea till last day of experiment. Symptoms of diarrhoea were prominent in EC group. These animals were weak and lean in comparison to other treated animal group. LE group was showing mild symptoms of diarrhea but severities of symptoms were mild in comparison to the EC group animals. Animals of CON, LL and LD groups were healthy till last date of experiment. Effect of Cultures on Body weight In the first two weeks, the increment in body weight was almost similar in all the rats of all groups. However after two weeks, weight was gained by LL (211 ± 3.91 gm) and LD (203 ± 9.20 gm). It was significantly higher in LD in comparison to control group at P
Serum Glutamic Pyruvic Transaminase (SGPT or ALT) and Serum Glutamic Oxaloacetic Transaminase (SGOT or AST) levels were assayed. These are enzymes of the liver and their presence in the blood stream indicates that the walls of the liver have been compromised and that these enzymes are leaking into the blood stream. Value of SGPT were significantly highest in case of EC (143 ± 1.09 IU/L) group animals. SGOT levels were significantly different in case of LD and LE group and these were found to be (103.28 ± 1.06 IU/L) and (123.94 ± 0.90 IU/L) respectively. SGPT level was significantly different in case of LL group and it was (28.25 ± 0.84 IU/L) as compared to CON group. Concentration of Total Protein was significantly different in case of EC group (4.19 ± 2.06 gm %) and LL (5.55 ± 0.06 gm %) at the level of P< 0.0001. [Fig 2] Cytokines assay Serum was used for the assay of Cytokines IL-6, IL-10 and TNF α for all respective groups. It was found that for LL, LD, EC group, IL-6 values and TNF α were significantly different as compared to CON group. However in case of IL-10, only LL group was showing significantly different values as compared with the CON group. For all other groups concentration of IL-10 was not significantly different at the level of P
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