Survival and retention of the probiotic Lactobacillus casei LAFTI L26 in the gastrointestinal tract of the mouse

AIMS: This study aimed to develop methods for the detection of the probiotic Lactobacillus casei LAFTI L26 (L26) from mouse faeces, and to determine the survival and retention time of L26 in the mouse gastrointestinal tract. METHODS AND RESULTS: A selective medium, de Man Rogosa Sharpe (MRS) + bromocresol green + vancomycin (MGV), was designed for the isolation and enumeration of L26 from faecal samples of mice. PCR primers were designed to confirm the identity of L26-like colonies on MGV. These primers did not produce PCR products from related organisms that grew on MGV. Following the administration of L26 to BALB/c mice, faecal samples were collected and analysed using the designed methods. Survival studies showed viable L26 cells to be present in the faeces of mice for >48 h. CONCLUSIONS: Our results suggest that L26 is able to survive and be retained within the digestive tract of mice for at least 48 h following oral administration. SIGNIFICANCE AND IMPACT OF THE STUDY: MGV allows effective recovery of L26 from the background microbiota, including lactobacilli of mice. PCR was used to confirm that L26-like colonies were correctly identified as L26. Given the long retention time of L26 in the gastrointestinal tract of mice, it would appear that this probiotic strain may survive in the human gastrointestinal tract.     

Comparative survival of probiotic lactobacilli spray-dried in the presence of prebiotic substances

AIMS: Probiotic milk-based formulations were spray-dried with various combinations of prebiotic substances in an effort to generate synbiotic powder products. METHODS AND RESULTS: To examine the effect of growth phase and inclusion of a prebiotic substance in the feed media on probiotic viability during spray-drying, Lactobacillus rhamnosus GG was spray-dried in lag, early log and stationary phases of growth in reconstituted skim milk (RSM) (20% w/v) or RSM (10% w/v), polydextrose (PD) (10% w/v) mixture at an outlet temperature of 85-90 degrees C. Stationary phase cultures survived best (31-50%) in both feed media and were the most stable during powder storage at 4-37 degrees C over 8 weeks, with 30-140-fold reductions in cell viability at 37 degrees C in RSM and PD/RSM powders, respectively. Stationary phase Lact. rhamnosus GG was subsequently spray-dried in the presence of the prebiotic inulin in the feed media, composed of RSM (10% w/v) and inulin (10% w/v), and survival following spray-drying was of the order 7.1-43%, while viability losses of 20,000-90,000-fold occurred in these powders after 8 weeks' storage at 37 degrees C. Survival of the Lactobacillus culture after spray-drying in powders produced using PD (20% w/v) or inulin (20% w/v) as the feed media was only 0.011-0.45%. To compare different probiotic lactobacilli during spray-drying, stationary phase Lact. rhamnosus E800 and Lact. salivarius UCC 500 were spray-dried using the same parameters as for Lact. rhamnosus GG in either RSM (20% w/v) or RSM (10% w/v) and PD (10% w/v). Lact. rhamnosus E800 experienced approx. 25-41% survival, yielding powders containing approximately 10(9) CFU g(-1), while Lact. salivarius UCC 500 performed poorly, experiencing over 99% loss in viability during spray-drying in both feed media. In addition to the superior survival of Lact. rhamnosus GG after spray-drying, both strains experienced higher viability losses (570-700-fold) during storage at 37 degrees C over 8 weeks compared with Lact. rhamnosus GG. CONCLUSIONS: Stationary phase cultures were most suitable for the spray-drying process, while lag phase was most susceptible. The presence of the prebiotics PD and inulin did not enhance viability during spray-drying or powder storage. SIGNIFICANCE AND IMPACT OF THE STUDY: High viability (approximately 10(9) CFU g(-1)) powders containing probiotic lactobacilli in combination with prebiotics were developed, which may be useful as functional food ingredients for the manufacture of probiotic foods.     

Survivability of a probiotic Lactobacillus casei in the gastrointestinal tract of healthy human volunteers and its impact on the faecal microflora

AIM: The aim of this study was to measure the gastrointestinal survival of Lactobacillus casei and its impact on the gut microflora in healthy human volunteers. METHODS AND RESULTS: Twenty healthy volunteers took part in a double-blind placebo-controlled probiotic feeding study (10 fed probiotic, 10 fed placebo). The probiotic was delivered in two 65 ml aliquots of fermented milk drink (FMD) daily for 21 days at a dose of 8.6 +/- 0.1 Log(10)Lact. casei CFU ml(-1) FMD. Faecal samples were collected before, during and after FMD or placebo consumption, and important groups of faecal bacteria enumerated by fluorescent in situ hybridization (FISH) using oligonucleotide probes targeting the 16S rRNA. The fed Lact. casei was enumerated using selective nutrient agar and colony identity confirmed by pulsed field gel electrophoresis. Seven days after ingestion of FMD, the Lact. casei was recovered from faecal samples taken from the active treatment group at 7.1 +/- 0.4 Log(10) CFU g(-1) faeces (mean +/- SD, n = 9) and numbers were maintained at this level until day 21. Lact. casei persisted in six volunteers until day 28 at 5.0 +/- 0.9 Log(10) CFU g(-1) faeces (mean +/- SD, n = 6). Numbers of faecal lactobacilli increased significantly upon FMD ingestion. In addition, the numbers of bifidobacteria were higher on days 7 and 21 than on days 0 and 28 in both FMD fed and placebo fed groups. Consumption of Lact. casei had little discernible effect on other bacterial groups enumerated. CONCLUSIONS: Daily consumption of FMD enabled a probiotic Lact. casei strain to be maintained in the gastrointestinal tract of volunteers at a stable relatively high population level during the probiotic feeding period. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has confirmed that this probiotic version of Lact. casei survives well within the human gastrointestinal tract.     

儿童肠道双歧杆菌和乳杆菌种群结构分析

以21例2～5岁中国儿童的肠道菌群为研究对象,利用传统培养计数法和分子生物学技术,对此年龄段健康儿童的肠道菌群分布及其中关键益生菌的种群结构进行了定量研究。实验表明,儿童肠道厌氧菌的数量高达109CFUg(湿重),其肠道菌群的定植抗力(平均BE=2.38)较强;不同的个体之间所能检测到的关键益生菌的种类有所不同,一般能检测到其中的1～4种双歧杆菌和1～5种乳杆菌;长双歧杆菌和假小链双歧杆菌的平均数量多达107CFUg(湿重),检出率分别为90.48%和85.71%,为儿童肠道内双歧杆菌的优势菌种;L.mucosae和发酵乳杆菌的数量较多,平均为3.68log10CFUg(湿重)和3.97log10CFUg(湿重),检出率分别为71.43%和52.38%,为稳定定植于儿童肠道内的优势乳杆菌;不同种类的益生菌在不同样本之间的数量组成均存在有很大差异,双歧杆菌的样本差异为1.86～3.85,乳杆菌的为2.43～4.07。     

Importance of probiotics in the prevention and treatment of colorectal cancer

Colorectal cancer (CRC) remains one of the most common and deadly cancers. Intestinal gut microflora is important to maintain and contributes to several intestinal functions, including the development of the mucosal immune system, absorption of complex macromolecules, synthesis of amino acids/vitamins and the protection against pathogenic microorganisms. It is well known that the gut microbiota changes or dysbiosis may have an essential impact in the initiation and promotion of chronic inflammatory pathways and also have a profound different genetic and epigenetic alterations leading to dysplasia, clonal expansion, and malignant transformation. Probiotic bacteria has antitumor activity with various mechanisms such as nonspecific physiological and immunological mechanisms. This review evaluates the effects of microbiota and probiotics in clinical trials, in vitro and animal model studies that have explored how probiotic against cancer development and also discusses the possible immunomodulatory mechanisms. Several mechanisms alteration of the intestinal microflora; inactivation of cancerogenic compounds; competition with putrefactive and pathogenic microbiota; improvement of the host's immune response; antiproliferative effects via regulation of apoptosis and cell differentiation; fermentation of undigested food; inhibition of tyrosine kinase; reduces the enteropathogenic complications before and after colon cancer surgery and improve diarrhea and it's have been able to create the integrity of gut mucosal and have stimulatory effects on the systemic immune system and prevent the CRC metastasis. Research in clinical trials encouraging findings that support a role of probiotics in CRC prevention and improve the safety and effectiveness of cancer therapy even though additional clinical research is still necessary.     

Tracing location by applying Emerald luciferase in an early phase of murine endometriotic lesion formation

The pathogenesis of endometriosis has not been fully elucidated. We focused on the behavior of the ectopic endometrium, that is, the origin of the endometriotic lesion, before adhering to the peritoneal cavity. To observe lesion formation in the very early phase, we developed a novel endometriosis animal model using bioluminescence technology. We established a new transgenic mouse that expressed Emerald luciferase (ELuc) under the control of the CAG promoter. This transgenic mouse, called the CAG-ELuc mouse, showed strong bioluminescence emission; we succeeded in tracing the lesion location by the emission of ELuc. The accuracy of tracing by ELuc was high (57.7–100% of correspondence) and depended on the dosage of E2 administration. In the very early phase after transplantation, the process of lesion formation can be observed non-invasively and chronologically. We have verified that the preferred location of the uterus (transplanted grafts) was fixed immediately after the transplantation of the grafts.     

3种微生态制剂对大型溞存活、生殖和种群增长的影响

研究了3种微生态制剂(复合微生态制剂Ⅰ、复合微生态制剂Ⅱ和芽孢杆菌)对大型溞的急、慢性毒性影响,分析比较了3种微生态制剂的安全使用剂量以及对大型溞生殖和种群增长的影响.试验结果表明,3种微生态制剂对大型溞死亡率影响均显著(P < 0.05),从安全浓度来看,对大型溞的毒性作用依次为:复合微生态制剂Ⅱ>复合微生态制剂Ⅰ>芽孢杆菌;在安全浓度范围内,3种微生态制剂对大型溞亲代(P)的生殖和种群增长均有明显的促进作用,表现在随微生态制剂浓度的升高,P代的净生殖率(R0)和内禀增长率(rm)均显著增加(P < 0.05),其中以复合微生态制剂Ⅰ的效果最好,R0最高值为对照组的7倍,rm最高值为对照组的1.7倍;3种微生态制剂对大型溞子一代(F1)和子二代(F2)的生殖和种群增长均没有促进作用.     

Mesenchymal stem cells preconditioned by staphylococcal enterotoxin B enhance survival and bacterial clearance in murine sepsis model

Sepsis, a health-threatening progressive infectious disease, is the major cause of morbidity and mortality worldwide. Cell therapy using mesenchymal stromal cells (MSCs) is an innovative strategy with excessive therapeutic potential in the treatment of sepsis. Staphylococcal enterotoxin B (SEB) preconditioning aims to prolong the interval of survival of transplanted MSCs which induces the production of cytoprotective agents, anti-apoptotic and anti-inflammatory factors. The MSCs were preconditioned with an optimum dose of SEB (470 μmol/L). The expression levels of apoptosis genes and antibacterial activity of MSC and SEB-MSC and their conditioned medium (CM), as well as cell survival, were studied in vitro in an oxidative stress and serum deprivation condition. Following treatment of the septic mice with MSCs and SEB-MSCs, pro/anti-inflammatory cytokines, hematological factors, bacterial clearance and animal survival were assessed. The apoptotic and pro-inflammatory cytokine's genes expression was down-regulated while antibacterial peptides and anti-inflammatory cytokines were up-regulated in SEB-MSC–treated mice. The animal survival rates were improved; bacterial clearance was enhanced in the peritoneal fluids, blood and organs; aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were reduced in blood, compared with saline and MSCs alone. This research concludes that transplantation of SEB-MSCs presents improved therapeutic effects on a live bacterial model of sepsis.     

Predominance of a bacteriocin-producing Lactobacillus salivarius component of a five-strain probiotic in the porcine ileum and effects on host immune phenotype

Relative predominance of each of five probiotic strains was investigated in the ileum of weaned pigs, compared with that in feces, when administered in combination at c. 5 x 10(9) CFU day(-1) for 28 days. Probiotic was excreted at 10(6)-10(9) CFU g(-1) feces, while ileal survival ranged from 10(2) to 10(6) CFU g(-1) digesta. In contrast to the feces, where Lactobacillus murinus DPC6002 predominated, the bacteriocin-producing Lactobacillus salivarus DPC6005 dominated over coadministered strains both in the ileum digesta and in mucosa. Probiotic administration did not alter counts of culturable fecal Lactobacillus or Enterobacteriaceae but higher ileal Enterobacteriaceae were observed in the ileal digesta of probiotic-fed pigs (P<0.05). We observed decreased CD25 induction on T cells and monocytes (P<0.01) and decreased CTLA-4 induction (P<0.05) by the mitogen phytohemagglutinin on CD4 T cells from the probiotic group. Probiotic treatment also increased the proportion of CD4+ CD8+ T cells within the peripheral T-cell population and increased ileal IL-8 mRNA expression (P<0.05). In conclusion, superior ileal survival of L. salivarius compared with the other coadministered probiotics may be due to a competitive advantage conferred by its bacteriocin. The findings also suggest that the five-strain combination may function as a probiotic, at least in part, via immunomodulation.     

Generation of a syngeneic mouse model to study the intraperitoneal dissemination of ovarian cancer with in vivo luciferase imaging

In order to facilitate the discovery and investigation of anti‐cancer therapeutics under physiological conditions, we have engineered the ovarian cancer cell line, HM‐1/luc, in mice. This cell stably expresses firefly luciferase and produces light that can be detected using an in vivo imaging system (IVIS). Parental HM‐1 cells cause severe carcinomatous peritonitis to B6C3F1 mice, but not to C57BL6 mice. Established HM‐1/luc cells showed pathologically similar findings to HM‐1 cells. HM‐1/luc cells were injected into the peritoneal cavity of B6C3F1 mice and IVIS 2000 was conducted weekly after inoculation to monitor intra‐peritoneal tumor growth. The mice were divided into three groups: non‐CDDP‐treated (control) and CDDP‐treated (0.2 and 0.4 mg). A disease‐suppressive effect of the CDDP was reflected by the significantly prolonged survival of the CDDP‐treated mice (control 23 ± 1.9 days, CDDP 0.2 mg 29.6 ± 2.9 days; p < 0.05); the total photon and area of flux were decreased. The optical imaging of intraperitoneal tumors via in vivo bioluminescence is effective for noninvasive monitoring and semi‐quantitative analysis. Our syngeneic mouse model has the relevant clinical features of ovarian cancer, which makes it a useful model for developing new ovarian cancer therapies. Copyright © 2009 John Wiley & Sons, Ltd.     

小鼠体内外受精植入前胚胎全基因组甲基化模式比较

为考察体外受精、操作及培养环境对体外受精的小鼠植入前胚胎全基因组DNA甲基化模式的影响,本研究以体内受精的植入前胚胎作为对照,采用间接免疫荧光法检测小鼠体内外受精植入前胚胎基因组DNA甲基化模式.实验结果表明,体外受精各期植入前胚胎呈现出与之相应时期的体内受精植入前胚胎不同的DNA甲基化模式和水平,原核期甲基化水平较高,2-4-、8-细胞期明显降低,而桑葚胚和囊胚期又略有升高.各期体外受精植入前胚胎的基因组DNA甲基化水平都比同时期体内受精胚胎的甲基化水平低.本实验结果部分显示了体外受精、操作及培养环境可能对正常的DNA甲基化模式产生影响,造成体外受精植入前胚胎甲基化模式异常.     

Development of an insect model for the in vivo pathogenicity testing of yeasts

Conventional in vivo assays to determine the relative pathogenicity of yeast isolates rely upon the use of a range of mammalian species. The purpose of the work presented here was to investigate the possibility of using an insect (Galleria mellonella) as a model system for in vivo pathogenicity testing. The haemolymph of G. mellonella larvae was inoculated with PBS containing different concentrations of stationary phase yeasts of the genus Candida by injection at the last pro-leg. Larvae were incubated at 30 degrees C and monitored over 72 hours. Results indicate that G. mellonella can be killed by the pathogenic yeast Candida albicans and by a range of other Candida species but not to a significant extent by the yeast Saccharomyces cerevisiae. The kill kinetics for larvae inoculated with clinical and laboratory isolates of C. albicans indicate the former class of isolates to be more pathogenic. Differences in the relative pathogenicity of a range of Candida species may be distinguished using G. mellonella as a model. This work indicates that G. mellonella may be employed to give results consistent with data previously obtained using mammals in conventional in vivo pathogenicity testing. Larvae of G. mellonella are inexpensive to culture, easy to manipulate and their use may reduce the need to employ mammals for routine in vivo pathogenicity testing with a concomitant reduction in mammalian suffering.     

Dolichyl phosphate linked sugars as intermediates in the synthesis of yeast mannoproteins: an in vivo study

Freshly prepared protoplasts of Saccharomyces cerevisiae X 2180 incorporate [³H]mannose and [¹⁴C]glucose for about 30 min into glycolipids and mannoproteins. Among the radioactive glycolipids formed dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides have been identified. The oligosaccharides released by weak acid from the dolichyl pyrophosphate were treated with endo-N-acetylglucosaminidase H and separated by gel filtration on Bio-Gel P-4. The largest oligosaccharide obtained corresponded exactly in size to Glc₃Man₉GlcNAc₁ the compound formed also in animal tissues. Other oligosaccharides released from dolichyl pyrophosphate in addition to the glucose containing ones were mainly Man₉GlcNAc₁ and Man₈GlcNAc₁. No mannosyl oligosaccharide corresponding in size to the total inner core region found in native mannoproteins could be detected in a lipid-bound form.The radioactive dolichyl pyrophosphate oligosaccharides were formed transiently; after 40 min only about 40% of the maximal radioactivity was observed in this fraction. In the presence of cycloheximide this decrease did not take place.It is concluded that the dolichol pathway of N-glycosylation of glycoproteins in yeast cells is very similar, if not identical, to the reaction sequence worked out for animal cells.Dedicated to Professor Dr. Otto Kandler on his 60th birthday     

Two new C3H mouse ascites tumor cell lines capable of proliferation in vivo and in suspension culture: Morphological,karyological, kinetic,and immunological properties

Summary The establishment of mouse tumor cell lines capable of proliferating in vivo and in suspension culture was undertaken. The MM-46 tumor line, initiated from primary mammary carcinoma arising in a C3H/He mouse, was maintained for over 100 generations in the peritoneal cavities of syngenic mice. At the 50th generation of the tumor suspension, cultures were initiated. The established cell lines, designated TMT-1 and TMT-2, were characterized in vitro and in vivo. The morphological finding indicated that TMT-1 and TMT-2 cells from mice closely resembled the MM-46 tumor cells. The oncogenic potential of the cultured cells was comparable to that of the original ascites tumor. The population doubling time of TMT-1 and TMT-2 cell lines was about 12 hr in mice, whereas the population doubling time of both cell lines lengthened to 20 hr in suspension culture. The increase of doubling time in culture was due to the prolongation of the G₁ period. The cell lines, TMT-1 and TMT-2, whether from culture or mice, possessed colony forming ability in soft agar medium. The colony forming ability of the cells decreased gradually through in vivo passages but it recovered upon recultivation of the cells from mouse to culture. Chromosome analysis and cytotoxicity test by anti-MM antiserum indicated that TMT-1 and TMT-2 cell lines closely resembled and had been derived from MM-46 tumor line. Therefore, it is possible to assay cell survival in vitro after in vivo experiments on these cells.     

Validation of the dorsal air pouch model to predict and examine immunostimulatory responses in the gut

Aims:  To validate the use of the air pouch system to predict and examine early immune responses induced by the presumptive probiotics Lactobacillus paracasei subsp. paracasei B112, DC205, DC215 and DC412 strains in the gut mucosa.
Methods and Results:  Only the DC412 strain interacted strongly with the cells forming the air pouch lining tissue and induced early innate immune responses such as polymorphonuclear (PMN) cell recruitment, phagocytosis and tumour necrosis factor alpha (TNF-α) production that equal the respective responses induced by the probiotic Lactobacillus acidophilus NCFB 1748. The strains exhibiting strong immunoregulatory activity in the air pouch also interacted strongly with the gut-associated lymphoid tissue (GALT). The strain DC412 exerts its effect on the intestine through stimulation of Toll-like receptor (TLR)2/TLR4-mediated signalling events leading to secretion of a certain profile of cytokines in which gamma interferon (IFN-γ), TNF-α, interleukin (IL)-6 and IL-10 are included. The probiotic Lact. acidophilus NCFB 1748 induces the same cytokine profile in addition to IL-12B, and this response is potentially mediated by the synergy of TLR2 and TLR9.
Conclusion:  The strain DC412 possesses the in vitro and in vivo characteristics of a probiotic micro-organism.
Significance and Impact of the Study:  The dorsal mouse or rat air pouch may be used as an alternative and rapid method for the initial discrimination and selection of potential probiotic Lactobacillus strains.     

Evaluation of the anti-Toxoplasma gondii Activity of Hederagenin in vitro and in vivo

Toxoplasma gondii infection is widespread worldwide, not only posing a serious threat to human food safety and animal husbandry, but also endangering human health. The selectivity index was employed to measure anti-T. gondii activity. Hederagenin (HE) exhibited potent anti-T. gondii activity and low cytotoxicity. For this reason, HE was selected for in vivo experiments. HE showed 64.8%±13.1% inhibition for peritoneal tachyzoites in mice, higher than spiramycin 56.8%±6.0%. Biochemical parameters such as alanine aminotransferase, aspartate aminotransferase, glutathione, and malondialdehyde, illustrated that HE was a good inhibitor of T. gondii in vivo. This compound was also effective in relieving T. gondii-induced liver damage. Collectively, it was demonstrated that HE had potential as an anti-T. gondii agent.