Protective efficacy of a hydroxy fatty acid against gastric Helicobacter infections.

BACKGROUND: We have previously revealed that omega-3 polyunsaturated fatty acids can prevent Helicobacter pylori infection by blocking the futalosine pathway, an alternative route for menaquinone (MK) biosynthesis. MATERIALS AND METHODS: 1, Different H. pylori strains were grown in liquid media supplemented with linoleic acid, an omega-6 fatty acid, or its 10-hydroxy derivative, 10-hydroxy-cis-12-octadecenoic acid (HYA), in the presence or absence of MK. The bacterial numbers in the media were estimated by plating; 2, C57BL/6NCrl mice received drinking water supplemented with different fatty acids starting from 1 week before infection with H. pylori or Helicobacter suis until the end of the experiment. The gastric colonization levels of H. pylori or H. suis were determined 2 weeks after infection by plating or quantitative PCR, respectively; 3, Mice were given HYA, starting 1 week before infection with H. suis and continuing until 6 months after infection, for analysis of the gastric conditions. RESULTS: 1, A low concentration (20 µmol/L) of HYA in culture broth suppressed the growth of H. pylori, and this inhibition was reduced by MK supplementation; 2, HYA treatment protected mice against H. pylori or H. suis infection; 3, HYA treatment suppressed the formation of lymphoid follicles in the gastric mucus layer after H. suis infection. CONCLUSIONS: HYA prevents gastric Helicobacter infections by blocking their futalosine pathways. Daily HYA supplementation is effective for the prevention of gastric mucosa-associated lymphoid tissue lymphoma induced by persistent infection with H. suis.

Draft Genome Sequence of Helicobacter suis Strain SNTW101, Isolated from a Japanese Patient with Nodular Gastritis.

We present here the draft whole-genome shotgun sequence of an uncultivated strain SNTW101 of Helicobacter suis, which has been maintained in the stomachs of mice. This strain was originally isolated from gastric biopsy specimens of a urea breath test-negative Japanese patient suffering from nodular gastritis.

Narrow-spectrum inhibitors targeting an alternative menaquinone biosynthetic pathway of Helicobacter pylori.

We aimed to identify narrow-spectrum natural compounds that specifically inhibit an alternative menaquinone (MK; vitamin K2) biosynthetic pathway (the futalosine pathway) of Helicobacter pylori. Culture broth samples of 6183 microbes were examined using the paper disc method with different combinations of 2 of the following 3 indicator microorganisms: Bacillus halodurans C-125 and Kitasatospora setae KM-6054(T), which have only the futalosine pathway of MK biosynthesis, and Bacillus subtilis H17, which has only the canonical MK biosynthetic pathway. Most of the active compounds isolated from culture broth samples were from the families of polyunsaturated fatty acids (PUFAs). Only one compound isolated from the culture broth of Streptomyces sp. K12-1112, siamycin I (a 21-residue lasso peptide antibiotic), targeted the futalosine pathway. The inhibitory activities of representative PUFAs and siamycin I against the growth of B. halodurans or K. setae were abrogated by supplementation with MK. Thereafter, the growth of H. pylori strains SS1 and TN2GF4 in broth cultures was dose-dependently suppressed by eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or siamycin I, and these inhibitory effects were reduced by supplementation with MK. Daily administration of EPA (100 µM), DHA (100 µM), or siamycin I (2.5 µM) in drinking water reduced the H. pylori SS1 colonization in the gastric mucosa of C57BL/6 mice by 96%, 78%, and 68%, respectively. These data suggest that EPA, DHA, and siamycin I prevented H. pylori infection by inhibiting the futalosine pathway of MK biosynthesis.

A highly susceptible CD46 transgenic mouse model of subcutaneous infection with Streptococcus dysgalactiae subspecies equisimilis.

The Streptococcus dysgalactiae subspecies equisimilis (SDSE) possesses clinical similarities to group A streptococcus (GAS) and has recently been recognized as a causative pathogen of life-threatening streptococcal infections. Human membrane cofactor protein (CD46), a complement regulatory protein ubiquitously expressed on every cell type except for erythrocytes, has been implicated as a receptor for human-specific pathogens including GAS. In the present report, SDSE strain GGS_124 was isolated from a patient suffering from streptococcal toxic shock syndrome. When CD46-expressing transgenic (Tg) and non-Tg mice were infected subcutaneously into a hind footpad with 1 × 10(7) colony-forming units of GGS_124, both CD46 Tg and non-Tg mice showed similar levels of colonization in the popliteal lymph nodes at day 3 after infection. However, the following differences were found between CD46 Tg and non-Tg mice after infection. First, there was a statistically significant difference in mortality rates between CD46 Tg (33%) and non-Tg (0%) mice within 35 days after infection. Second, all surviving CD46 Tg mice developed ankle arthritis at day 35 after infection, whereas non-Tg mice did not develop ankle arthritis on the infected hind paws. Finally, CD46 Tg mice developed a pus-filled abscess accompanied by renal failure at day 6 or later after infection. These observations suggest that CD46, the host cell-surface pathogen receptor, functioned to attract GGS_124 into deep tissues, so that the subcutaneous infection with GGS_124 induced invasive streptococcal diseases in CD46 Tg mice.

Mouse Models for Assessing the Protective Efficacy of Lactobacillus gasseri SBT2055 against Helicobacter suis Infection Associated with the Development of Gastric Mucosa-Associated Lymphoid Tissue Lymphoma.

BACKGROUND: Helicobacter suis strain TKY infection has been strongly associated with the development of gastric mucosa-associated lymphoid tissue (MALT) lymphoma in a C57BL/6J mouse model. MATERIALS AND METHODS: 1. C57BL/6J mice were intragastrically administered Lactobacillus strains once daily with 10(8)-10(9) colony-forming units (CFU), starting 2 days before intragastric infection with H. suis TKY (approximately 1 × 10(4) copies of 16S rRNA genes) or H. pylori Sydney strain 1 (SS1; 3 × 10(8) CFU) and continuing for 14 days after infection. 2. C57BL/6J mice were given powdered feed mixed with lyophilized L. gasseri SBT2055 (LG2055) cells (5 × 10(8) CFU/g), starting 2 weeks before intragastric infection with H. suis TKY and continuing 12 months after infection. RESULTS: 1. Among the 5 Lactobacillus strains that we examined, only LG2055 exhibited significantly preventive efficacy against both H. suis TKY and H. pylori SS1 at day 15 after infection. 2. Dietary supplementation with LG2055 protected mice from the formation of round protrusive lesions in the gastric fundus 12 months after infection with H. suis TKY, whereas such lesions had developed in the gastric fundus of nonsupplemented mice 12 months after infection. In addition, the formation of lymphoid follicles in gastric mucus layers was suppressed by dietary LG2055 at 3 months after infection. CONCLUSIONS: LG2055 administration is effective for suppressing the progression of gastric MALT lymphoma by reducing H. suis colonization.

Alteration of angiogenesis in Helicobacter heilmannii-induced mucosa-associated lymphoid tissue lymphoma: interaction with c-Met and hepatocyte growth factor.

BACKGROUND AND AIM: The hepatocyte growth factor (HGF)/c-Met pathway has attracted attention in the formation of malignant tumors, as HGF secreted from the microcirculatory components as well as residing macrophages has been suggested to act on the c-Met receptors of cancer cells to decrease apoptosis and increase proliferation, invasion, and metastasis. The present study was undertaken to elucidate the interaction of the gastric, hepatic, and pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma induced by Helicobacter heilmannii infection with c-Met and HGF. METHODS: C57BL/6 female mice, infected with H. heilmannii for 3 months were used. The localization of the HGF, c-Met, and HGF activator immunoreactivities was observed by the indirect immunohistochemical methods. In addition, the effect of c-Met antibody and c-Met inhibitor, PHA-665752, was also investigated. RESULTS: c-Met immunoreactivity was found in the lymphocytes composing the MALT lymphoma, and HGF immunoreactivity was recognized mostly in the endothelial cells and macrophages in the MALT lymphoma. HGFA was localized on mesenchymal cells other than the lymphocytes. The administration of the antibody against c-Met or the c-Met inhibitor to the infected mice induced the significant suppression of hepatic and pulmonary MALT lymphoma, while the gastric MALT lymphoma showed only a tendency to decrease in size, while the active caspase 3 positive cells markedly decreased in the gastric, hepatic, and pulmonary MALT lymphoma after the treatment with the c-Met antibody or the c-Met antagonist. CONCLUSIONS: HGF and c-Met pathway were suggested to contribute to the lymphomagenesis in the MALT lymphoma after H. heilmannii infection.

Development of new PCR primers by comparative genomics for the detection of Helicobacter suis in gastric biopsy specimens.

BACKGROUND: Although the infection rate of Helicobacter suis is significantly lower than that of Helicobacter pylori, the H. suis infection is associated with a high rate of gastric mucosa-associated lymphoid tissue (MALT) lymphoma. In addition, in vitro cultivation of H. suis remains difficult, and some H. suis-infected patients show negative results on the urea breath test (UBT). MATERIALS AND METHODS: Female C57BL/6J mice were orally inoculated with mouse gastric mucosal homogenates containing H. suis strains TKY or SNTW101 isolated from a cynomolgus monkey or a patient suffering from nodular gastritis, respectively. The high-purity chromosomal DNA samples of H. suis strains TKY and SNTW101 were prepared from the infected mouse gastric mucosa. The SOLiD sequencing of two H. suis genomes enabled comparative genomics of 20 Helicobacter and 11 Campylobacter strains for the identification of the H. suis-specific nucleotide sequences. RESULTS: Oral inoculation with mouse gastric mucosal homogenates containing H. suis strains TKY and SNTW101 induced gastric MALT lymphoma and the formation of gastric lymphoid follicles, respectively, in C57BL/6J mice. Two conserved nucleotide sequences among six H. suis strains were identified and were used to design diagnostic PCR primers for the detection of H. suis. CONCLUSIONS: There was a strong association between the H. suis infection and gastric diseases in the C57BL/6 mouse model. PCR diagnosis using an H. suis-specific primer pair is a valuable method for detecting H. suis in gastric biopsy specimens.

New pharmaceutical treatment of gastric MALT lymphoma: anti-angiogenesis treatment using VEGF receptor antibodies and celecoxib.

In addition to eradication of Helicobacter pylori, chemotherapy with anticancer agents, and radiation therapy, the treatment with molecular target drugs including rituximab, a CD20 antagonist, is one of the promising new regimens. The mucosa-associated lymphoid tissue (MALT) lymphoma is histologically characterized by rich distribution of the microvascular network consisting of the immature capillaries, lymphatics and venules, and this microvascular network could be the target of the new pharmacotherapy in addition to the direct action on the accumulated B lymphocytes. We have established the animal model of the gastric MALT lymphoma by the Helicobacter heilmannii (H. heilmannii) peroral infection of C57BL/6 mice. The disease induced by this model is very similar to the human counterpart, because of the lymphoepithelial lesion characteristic of the human MALT lymphoma as well as the rich vascularization and localization of vascular endothelial growth factor (VEGF) and its receptors, Flt-1, Flk-1 and Flt-4. By administering VEGF receptor antibodies or celecoxib, one of the cyclooxygenase 2 inhibitors, we were able to induce a significant decrease in the size of the tumor and the apoptotic changes of the endothelial cells of the microvascular network. These antiangiogenic strategies were suggested to be candidates for the new pharmacological treatment of gastric MALT lymphoma, when other treatments are not effective.

Musashi-1 post-transcriptionally enhances phosphotyrosine-binding domain-containing m-Numb protein expression in regenerating gastric mucosa.

OBJECTIVE: Upregulation of the RNA-binding protein Musashi-1 (Msi1) has been shown to occur in rat gastric corpus mucosa after ethanol-induced mucosal injury. However, there is no direct evidence linking Msi1 with gastric regeneration. We examined the process of tissue repair after acute gastric mucosal injury with Msi1-knock-out (KO) mice to clarify the role of Msi1 and Msi1-dependent regulation of m-Numb expression in regenerating gastric mucosa. METHODS: Acute gastric injury was induced in Msi1-KO and wild-type ICR mice by administering absolute ethanol. Expression of the splicing variants of m-Numb mRNA and protein in the gastric mucosa were analyzed by quantitative RT-PCR and western blotting, respectively. RESULTS: We demonstrated that phosphotyrosine-binding domain-containing m-Numb expression was significantly upregulated at both the mRNA and protein levels in wild-type mice at 3 h after ethanol-induced acute gastric injury. In contrast, in Msi1-KO mice, the m-Numb protein was expressed weakly, and was associated with delayed regeneration of the injured gastric mucosal epithelium. In the Msi1-KO mouse, the ratio of m-Numb mRNA to total m-Numb mRNA in the heavy polysome fractions was lower than that in the wild-type mouse. Further, we showed that m-Numb-enhancement in gastric mucous cells induced the expression of prostate stem cell antigen and metallothionein-2. Under the m-Numb enhancing condition, the gastric cells exhibited enhanced cell proliferation and were significantly more resistant to H(2)O(2)-induced cell death than control cells. CONCLUSIONS: Msi1-dependent post-transcriptional enhancement of m-Numb is crucial in gastric epithelial regeneration.

A CD46 transgenic mouse model for studying the histopathology of arthritis caused by subcutaneous infection with Streptococcus dysgalactiae subspecies equisimilis.

Ankle arthritis was induced by a single subcutaneous (s.c.) infection of 1×10(7) c.f.u. of the Streptococcus dysgalactiae subspecies equisimilis strain RE378, which was isolated from a patient suffering from multiple organ failure due to septicaemia, into both hind footpads of human CD46-expressing transgenic (Tg) mice. In contrast, in non-Tg mice, the incipient foot lesions (swelling and redness) resolved before arthritis developed. The number of viable bacteria in tissue samples and the arthritis frequency on days 3 and 28 after infection were higher in CD46 Tg mice than in non-Tg mice. The histopathological findings in the hind ankle sections of CD46 Tg mice showed the stimulation of osteoclast formation associated with inflammation of the synovial membrane and the development of aggressive granulation tissue (pannus). In addition, increased expression levels of interleukin (IL)-6, receptor activator of NF-κB ligand, IL-1ß and tumour necrosis factor alpha were detected in the foot bones of CD46 Tg mice but not in those of non-Tg mice. These observations suggest that the s.c. infection with S. dysgalactiae subsp. equisimilis induced arthritis in the ankle joints of CD46 Tg mice as a consequence of the prolonged inflammation associated with focal bone loss.

Dermal mast cells reduce progressive tissue necrosis caused by subcutaneous infection with Streptococcus pyogenes in mice.

A single subcutaneous (s.c.) infection with 1×10(7) c.f.u. GAS472, a group A streptococcus (GAS) serotype M1 strain isolated from the blood of a patient suffering from streptococcal toxic shock syndrome, led to severe damage of striated muscle layers in the feet of mast cell (MC)-deficient WBB6F(1)-Kit(W)/Kit(W-v) (W/W(v)) mice 72 h after infection. In contrast, no damage was recognized in striated muscle layers in the feet of the control WBB6F(1)-Kit(+/+) (+/+) mice 72 h after infection. In addition, adoptively transferred MCs reduced progressive tissue necrosis of the feet of W/W(v) mice after infection. However, there was no significant difference in the mortality rates between the W/W(v) and +/+ mice, or between the human CD46-expressing transgenic (Tg) mouse bone marrow-derived cultured MC-reconstituted W/W(v) and non-Tg mouse bone marrow-derived cultured MC-reconstituted W/W(v) mice after infection. Consequently, although MCs can help to reduce the severity of necrosis of the feet caused by s.c. infection with GAS472, such reduction of tissue necrosis scarcely improves the mortality rates of these mice. Moreover, human CD46 does not play a crucial role in the MC-mediated innate immune defence against GAS infection.

Suppression of lymphangiogenesis induced by Flt-4 antibody in gastric low-grade mucosa-associated lymphoid tissue lymphoma by Helicobacter heilmannii infection.

BACKGROUND AND AIMS: Our recent study revealed that per oral infection with Helicobacter heilmannii induced low-grade mucosa-associated lymphoid tissue (MALT) lymphoma in the gastric fundus of C57BL/6 mice after a period of 6 months, although the pathophysiological mechanism of lymphoma expansion remains to be clarified. The present study was undertaken to elucidate the interaction of this tumor with angiogenesis and lymphangiogenesis. In addition, the effect of Flt-4 antibodies on lymphoma expansion was investigated. METHODS: C57BL/6 female mice infected with H. heilmannii for 3 months were used in the experiments. Localization of vascular endothelial growth factor C (VEGF-C) and Flt-4 immunoreactivity were detected by indirect immunohistochemical methods. Localization of lymphatic and vascular endothelial cells was investigated by localization of prox-1. In addition, Flt-4 antibody with and without Flt-1 or Flk-1 antibodies was administered i.p. to clarify their effects on tumor size. RESULTS: MALT lymphoma has a rich microvascular network consisting of immature capillaries, lymphatics and venules. By immunohistochemical analysis, prox-1 immunoreactivity was observed mostly in the marginal area of the lymphoma, where VEGF-C and Flt-4 immunoreactivities were also seen. Stereomicroscopic study revealed that administration of Flt-4 and Flt-1 antibodies significantly reduced the surface area of the lymphoma in the mouse stomach. CONCLUSION: A VEGF-C-mediated mechanism plays an important role in the expansion of MALT lymphoma and the administration of VEGF receptor antibodies had a suppressive effect on tumor growth.

CD46 transgenic mouse model of necrotizing fasciitis caused by Streptococcus pyogenes infection.

We developed a human CD46-expressing transgenic (Tg) mouse model of subcutaneous (s.c.) infection into both hind footpads with clinically isolated 11 group A streptococcus (GAS) serotype M1 strains. When the severity levels of foot lesions at 72 h and the mortality rates by 336 h were compared after s.c. infection with 1x10(7) CFU of each GAS strain, the GAS472 strain, isolated from the blood of a patient suffering from streptococcal toxic shock syndrome (STSS), induced the highest severity levels and mortality rates. GAS472 led to a 100% mortality rate in CD46 Tg mice after only 168 h postinfection through the supervention of severe necrotizing fasciitis (NF) of the feet. In contrast, GAS472 led to a 10% mortality rate in non-Tg mice through the supervention of partial necrotizing cutaneous lesions of the feet. The footpad skin sections of CD46 Tg mice showed hemorrhaging and necrotic striated muscle layers in the dermis, along with the exfoliation of epidermis with intracellular edema until 48 h after s.c. infection with GAS472. Thereafter, the bacteria proliferated, reaching a 90-fold or 7-fold increase in the livers of CD46 Tg mice or non-Tg mice, respectively, for 24 h between 48 and 72 h after s.c. infection with GAS472. As a result, the infected CD46 Tg mice appeared to suffer severe liver injuries. These findings suggest that human CD46 enhanced the progression of NF in the feet and the exponential growth of bacteria in deep tissues, leading to death.

A novel approach for screening of new anti-Helicobacter pylori substances.

Gastric mucosal cell-derived L-lactic acid strongly enhances proliferation of Helicobacter pylori, and may contribute to the long-term colonization of H. pylori in the stomach. Therefore it is assumed that inhibitory substances active against L-lactic acid-dependent growth of H. pylori will be useful candidates as novel therapeutic agents for H. pylori infection. In this study, we developed a new assay system for screening anti-H. pylori substances, and baicalein and glycyrrhetinic acid were found as potent inhibitory substances against L-lactic acid-dependent H. pylori growth but not L-lactic acid-independent growth. The newly developed assay system described in this study also may facilitate the development of novel therapeutic agents for H. pylori infection.

L-lactic acid secreted from gastric mucosal cells enhances growth of Helicobacter pylori.

BACKGROUND: Helicobacter pylori mainly inhabit the mucus layer in the gastric mucosa. However, mechanisms involving H. pylori colonization and proliferation in gastric mucosa are not well established. This study focuses on elucidating the role of gastric mucosal cells on growth of H. pylori. MATERIALS AND METHODS: H. pylori was co-cultured with the murine gastric surface mucosal cells (GSM06), and the growth of H. pylori on the cells was assessed by enumerating the colony-forming units (CFU). The H. pylori growth factor in the culture media conditioned by GSM06 cell was purified by HPLC, and the chemical structure of the growth factor was identified by analyses of (1)H- and (13)C-NMR spectra. RESULTS: A marked increase in the number of CFU of H. pylori was observed in the GSM06 cells. The enhanced H. pylori growth was also observed when indirectly incubated with GSM06 cells through semi-permeable membrane. In addition, culture media conditioned by GSM06 cell stimulated H. pylori growth approximately one thousand-fold. By bioassay-guided purification, the H. pylori growth factor was isolated from the conditioned medium of GSM06 cells and identified as L-lactic acid. The H. pylori growth-enhancing activity under microaerobic condition was well correlated with L-lactic acid concentrations in the conditioned media. CONCLUSIONS: This study demonstrates that L-lactic acid secreted by gastric mucosal cells enhances the growth of H. pylori, and this L-lactic acid-dependent growth of H. pylori may be important to the long-term colonization of H. pylori in the stomach.

"Candidatus Helicobacter heilmannii" from a cynomolgus monkey induces gastric mucosa-associated lymphoid tissue lymphomas in C57BL/6 mice.

Both Helicobacter pylori and "Candidatus Helicobacter heilmannii" infections are associated with peptic ulcers, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphomas. However, good animal models of H. pylori clinical diseases are rare. In this study, we aimed to establish an animal model of "Candidatus Helicobacter heilmannii" gastric MALT lymphoma. We used a urease-positive gastric mucosal and mucus homogenate from a cynomolgus monkey maintained in C57BL/6 mouse stomachs. The bacterium in the homogenate was identified as "Candidatus Helicobacter heilmannii" based on a DNA sequence analysis of the 16S rRNA and urease genes. Mucosal and mucus homogenates were used to inoculate C57BL/6 mice, which were then examined for 24 months. We observed a gradual increase in the surface area of protrusive lesions in almost all infected C57BL/6 mouse fundic stomachs 6 months after infection. Light microscopic observations revealed an accumulation of B lymphocytes along with destruction of glandular elements and the presence of lymphoepithelial lesions consistent with low-grade MALT lymphomas. Electron microscopic observation revealed numerous "Candidatus Helicobacter heilmannii" bacilli in the fundic glandular lumen, the intracellular canaliculi, and the cytoplasm of intact cells, as well as damaged parietal cells. In conclusion, "Candidatus Helicobacter heilmannii" induced gastric MALT lymphomas in almost 100% of infected C57BL/6 mice after a 6-month period associated with the destruction of parietal cells.

Direct autoradiographic evidence that rebamipide interacts with neutrophils in dextran sulfate sodium induced colitis in rats.

To determine the effects of rebamipide during the early stages of colitits development, colitis was induced in rats by oral administration of dextran sulfate sodium for 3 or 7 days. The target sites of (3)H-rebamipide were examined by intra-aortic infusion of the radiolabeled compound followed by autoradiography. (3)H-rebamipide was localized in goblet cells in the colon of the control rat, whereas it accumulated in the cytoplasm of mesenchymal cells in dextran sulfate sodium treated rats, localized predominantly to polymorphonuclear leucocytes.

A novel in vitro infection model of Helicobacter pylori using mucin-producing murine gastric surface mucous cells.

BACKGROUND: Helicobacter pylori is found within the gastric surface mucous gel layer and in the epithelial surface. Gastric cancer cells have been used in experimental H. pylori infection in vitro, although cancer cells have some abnormalities in cellular properties. The aim of this study was to develop an in vitro H. pylori infection model using normal gastric surface cells that produce gastric mucin. MATERIALS AND METHODS: Normal murine gastric surface mucous cells (GSM06) were cultured by the liquid interface method using a serum-free medium and a collagen gel containing a fibroblast cell line (L929) and infected with H. pylori. Infection by H. pylori was assessed by enumerating the colony-forming units (CFU) of H. pylori adhered to GSM06 cells and by transmission electron microscopy. The production of mucin was determined by a lectin binding assay, sugar analysis, and MUC5AC gene expression. RESULTS: GSM06 cells cultured under these conditions produced mucin containing N-acetylgalactosamine and MUC5AC as the core protein. Significantly higher numbers of H. pylori adhered to GSM06 cells under mucin-producing conditions than under nonproducing conditions. Microscopic observation showed a filamentous structure resembling a type IV secretion system apparatus formed between the surface of GSM06 cells and H. pylori. CONCLUSIONS: This study demonstrates a novel in vitro H. pylori infection model using mucin-producing murine GSM06 cells for early stages of infection.