1. 2 3 4 5 6 7 Cervical cancer Aim of The Present Study Lactobacilli Cervical cancer 8.

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7 Cervical cancer

Aim of The Present Study Lactobacilli Cervical cancer 8

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Experimental Procedures 10

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Grade 1 Grade 2 Grade 3 12

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Lactobacilli Molecular Identification, Primer Design For Multiplex PCR Jensenii F crispatus F crispatus R gasseri R acidophilus F gasseri F Jen, acid R 16S rRNA 23S rRNA paracasei R rhamnosus R para, rham F salivarius F reuteri F fermentum F salivarius R plantarum F plantarum R reuteri R fermentum R ISR 14

Cell wall & Cytoplasmic Extract Separation Extract Separation

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Number Nugent score 54Grade I (Normal) 60Grade II (Intermediate) 64Grade III (BV infected) 178Total 22

Group I 450 bp Group II 300 bp Group III 400 bp Group IV 350 bp Multiplex PCR Results for Lactobacilli Identification Group III L. paracasei 312bp L. rhamnosus 113bp 23

The Prevalence of Lactobacilli in Different Grades

MTT Results (Hela cell) 25 OD Cell Number

MTT Results (HNCF cell) 26 OD Cell Number

5% Lactic acid + MRS pH= 4 HCl + MRS pH= 4 27 Supernatants of: LGS: L.gasseri, LCS: L.crispatus, LGSN: LGS + NaOH, LCSN: LCS + NaOH, MRH: MRS + HCl, MRL: MRS + Lactic Acid, MRS :lactobacilli Media 5% HNCF cell Hela cell viability Concentration (%)

Vaginal Lactobacilli & Commercial Probiotics Supernatant MTT Result Supernatants of: LGS: L.gasseri LCS: L.crispatus LRS: L.rhamnosus LPS: L.paracasei LHS: L.helveticus LNS: L.acidophilus NCFM LAS: L.acidophilus candisis and MRS :lactobacilli Media as control Commercial Probiotics 28 pH=4pH=5 pH=6.5 Hela cell HNCF cell viability Concentration (%) viability Concentration (%)

29 Hela cell viability Concentration (%)

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LDH Assay; Apoptotis & Necrosis Ratio 32 Hela cell HNCF cell

Real-time PCR Results (Quality Controls) HPRT MP=78°C Bcl2 MP=87°C Fas MP=81°C Bax MP=87°C Caspase3 MP=81°C 33

34 P= 0.35 P= 0.66 P= 0.39 P= 0.73 P= 0.67P= 0.39

35 P= 0.35P= 0.34 P= 0.84

36 P= P= 0.31

Biomarker Assay; Expression Analysis of hCGb by ReaL-Time PCR 37 hCGb MP= 89°C TissueCgb expression placenta+ Cervical cancer+ Hela+ Uterine myometrium- Normal cervix-

38 P= 0.29P= 0.04 P= P= 0.04 P= 0.29 P= P= 0.31

Caspase3 & cgb Expression, (Cytoplasmic Extract & Cell Wall) 39

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Snapshot of Results 41

Prevalence of Vaginal Lactobacilli of Healthy Women; Similarities Between Our Study & Recent Findings 42

Comparison of Healthy Vaginal Flora With BV Infected in Different Studies & Our Study 43

Most Potent Cytotoxic Component 44

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Choi et al., 2006: L. acidophilus inhibits cancer cell proliferation but less effect on normal cells 46

Anti-apoptotic Effect of Lactobacilli Supernatants 47

Lactobacilli supernatant MKP1 Autophagy TGF β receptor Cell death 48 (Brion et al., 2011) (Iles, 2007)

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