Search

Two Gram-stain-positive, rod-shaped, non-motile, non-spore-forming, catalase-negative bacteria, designated strains SG162T and NK01, were isolated from Japanese rice grain silage and total mixed ration silage, respectively. They were initially identified as Lactobacillus buchneri based on the 16S rRNA gene sequence similarities. However, the two strains were separated into a distinct clade from L. buchneri DSM 20057T (=JCM 1115T) through whole-genome sequence-based characterization, forming an infraspecific subgroup together with strains CD034 and S42, whose genomic sequences were available in the public sequence database. Strains within the subgroup shared 99.4–99.7 % average nucleotide identity (ANI) and 97.5–99.0 % digital DNA–DNA hybridization (dDDH) with each other, albeit 96.9–97.0 % ANI and 76.0–76.6 % dDDH against DSM 20057T. Strains SG162T and NK01 could utilize more substrates as sole carbon sources than DSM 20057T, potentially owing to the abundance of genes involved in carbon metabolism, especially the Entner–Doudoroff pathway. The inability of γ-aminobutyric acid (GABA) production was evidenced by the lack of glutamate decarboxylase and glutamate/GABA antiporter genes in the new subgroup strains. Strain SG162T grew at 10–45 °C (optimum, 30 °C), pH 3.5–8.0, and 0–8 % (w/v) NaCl. Its genomic DNA G+C content was 44.1 mol%. The predominant fatty acids were C16 : 0, C19 : 0 cyclo ω8c, and summed feature 8. On the basis of the polyphasic characterization findings, strains SG162T and NK01 represent a novel subspecies of L. buchneri , for which the name Lactobacillus buchneri subsp. silagei subsp. nov. is proposed. The type strain is SG162T (=JCM 32599T=DSM 107969T), and strains CD034 and S42 are also transferred to L. buchneri subsp. silagei.

More than 2,000 varieties of cheese currently exist in the world, and cheese manufacture continues to flourish. To develop the cheese ripening process, additional ingredients are used during cheese production. In this study, the effect of sake lees as an additional ingredient on the fermentation of cheese using Aspergillus oryzae (koji mold), known as koji cheese, was investigated. Aspergillus oryzae is used in the fermentation of Japanese traditional foods, such as sake and soy sauce, given its strong enzymatic activities, as well as in cheese production (i.e., koji cheese). Sake lees, a by-product of the fermentation of rice with A. oryzae and yeasts in the sake brewing process, contains various metabolites, such as amino acids. Here, supplementation with sake lees enhanced the activities of lactic acid bacteria and affected the color of the cheese. Metabolome analysis revealed that sake lees altered the balance of carbohydrates and fatty acids in the cheese. Remarkably, supplementation with sake lees enhanced the production of umami-enhancing γ-glutamyl (kokumi-active) peptides. This study suggests that a new type of cheese can be produced using A. oryzae and sake lees, and information on the synergistic effects of A. oryzae and sake lees will aid the development of cheese production.

In this follow-up of the R-NAC-01 study, we assessed the long-term oncological benefit of four courses of modified leucovorin, 5-fluorouracil (FU), and oxaliplatin (mFOLFOX6) chemotherapy before rectal surgery.In this prospective, multicenter study (UMIN 000012559) involving 11 hospitals in Japan, patients with lower rectal cancer underwent four cycles of mFOLFOX6 chemotherapy and subsequent surgery within four to six weeks. The 3-year recurrence-free survival and local recurrence rates were then reported.Of 41 patients (36 males, 5 females; mean age: 60.8 years old) who received 4 courses of chemotherapy, 40 underwent total mesorectal excision, and 1 underwent total pelvic exenteration. R0 resection was achieved in 40 patients, but none showed a pathological complete response. Twenty-nine patients received adjuvant chemotherapy for an average of 4 months. The 3 year recurrence-free survival and local recurrence rates in patients undergoing curable resection were 72.8% and 8.5%, respectively. cStage III patients with adjuvant chemotherapy had a significantly higher 3 year recurrence-free survival than those without adjuvant chemotherapy (76.6 vs. 40.0%, log-rank p = 0.03).Four courses of mFOLFOX6 chemotherapy before surgery may be a promising treatment strategy for locally advanced rectal cancer. Adjuvant chemotherapy might be needed for cStage III patients, even after four courses of neoadjuvant mFOLFOX6.

Whole-transcriptome analysis of aerobic stress response gene in Enterococcus gilvus was performed using RNA-sequencing to identify carotenoid-based stress response genes in lactic acid bacteria. The expression of gene responsible for pyruvate dehydrogenase complex synthesis was highly upregulated after aerobic treatment. In addition, the expression of transcriptional regulator spx and genes encoding UvrABC system protein was also upregulated.

CD4+T cells mediate antigen-specific allograft tolerance, but die in culture without activated lymphocyte derived cytokines. Supplementation of the media with cytokine rich supernatant, from ConA activated spleen cells, preserves the capacity of tolerant cells to transfer tolerance and suppress rejection. rIL-2 or rIL-4 alone are insufficient to maintain these cells, however. We observed that activation of naive CD4+CD25+FOXP3+Treg with alloantigen and the Th2 cytokine rIL-4 induces them to express interleukin-5 specific receptor alpha (IL-5Rα) suggesting that IL-5, a Th2 cytokine that is produced later in the immune response may promote tolerance mediating Treg. This study examined if recombinant IL-5(rIL-5) promoted survival of tolerant CD4+, especially CD4+CD25+T cells. CD4+T cells, from DA rats tolerant to fully allogeneic PVG heart allografts surviving over 100days without on-going immunosuppression, were cultured with PVG alloantigen and rIL-5. The ability of these cells to adoptively transfer tolerance to specific-donor allograft and suppress normal CD4+T cell mediated rejection in adoptive DA hosts was examined. Tolerant CD4+CD25+T cells' response to rIL-5 and expression of IL-5Rα was also assessed. rIL-5 was sufficient to promote transplant tolerance mediating CD4+T cells' survival in culture with specific-donor alloantigen. Tolerant CD4+T cells cultured with rIL-5 retained the capacity to transfer alloantigen-specific tolerance and inhibited naive CD4+T cells' capacity to effect specific-donor graft rejection. rIL-5 promoted tolerant CD4+CD25+T cells' proliferation in vitro when stimulated with specific-donor but not third-party stimulator cells. Tolerant CD4+CD25+T cells expressed IL-5Rα. This study demonstrated that IL-5 promoted the survival of alloantigen-specific CD4+CD25+T cells that mediate transplant tolerance.

Over the past few decades, beef producers in Japan have improved marbling in their beef products. It was recently reported that marbling is not well correlated with palatability as rated by Japanese consumers. This study sought to identify the consumer segments in Japan that prefer sensory characteristics of beef other than high marbling.; Results: Three Wagyu beef, one Holstein beef and two lean imported beef longissimus samples were subjected to a descriptive sensory test, physicochemical analysis and a consumer (n = 307) preference test. According to consumer classification and external preference mapping, four consumer segments were identified as 'gradual high-fat likers', 'moderate-fat and distinctive taste likers', 'Wagyu likers' and 'distinctive texture likers'. Although the major trend of Japanese consumers' beef preference was 'marbling liking', 16.9% of the consumers preferred beef samples that had moderate marbling and distinctive taste. The consumers' attitudes expressed in a questionnaire survey were in good agreement with the preference for marbling among the 'moderate-fat and distinctive taste likers'.; Conclusion: These results indicate that moderately marbled beef is a potent category in the Japanese beef market. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

Experimental autoimmune neuritis (EAN) induced by peripheral nerve myelin (PNM) is self-limiting and re-immunization with PNM does not re-activate disease. This study showed inhibition of EAN by CD4

Therapy with alloantigen-specific CD4+CD25+ T regulatory cells (Treg) for induction of transplant tolerance is desirable, as naïve thymic Treg (tTreg) are not alloantigen-specific and are weak suppressor cells. Naïve tTreg from DA rats cultured with fully allogeneic PVG stimulator cells in the presence of rIL-2 express IFN-gamma receptor (IFNGR) and IL-12 receptor beta2 (IL-12Rβ2) and are more potent alloantigen-specific regulators that we call Ts1 cells. This study examined additional markers that could identify the activated alloantigen-specific Treg as a subpopulation within the CD4+CD25+Foxp3+Treg. After culture of naïve DA CD4+CD8−CD25+T cells with rIL-2 and PVG alloantigen, or rIL-2 without alloantigen, CD8α was expressed on 10–20% and CD8β on

Fermented dairy products comprise many functional components. Our previous study using fermented milk showed that the γ-aminobutyric acid (GABA)-producing Lactococcus lactis 01-7 strain can produce unique metabolites such as antihypertensive peptides, whereas this study was designed to find the unique metabolites in GABA-rich cheese using the 01-7 strain. Metabolites between cheese ripening with the non-GABA-producing L. lactis 01-1 strain (control) and GABA-rich cheese ripening with a mixture of 01-1 and 01-7 strains were compared. GABA and ornithine were detected in GABA-rich cheese using an amino acid analyzer and citrate was detected in the control cheese using HPLC. Metabolome analysis using LC-MS showed that peptides with unknown function and those with antihypertensive activity were higher in the GABA-rich cheese than in the control cheese. Further analysis of the amount of the YLGY derivatives showed that the amount of YL in the GABA-rich cheese was lower than that in the control. PRACTICAL APPLICATIONS: Clarification of metabolites in cheese contributes to the improvement of cheese ripening, thereby providing consumers with unique cheese with good nutritional and functional characteristics. The use of the 01-7 strain as a cheese starter might provide a functional cheese with antihypertensive-, antioxidative-, and anxiolytic-like activities.

γ-Aminobutyric acid (GABA) is one of the most important functional components in fermented foods because of its physiological functions, such as neurotransmission and antihypertensive activities. However, little is known about components other than GABA in GABA-rich fermented foods. A metabolomic approach offers an opportunity to discover bioactive and flavor components in fermented food. To find specific components in milk fermented with GABA-producing Lactococcus lactis 01-7, we compared the components found in GABA-rich fermented milk with those found in control milk fermented without GABA production using capillary electrophoresis time-of-flight mass spectrometry. A principal component analysis score plot showed a clear differentiation between the control milk fermented with L. lactis 01-1, which does not produce GABA, and GABA-rich milk fermented with a combination of L. lactis strains 01-1 and 01-7. As expected, the amount of GABA in GABA-rich fermented milk was much higher (1,216-fold) than that of the control milk. Interestingly, the amount of Orn was also much higher (27-fold) than that of the control milk. Peptide analysis showed that levels of 6 putative angiotensin-I-converting enzyme (ACE)-inhibitory peptides were also higher in the GABA-rich fermented milk. Furthermore, ACE-inhibitory activity of GABA-rich fermented milk tended to be higher than that of the control milk. These results indicate that the GABA-producing strain 01-7 provides fermented milk with other functional components in addition to GABA.

We investigated the expression profiles of proteolysis-related genes in Lactobacillus paracasei EG9, a non-starter lactic acid bacterial strain used as an adjunct starter in Gouda-type cheese production. The amino acid sequences of previously reported proteolytic enzymes in lactic acid bacteria were matched against the EG9 genome by translated BLAST search, and 31 candidate genes, including those coding for peptidases, oligopeptide transporters, and cell-envelope proteinases, were listed. The relative expression levels of these genes were evaluated at 0 and 30 days of cheese ripening. On day 0, 27 genes showed significantly up-regulated expression compared with the genes in the stationary phase of the reference cultured cells. On day 30, 25 genes were found to be significantly down-regulated. Our findings suggest that the proteolysis-related genes in EG9 play an important role in the proteolytic process from casein degradation through peptide uptake to free amino acid production during cheesemaking.

Experimental autoimmune neuritis (EAN) induced by peripheral nerve myelin (PNM) is self-limiting and re-immunization with PNM does not re-activate disease. This study showed inhibition of EAN by CD4+CD25+T cells both from sensitized hosts or from naive hosts after ex-vivo activation by PNM and rIL-2. Transfer of naive CD4+CD25+T cells has no effect on EAN, nor did naive CD4+CD25+T cells activated with rIL-2 and renal tubular antigen. Culture of naive CD4+CD25+Treg with rIL-2 and PNM induced mRNA for the IFN-gamma receptor. We showed naive CD4+CD25+T cells activated by specific auto-antigen and rIL-2 produced more potent antigen-specific Treg that may have therapeutic potential.

The aim of this study was to assess the safety of rectal surgery after 5-fluorouracil–leucovorin–oxaliplatin chemotherapy (FOLFOX6). This was a prospective, multicenter study in 11 Japanese hospitals. We included patients with rectal cancer who received 4 courses of modified FOLFOX6 (mFOLFOX6) before rectal surgery and examined the postoperative complication rate, the clinicopathological response, and the rate of chemotherapy-related adverse events (UMIN 000012559). The study population included 36 men and 5 women. The average age of the patients was 60.8 years and the average body mass index was 23.1 kg/m2. After 4 courses of chemotherapy, grade 2 peripheral nerve disorder and other grade 3 adverse events were seen in 3 patients each (7.3%). Twenty-eight (73.7%) and 8 (21.1%) patients underwent low anterior resection and abdominoperineal resection, respectively. The pelvic nerves were preserved in 35 patients. Surgical morbidity (grade ≥ 3) occurred in 4 patients (10.5%). Anastomotic leakage occurred after surgery in 2 patients (7.1%). No patients achieved pathologically complete remission. However, downstaging of the clinical stage and N stage was seen in 17 (41.5%) and 22 (53.7%) patients, respectively. Surgery after four courses of mFOLFOX6 chemotherapy can be a safe and promising strategy for patients with locally advanced rectal cancer.

Enterococcus gilvus CR1, isolated from raw cow’s milk, can produce carotenoids. The complete genome sequence of this strain was determined using the PacBio RS II platform., Enterococcus gilvus CR1, isolated from raw cow’s milk, can produce carotenoids. The complete genome sequence of this strain was determined using the PacBio RS II platform. The assembly was found to contain a circular chromosome, including carotenoid biosynthesis genes, and comprises 2,863,043 bp, with a G+C content of 41.86% and three plasmids.

A 72-year-old woman noted a mass in the left breast about 5 years ago, but she did not consult a medical institution. She was taken in the ambulance and hospitalized to our department due to severe anemia and malnutrition. A computed tomography( CT)scan indicated an 18×12 cm tumor in her left breast. A fiborsarcoma protuberance was suspected based on needle core biopsy results. Simple mastectomy was performed to control hemorrhage and infection. The resected tumor weighed 2.6 kg. The pathological diagnosis was a malignant phyllodes tumor. We report a patient with giant malignant phyllodes tumor associated with severe anemia.

Lactococcus lactis subsp. lactis G50 is a strain with immunostimulating activity, isolated from Napier grass ( Pennisetum purpureum ). We determined the complete genome sequence of this strain using the PacBio RS II platform. The single circular chromosome consists of 2,346,663 bp, with 35.03% G+C content and no plasmids.

Various bacteria have been found in raw cow's milk, and identifying milk microflora and its functions is critical for maintaining cow health and farm hygiene. Although studies on pathogens and spoilage bacteria in milk have been widely reported, the relationship between milk bacteria, including nonpathogenic bacteria, and the bovine udder is poorly understood. We investigated milk microflora over 1 year using a culture-dependent method and culture-independent analysis by denaturing gradient gel electrophoresis. Among 240 isolates, Lactococcus lactis (81/240) was predominant. The predominant genera were Lactococcus, Stenotrophomonas, Microbacterium, Chryseobacterium, Serratia and Pseudomonas. Among seven strains belonging to these predominant genera, two strains of L. lactis (ssp. lactis and ssp. cremoris) exhibited the highest adherence to bovine mammary gland epithelial cells (BMECs) derived from the bovine udder; 3.4 % of the inoculated bacteria adhered to BMECs. This was followed by Serratia sp. (1.6 %), Microbacterium sp. (0.8 %), Stenotrophomonas maltophilia (0.5 %), Pseudomonas sp. (0.3 %) and Chryseobacterium sp. (0.1 %). The two L. lactis isolates exhibited higher adherence to BMECs than type strains and isolates of various origins.

Strains of Lactococcus lactis are present in various natural environment or fermented products, and have been used as starters. In this review, we firstly discuss the immunomodulatory effects of Lactococcus strains from the perspective of their strain-specificity. Forty-six different Lactococcus strains were investigated for their ability to induce cytokine production in the murine macrophage-like cell line, J774.1. Strain-specificity and heat stability related to the ability of Lactococcus cells to induce interleukin (IL)-6, IL-12 and TNF-α, and induce cell necrosis or apoptosis were discussed. Secondly, to examine the immunomodulatory effects of Lactococcus cells in vivo, the effects of oral administration of four strains were investigated for their ability to regulate IgE production in ovalbumin-sensitized mice. Among the four strains, only L. lactis strain C59 showed a significant reduction in the total IgE antibody levels in serum. This suppressive effect on IgE antibody production was lost when strain C59 was heat-killed. Experiments using splenocytes of mice administered with live strain C59 indicated that the suppression of IgE antibody production by live strain C59 was due to the suppression of IL-4 production. The strain-specific immunomodulatory effects of Lactococcus strains are discussed. Discipline: Food Additional key words: apoptosis, IgE antibody, interleukin, lactic acid bacteria, macrophage

CD4

Over the past few decades, beef producers in Japan have improved marbling in their beef products. It was recently reported that marbling is not well correlated with palatability as rated by Japanese consumers. This study sought to identify the consumer segments in Japan that prefer sensory characteristics of beef other than high marbling.Three Wagyu beef, one Holstein beef and two lean imported beef longissimus samples were subjected to a descriptive sensory test, physicochemical analysis and a consumer (n = 307) preference test. According to consumer classification and external preference mapping, four consumer segments were identified as 'gradual high-fat likers', 'moderate-fat and distinctive taste likers', 'Wagyu likers' and 'distinctive texture likers'. Although the major trend of Japanese consumers' beef preference was 'marbling liking', 16.9% of the consumers preferred beef samples that had moderate marbling and distinctive taste. The consumers' attitudes expressed in a questionnaire survey were in good agreement with the preference for marbling among the 'moderate-fat and distinctive taste likers'.These results indicate that moderately marbled beef is a potent category in the Japanese beef market. © 2017 Society of Chemical Industry.

In order to understand the relationship between lactic acid bacteria (LAB) species and silage fermentation, a total of 65 LAB strains isolated from mixed pasture of timothy (Phleum pratense L.) and orchardgrass (Dactylis glomerata L.), and its badly preserved silages were subjected to phenotypic and genetic analysis. According to these analyses, the isolates were divided into 13 groups, including Enterococcus gallinarum, Lactobacillus acidipiscis, L. coryniformis subsp. coryniformis, L. coryniformis subsp. torquens, L. curvatus, L. paraplantarum, L. plantarum subsp. argentoratensis, L. plantarum subsp. plantarum, L. sakei subsp. carnosus, Lactococcus garvieae, Lactococcus lactis subsp. cremoris, Leuconostoc pseudomesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus, Weissella hellenica, Weissella paramesenteroides and Carnobacterium divergens. This is the first report to document that C. divergens, L. acidipiscis, L. sakei subsp. carnosus, L. garvieae, phenotypically novel L. lactis subsp. cremoris, E. gallinarum and W. hellenica are present in vegetative forage crops. L. plantarum group strains were most frequently isolated from the badly preserved silages. Some isolates showed a wide range of growth preferences for carbohydrate utilization, optimal growth pH and temperature in vitro, indicating that they have a high growth potential. These results are useful in understanding the diversity of LAB associated with decayed silage of timothy and orchardgrass.

Twenty-three lactic acid bacteria (LAB) isolated from three cultivars (Akiaoba, Nagahahikari and Tachiwase) of Italian ryegrass (Lolium multiflorum Lam.) silage were precisely characterized by a combination of phenotypic tests, genotypic 16S ribosomal DNA sequencing and rapid PCR-based analyses, focusing on their useful phenotypes for silage preparation as inoculants. We successfully identified both at the species and subspecies levels: phenotypically novel Lactococcus lactis subsp. lactis, Lactobacillus brevis, Lactobacillus coryniformis subsp. torquens, Lactobacillus curvatus, Lactobacillus plantarum subsp. plantarum, Lactobacillus sakei subsp. carnosus, Leuconostoc mesenteroides subsp. dextranicum and Pediococcus parvulus. This is the first report to elucidate the presence of Lactobacillus coryniformis ssp. torquens and Leuconostoc mesenteroides subsp. dextranicum in Italian ryegrass silages. Physiological and biochemical tests revealed that phenotypic characteristics are different among the different strains of the same species and subspecies, and that the isolates show unique and diverse phenotypes related to fermentation factors, such as available carbohydrates, optimal growth pH and temperature. These results suggest that, for various well-preserved silage preparations, the isolates may be useful in producing novel inoculants corresponding to their optimally climatic and ecological niches.

Lactobacillus paracasei EG9 is a strain isolated from well-ripened cheese and accelerates free amino acid production during cheese ripening. Its complete genome sequence was determined using the PacBio RS II platform, revealing a single circular chromosome of 2,927,257 bp, a G+C content of 46.59%, and three plasmids.

Lactobacillus plantarum LQ80 is a strain isolated from liquid feed for pigs. We determined the complete genome sequence of this strain using the PacBio RS II platform. LQ80 contained a single circular chromosome of 3,230,192 bp, with 44.66% G+C content and seven plasmids.

Variations in milk microflora caused by changes in the cow feeding environment (from inside to outside grazing) were analyzed using a molecular-based approach comprising denaturing gradient gel electrophoresis and real-time PCR. After 8 d of outside grazing, changes in milk microflora were observed. Denaturing gradient gel electrophoresis analysis showed that the predominant bacterial group in the milk belonged to the Lactobacillus species during the experimental period, whereas the genus Staphylococcus gained in numbers during the outside grazing period in addition to Lactobacillus. To investigate the quantitative dynamics of staphylococci, real-time PCR was performed using staphylococcal-specific primers. Real-time PCR analysis revealed that the population of staphylococci increased during the outside grazing period. Our combined denaturing gradient gel electrophoresis and real-time PCR approach enables precise monitoring of the dynamics of both total bacteria and specific bacterial species in milk. Our results provide information on grazing management and the manufacture of dairy products.

Meat 'reddening' by bacteria was observed in chilled beef. To identify the reddening bacteria, isolates were inoculated onto beef and the changes in CIE L*a*b* values monitored. As a result, two Pseudomonas spp., including Pseudomonas fragi which is commonly observed in raw meat, were selected and identified as reddening bacteria. The reddening was coincidentally occurred with the appearance of slime, and the increase in thiobarbituric acid-reactive substances (TBARS) was simultaneously suppressed. In myoglobin-containing nutrient broth, it is shown spectroscopically that P. fragi converted metmyoglobin into deoxymyoglobin. It was concluded that the meat reddening was due to the formation of deoxymyoglobin, induced by the very-low-oxygen tension brought about by Pseudomonad's oxygen consumption: This oxygen depletion simultaneously suppressed TBARS increase.

Bile resistance is one of the basic characteristics of probiotic bacteria. The aim of this study was to investigate the characteristics of bile resistance in lactococci by studying the relationship between bile resistance and cellular fatty acid composition in lactococcci grown on different media. We determined the bile resistance of 14 strains in lactose-free M17 medium supplemented with either glucose only (GM17) or lactose only (LM17). Gas chromatographic analyses of free lipids extracted from the tested strains were used for determining their fatty acid composition. A correlation analysis of all strains grown in both media revealed significant positive correlations between bile resistance and relative contents of hexadecanoic acid and octadecenoic acid, and negative correlations between bile resistance and relative contents of hexadecenoic acid and C-19 cyclopropane fatty acid. It is also a fact that the fatty acids associated with bile resistance depended on species, strain, and/or growth medium. In L. lactis subsp. cremoris strains grown in GM17 medium, the bile-resistant strains had significantly more octadecenoic acid than the bile-sensitive strains. In LM17 medium, bile-resistant strains had significantly more octadecenoic acid and significantly less C-19 cyclopropane fatty acid than the bile-sensitive strains. In L. lactis subsp. lactis strains, bile resistances of some of the tested strains were altered by growth medium. Some strains were resistant to bile in GM17 medium but sensitive to bile in LM17 medium. Some strains were resistant in both media tested. The strains grown in GM17 medium had significantly more hexadecanoic acid and octadecenoic acid, and significantly less tetradecanoic acid, octadecadienoic acid and C-19 cyclopropane fatty acid than the strains grown in LM17 medium. In conclusion, the fatty acid compositions of the bile-resistant lactococci differed from those of the bile-sensitive ones. More importantly, our data suggest that altering their fatty acid composition (i.e. increased hexadecanoic acid and octadecenoic acid and decreased hexadecenoic acid and C-19 cyclopropane fatty acid) by changing growth conditions may be a useful way to enhance their bile resistance in lactococci.

In a series of in vitro culture experiments using the murine macrophage-like cell line, J774.1, we investigated the ability of 46 different Lactococcus lactis strains to induce production of the cytokines interleukin (IL)-6, IL-12 and tumor necrosis factor (TNF)-alpha. The extent of induction of IL-6, IL-12 and TNF-alpha was strain-specific and was not related to subspecies, biovariety, or the source of the isolate. When incubated with a high concentration of viable cells of some lactococcal strains, J774.1 cells hardly produced cytokines in which case the percentage of J774.1 cells that were double-stained with the apoptosis probe FITC-labeled annexin V and propidium iodide was significantly increased. This finding suggests that perturbation of cytokine induction is due to the cytotoxic effects of these strains. On the other hand, when incubated with living cells of other strains, even at a high concentration, J774.1 cells produced IL-6, IL-12 and TNF-alpha. In these cases, FITC-labeled annexin V interacted with these cells, suggesting that incubation with these strains causes phosphatidylserine to be exposed at the cell surface. The ability of these strains to induce TNF-alpha, but not IL-6 and IL-12, was lost after heat treatment, suggesting that the stimulus required for TNF-alpha induction is heat sensitive and is different from those required for IL-6 and IL-12 induction. The specificity of cytokine induction by different lactococci is discussed in terms of interaction of non-pathogenic bacteria with macrophages, as well as the implications for the use of lactococci as probiotics.

Background. The mechanisms by which CD4 + T cells, especially CD4 + CD25 + T cells, transfer allograft specific tolerance are poorly defined. The role of cytokines and the effect on antigen-presenting cells is not resolved. Methods. Anti-CD3 monoclonal antibody (mAb) therapy induced tolerance to PVG heterotopic cardiac transplantation in DA rats. Peripheral CD4 + T cells or CD4 + CD25 + and CD4 + CD25 - T cell subsets were adoptively transferred to irradiated DA hosts grafted with PVG heart grafts. For specificity studies, tolerant CD4 + T cells were transferred to hosts with Lewis or (PUG×Lewis)F 1 heart grafts. Cytokine mRNA induction and the requirement for interleukin (IL)-4 and transforming growth factor (TGF)-β in the transfer of tolerance was assessed. Results. CD4 + T cells transferred specific tolerance and suppressed naive CD4 + T cells capacity to effect rejection of PVG but not Lewis grafts. (PVG×Lewis)F 1 grafts had a major rejection episode but recovered. Later these hosts accepted PVG but not Lewis skin grafts. Adoptive hosts restored with tolerant or naive cells had similar levels of mRNA expression for all Th1 and Th2 cytokines and effector molecules assayed. Transfer of tolerance by CD4 + T cells was not blocked by mAb to IL-4 or TGF-β. CD4 + CD25 - T cells from either naive or tolerant hosts effected rejection. In contrast neither tolerant nor naive CD4 + CD25 + T cells restored rejection. Conclusions. Specific tolerance transfer required CD4 + containing CD4 + CD25 + T cells. An inflammatory response with induction of mRNA for Thi and Th2 cytokines plus cytotoxic effector molecules occurred, but IL-4 and TGF-β were not essential. Inhibition of antigen presenting cells was not the sole mechanism as there was no linked tolerance.

The most widely used probiotic bacteria are lactobacilli and bifidobacteria, which have been isolated from the human gastrointestinal tract. The development of new probiotic strains, which are more feasible and beneficial organisms, is awaited in the dairy industry. Lactococci would be promising species because of their extensive usage in manufacturing dairy products such as cheese and fermented milk. However, there have been few studies on the probiotic activity of lactococci since they are traditionally not considered to be natural inhabitants of the human gastrointestinal tract. Recently, several works showed the possibility of the presence of lactococci in the flora of the human or animal gastrointestinal tract. In this review, we would like to propose Lactococcus sp. as new probiotic bacteria.

Some lactic acid bacteria that harbour carotenoid biosynthesis genes ( crtNM ) can produce carotenoids. Although aerobic conditions can increase carotenoid production and crtNM expression levels, their effects on the pathways that synthesize carotenoid precursors such as mevalonate and isoprene are not completely understood. In this study, we investigated whether aerobic conditions affected gene expression levels involved in the isoprenoid biosynthesis pathway that includes the mevalonate and isoprene biosynthesis pathways in Enterococcus gilvus using real-time quantitative reverse transcription PCR. NADH oxidase ( nox ) and superoxide dismutase ( sod ) gene expression levels were investigated as controls for aerobic conditions. The expression levels of nox and sod under aerobic conditions were 7.2- and 8.0-fold higher, respectively, than those under anaerobic conditions. Aerobic conditions concomitantly increased the expression levels of crtNM carotenoid biosynthesis genes. HMG-CoA synthase gene expression levels in the mevalonate pathway were only slightly increased under aerobic conditions, whereas the expression levels of HMG-CoA reductase and five other genes in the isoprene biosynthesis pathways were 1.2–2.3-fold higher than those under anaerobic conditions. These results demonstrated that aerobic conditions could increase the expression levels of genes involved in the isoprenoid biosynthesis pathway via mevalonate in E . gilvus .

Three different types of treatment were conducted to clarify the properties of a novel immunomodulator, FTY720, in canine kidney allograft models. Survival, biochemical and hematological tests, pharmacokinetics, and histopathology of grafts and extra-renal organs were analyzed. Accompanying a remarkable reduction in circulating lymphocytes, single-drug treatment of FTY720, ranging from 0.05 to 10 mg/kg, exhibited significant prolongation of graft survival without a dose-dependent effect. Short-course induction with FTY720 at 5 mg/kg per day exhibited similar anti-rejection effects as did single-drug treatment but no advantage in rescuing ongoing rejection. In combination with cyclosporine (CsA; 5 mg/kg) or tacrolimus (FK; 0.5 mg/kg), FTY720 had an additive effect. Trough blood concentrations of FTY720 were linearly correlated with dose. No animal showed critical adverse effects at any point. FTY720 holds promise as a candidate in a new category of drugs that can be combined with conventional agents for induction and maintenance immunosuppression in clinical organ transplantation.

Probiotics are viable microorganisms that exhibit beneficial effects on the health of the host when they are ingested. In Asian areas including Japan, various fermented vegetable foods are produced and actively consumed in our meals. In the present study, we isolated lactococci from plant materials (fermented vegetables, silage and plants) and investigated their probiotic activities, such as tolerance to bile and the ability to remove cholesterol. A total of 411 lactic acid bacteria strains were isolated from plant materials. Out of the strains, 27 strains were identified as Lactococcus lactis subsp. lactis by phenotypic tests and a PCR-based method. Among them, 12 strains were subjected to further study. All lactococci tested could grow in broth containing 0.3% bile, and removed cholesterol from media during growth. These results showed that the lactococcal strains isolated from plant materials had probiotic activities.

One of the important properties of probiotics is the ability to survive in the intestine. There have been few studies on the probiotic property of lactococci, since they are formally not considered to be natural inhabitants of the intestine. To evaluate lactococci as probiotic bacteria, we investigated their ability to survive during gastric transit by in vitro and in vivo tests. When exposed to an in vitro simulated gastrointestinal environment, such as low pH and bile, only Lactococcus lactis subsp. lactis bv. diacetylactis N7 showed a moderate survival rate among the four strains tested. The tested strains were orally administered to mice, and intestinal passage of the ingested strains was monitored by two methods: antibiotics and PCR. Viable cells of strain N7 were recovered from feces within 24–48 h after administration but not at 72 h. Lactococcus lactis subsp. cremoris ATCC 19257, which had a poor survival rate in vitro test, was also detected at 12 h but not at 24 h. These results indicate that lactococci can reach the mouse intestine alive, but not colonize it. If administered daily, viable strain N7 may exist continuously in the intestine. The effect of strain N7 on intestinal microbial balance and on animal health will be the subject of a further study.Key words: Lactococcus, survival, gastrointestinal tract, probiotics.

Lactococcus Lactis subsp. lactis biovar. diacetylactis DRC1 carries more than 6 plasmids, including a 7.4 kb cryptic plasmid, which was designated as pDR1-1. The pDR1-1 plasmid was found to significantly affect the maximum specific growth rate (μ m a x ) of the host cells because of its limiting effect on growth. To investigate the properties of the limiting effect, the entire nucleotide sequence of pDR1-1 was determined. It consisted of 7412 bp, and 6 open reading frames (ORFs) were identified. The first ORF showed a high degree of similarity to a family of replication genes (rep) that are commonly found in lactococcal strains. The rep gene in pDR1-1was followed by a second ORF of unknown function. Directly downstream of the second ORF, a third ORF was found, that showed homology to the S subunit from type 1 restriction/modification systems. No significant similarity to the contents of the database was found for the other ORFs. PCR analysis was carried out in order to detect pDR1-1 in the other L lactis strains. The μ m a x of the pDR1-1-positive strain was the same as that of DRC1. These results suggest that the load of pDR1-1 (or pDR1-1-like plasmid) is a major factor influencing the μ m a x of DRC1 because of its limiting effect on growth, an effect which is much more pronounced than that produced by the overall load of other coexisting plasmids.

Background. Blockade of CD40-CD40 ligand (CD154) costimulatory pathway with anti-CD154 antibody (Ab) prolongs allograft survival in experimental organ transplantations; however, repeated agent administration is needed to provide an adequate immunosuppression. Seeking for simple and effective approach to interfere this signaling, we applied adenovirus-mediated gene therapy by encoding CD40Ig gene (AdCD40Ig). Methods. Liver graft from ACI (RT1av1) rat was transplanted orthotopically into LEW (RT1l) rat, and AdCD40Ig was given to animals via the penile vein immediately after grafting (n=6). Results. A single treatment with AdCD40Ig at 1×109 plaque forming units induced specific expression of CD40Ig gene on allograft liver, produced substantial amount of the protein in the sera, and allowed indefinite graft survival. Whereas, LEW recipients given no treatment or control adenovirus vector (AdLacZ) promptly rejected ACI liver. In addition, AdCD40Ig-treated, long-term survivors accepted skin graft from the donor strain but not the third party graft. Histopathology revealed that liver structure of the long-term surviving animals was completely preserved in normal with no infiltration of mononuclear cells. Conclusion. Blockade of CD40-CD154 pathway by CD40Ig gene therapy is a potent alloantigen-specific immunosuppressive strategy to induce permanent acceptance of liver allograft and would be a new therapeutic candidate in a clinical liver transplantation.

Certain strains of lactic acid bacteria present in commercial cheese starters, characterized by faint transparent colonies on an agar plate containing 1 mg kg −1 crystal violet (CVT), were identified as Lactococcus lactis subsp. (ssp) lactis biovar diacetylactis. The effect of citrate on the growth of these strains (CVT strains) in the presence of glucose was studied, in comparison with L. lactis strains. Molar growth yield from glucose ( Y G , g dry weight/mole of glucose consumed) for CVT strains grown on glucose plus citrate was significantly higher than the control (i.e. without citrate), but not for other L. lactis strains tested. Enhanced Y G was also observed at a pH-controlled experiment, indicating that enhanced Y G did not result from a buffering effect of citrate. CVT strains, in contrast to other strains of the same species, were shown to obtain enough energy to enhance Y G on glucose–citrate mixtures.