Desulfovibrio subterraneus sp. nov., a mesophilic sulfate-reducing deltaproteobacterium isolated from a deep siliceous mudstone formation.

A novel mesophilic sulfate-reducing bacterium, strain HN2T, was isolated from groundwater sampled from the subsurface siliceous mudstone of the Wakkanai Formation located in Horonobe, Hokkaido, Japan. The bacterium was Gram-negative and vibrio-shaped, and its motility was conferred by a single polar flagellum. Cells had desulfoviridin. Catalase and oxidase activities were not detected. It grew in the temperature range of 25-40 °C (optimum, 35 °C) and pH range of 6.3-8.1 (optimum, pH 7.2-7.6). It used sulfate, thiosulfate, dimethyl sulfoxide, anthraquinone-2,6-disulfonate, Fe3+, and manganese oxide, but not elemental sulfur, nitrite, nitrate, or fumarate as electron acceptors. The strain showed weak growth with sulfite as the electron acceptor. Fermentative growth with pyruvate, lactate and cysteine was observed in the absence of sulfate, but not with malate or fumarate. NaCl was not required, but the strain tolerated up to 40 g l-1. Strain HN2T did not require vitamins. The major cellular fatty acids were iso-C15 : 0 (23.8 %), C18 : 1 ω9t (18.4 %), C18 : 0 (15.0 %), C16 : 0 (14.5 %), and anteiso-C17 :0 (10.1 %). The major respiratory quinone was menaquinone MK-6(H2). The G+C content of the genomic DNA was 56.7 mol%. Based on 16S rRNA gene sequence analysis, the closest phylogenetic relative of strain HN2T is Desulfovibrio psychrotolerans JS1T (97.0 %). Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values of the strains HN2T and D. psychrotolerans JS1T were 22.2 and 79.8 %, respectively. Based on the phenotypic and molecular genetic evidence, we propose a novel species, D. subterraneus sp. nov. with the type strain HN2T (=DSM 101010T=NBRC 112213T).

A Proposed Method to Estimate In Situ Dissolved Gas Concentrations in Gas-Saturated Groundwater.

Gas-saturated groundwater forms bubbles when brought to atmospheric pressure, preventing precise determination of its in situ dissolved gas concentrations. To overcome this problem, a modeling approach called the atmospheric sampling method is suggested here to recover the in situ dissolved gas concentrations of groundwater collected ex situ under atmospheric conditions at the Horonobe Underground Research Laboratory, Japan. The results from this method were compared with results measured at the same locations using two special techniques, the sealed sampler and pre-evacuated vial methods, that have been developed to collect groundwater under its in situ conditions. In gas-saturated groundwater cases, dissolved methane and inorganic carbon concentrations derived using the atmospheric sampling method were mostly within ±4 and ±10%, respectively, of values from the sealed sampler and pre-evacuated vial methods. In gas-unsaturated groundwater, however, the atmospheric sampling method overestimated the in situ dissolved methane concentrations, because the groundwater pressure at which bubbles appear (Pcritical ) was overestimated. The atmospheric sampling method is recommended for use where gas-saturated groundwater can be collected only ex situ under atmospheric conditions.

Anaerobic decomposition of humic substances by Clostridium from the deep subsurface.

Decomposition of humic substances (HSs) is a slow and cryptic but non-negligible component of carbon cycling in sediments. Aerobic decomposition of HSs by microorganisms in the surface environment has been well documented; however, the mechanism of anaerobic microbial decomposition of HSs is not completely understood. Moreover, no microorganisms capable of anaerobic decomposition of HSs have been isolated. Here, we report the anaerobic decomposition of humic acids (HAs) by the anaerobic bacterium Clostridium sp. HSAI-1 isolated from the deep terrestrial subsurface. The use of (14)C-labelled polycatechol as an HA analogue demonstrated that the bacterium decomposed this substance up to 7.4% over 14 days. The decomposition of commercial and natural HAs by the bacterium yielded lower molecular mass fractions, as determined using high-performance size-exclusion chromatography. Fourier transform infrared spectroscopy revealed the removal of carboxyl groups and polysaccharide-related substances, as well as the generation of aliphatic components, amide and aromatic groups. Therefore, our results suggest that Clostridium sp. HSAI-1 anaerobically decomposes and transforms HSs. This study improves our understanding of the anaerobic decomposition of HSs in the hidden carbon cycling in the Earth's subsurface.

Methanosarcina subterranea sp. nov., a methanogenic archaeon isolated from a deep subsurface diatomaceous shale formation.

A methanogenic archaeon, strain HC-2(T), was isolated from a deep diatomaceous shale formation. The strain grew on methanol, monomethylamine, dimethylamine, trimethylamine and dimethylsulphide, but not on acetate, H2/CO2, formate, 2-propanol, 2-butanol or cyclopentanol. Cells were Gram-stain-negative, non-motile, and coccus-like, 0.9-1.4 µm in diameter, and occurred singly, in pairs, or as aggregates. The strain grew at 10-40 °C (optimum 35 °C), pH 5.9-7.4 (optimum pH 6.6-6.8) and in 0-0.6 M NaCl (optimum 0.1-0.2 M). The genomic DNA G+C content was 41.5 mol% and the 16S rRNA gene sequence was closely related to those of Methanosarcina lacustris DSM 13486(T) (99.1%) and Methanosarcina siciliae DSM 3028(T) (98.3%). Values for DNA-DNA hybridization with these strains were less than 30%. The phenotypic and phylogenetic features of HC-2(T) indicate that it represents a novel species of the genus Methanosarcina , for which the name Methanosarcina subterranea sp. nov. is proposed. The type strain is HC-2(T) ( = DSM 22503(T) = JCM 15540(T) = NBRC 102578(T)).

Methanoculleus horonobensis sp. nov., a methanogenic archaeon isolated from a deep diatomaceous shale formation.

A methanogenic organism from the domain Archaea, designated strain T10(T), was isolated from groundwater sampled from a deep diatomaceous shale formation located in Horonobe, Hokkaido, Japan. The strain utilized H2/CO2 and formate as substrates for methanogenesis. Cells were strictly anaerobic, Gram-negative-staining, flagellated, irregular coccoids, 0.7-1.6 µm in diameter, and occurred singly. The strain grew at 25-45 °C (optimum 37-42 °C), at pH 5.8-8.2 (optimum pH 6.7-6.8) and in the presence of 0-1.3 M NaCl (optimum 0.1-0.2 M NaCl). The G+C content of the genomic DNA was 62.9 mol%. 16S rRNA gene sequencing revealed that, although the strain is a member of the genus Methanoculleus, it clearly differed from all described species of this genus (95.5-98.3 % sequence similarity). Values for DNA-DNA hybridization with type strains of closely related Methanoculleus species were less than 50 %. Phenotypic and phylogenetic features of strain T10(T) clearly indicate that it represents a novel species of the genus Methanoculleus, for which the name Methanoculleus horonobensis sp. nov. is proposed. The type strain is T10(T) ( = DSM 21626(T) = JCM 15517(T)).

Influence of Fracture Width on Sealability in High-Strength and Ultra-Low-Permeability Concrete in Seawater.

For cementitious composites and materials, the sealing of fractures can occur in water by the precipitation of calcium compounds. In this study, the sealing behavior in a macro-fractured high-strength and ultra-low-permeability concrete (HSULPC) specimen was investigated in simulated seawater using micro-focus X-ray computed tomography (CT). In particular, the influence of fracture width (0.10 and 0.25 mm) on fracture sealing was investigated. Precipitation occurred mainly at the outermost parts of the fractured surface of the specimen for both fracture widths. While significant sealing was observed for the fracture width of 0.10 mm, sealing was not attained for the fracture width of 0.25 mm within the observation period (49 days). Examination of the sealed regions on the macro-fracture was performed using a three-dimensional image registration technique and applying image subtraction between the CT images of the HSULPC specimen before and after maintaining the specimen in simulated seawater. The temporal change of the sealing deposits for the fracture width of 0.10 mm was much larger than that for the fracture width of 0.25 mm. Therefore, it is concluded that the sealability of the fracture in the HSULPC is affected by the fracture width.

Alloantigen presentation by ethylcarbodiimide-treated dendritic cells induces T cell hyporesponsiveness, and prolongs organ graft survival.

Ethylcarbodiimide (ECDI) couples soluble antigens (Ag) to lymphoid cells bestowing tolerizing potential. We examined whether ECDI-treated allogeneic dendritic cells (DC) could promote Ag-specific T cell unresponsiveness and prolong graft survival. Exposure of murine myeloid DC to ECDI did not affect surface immunophenotype but reduced their ability to cluster with T cells, enhanced their apoptotic death, and markedly reduced their allostimulatory activity. Anti-donor proliferative and cytotoxic T cell responses of mice primed with ECDI-treated DC were markedly inhibited. Secretion of both Th1 (IFNgamma) and Th2 cytokines (IL-5, IL-10) was suppressed. Cardiac allograft survival in mice preconditioned with a single injection of ECDI-DC was prolonged significantly. These results indicate that ECDI-treated DC promote T cell unresponsiveness to donor alloAgs and prolong transplant survival. The effects are not associated with sparing of Th2 responses, but may reflect inhibitory effects of apoptotic donor DC on host immune reactivity.

Parenteral nutrition decreases hepatic dihydropyrimidine dehydrogenase activity and modulates catabolism of 5-fluorouracil in rats.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU). The effect of parenteral nutrition (PN) on hepatic DPD activity and metabolism of 5-FU remains unknown. MATERIALS AND METHODS: Rats were divided into two groups: a sham-operated oral feeding group (FED) and a PN group. After 7-day PN infusion, hepatic DPD activity, serum 5-FU levels and thymidylate synthase (TS) levels in the jejunum and tumor were measured. RESULTS: PN administration significantly decreased hepatic DPD activities. After infusion of 5-FU (40 mg/kg body), the serum 5-FU concentration and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP)-bound TS levels in the jejunum were significantly higher in the PN group than the FED group (156.8 +/- 51.9 vs 100.5 +/- 51.9 ng/ml, p < 0.001 and 38.55 +/- 7.61 vs 22.89 +/- 4.46 pmol/g of tissue, p < 0.01, respectively). In Yoshida sarcoma-bearing rats, the FdUMP-bound TS level in the tumor did not differ significantly between the PN and FED rats. CONCLUSION: PN decreases hepatic DPD activity, which may lead to increased toxicity of 5-FU.

Marked inhibition of transplant vascular sclerosis by in vivo-mobilized donor dendritic cells and anti-CD154 mAb.

BACKGROUND: Combination of donor dendritic cells (DC) and anti-CD40 Ligand (L) (CD154) monoclonal antibody (mAb) markedly prolongs heart or skin allograft survival, but the influence of this strategy in models of chronic rejection is unknown. Our aim was to ascertain the influence of in vivo-mobilized immature donor DC plus anti-CD40L mAb on vascular sclerosis in functional murine aortic allografts. METHODS: C3H He/J (C3H;H2k) mice received 2 x 106 freshly isolated, immunobead-purified (>90%) fms-like tyrosine kinase 3 ligand-mobilized C57BL/10 (B10;H2b) CD11c+ DC intravenously (IV), together with 500 microg of anti-CD40L mAb (MR1) intraperitoneally (IP) on days -7, 0, 4, and 10. Controls received either no donor cells, no mAb, or were untreated. B10 aortic grafts were transplanted in the abdominal aorta on day 0. At day 30, antidonor T-cell proliferative and cytotoxic responses and both complement fixing and immunoglobulin (Ig)G alloantibody levels were determined. Grafts were harvested on days 30 and 60 and examined by histology and immunohistochemistry. RESULTS: DC infusion alone enhanced ex vivo antidonor proliferative and cytotoxic T-cell activity. By contrast, complement-fixing alloantibody levels were reduced. Anti-CD40L mAb alone strongly suppressed each of these responses. Graft inflammatory cell infiltration, intimal smooth muscle cell proliferation, fibrosis, and elastic lamina disruption observed in untreated animals were reduced in response to anti-CD40L mAb or donor DC alone. Antidonor immune reactivity, including IgG levels, and intimal proliferation were all markedly suppressed to an overall greater extent in mice given both treatments. CONCLUSION: Whereas blockade of the CD40-CD40L pathway ameliorated transplant vasculopathy, preservation of near-normal vessel architecture was achieved by simultaneous administration of donor DC. This strategy represents a novel application of DC for suppression of chronic rejection.

Pharmacologic, biologic, and genetic engineering approaches to potentiation of donor-derived dendritic cell tolerogenicity.

There are various approaches to the enhancement of dendritic cell (DC) tolerogenicity for the promotion of cell or organ allograft survival. Both pharmacologic and biologic agents, including several commonly used immunosuppressive drugs, and specific anti-inflammatory cytokines inhibit DC maturation, whereas co-stimulation-blocking agents can also promote the induction of antigen-specific T-cell unresponsiveness by DC. Delivery of genes encoding molecules that subvert T-cell responses by various mechanisms, and targeting of DC migration by selective manipulation of chemokine and chemokine receptor expression, represent additional promising strategies. In this short review, the authors consider those approaches that have been used to promote the tolerogenicity of donor-derived DC in experimental models. Whereas most work to date has focused on myeloid DC, manipulation of other DC subsets may also offer potential for improving the outcome of transplantation and enhancing tolerance induction.

Treatment planning in a case of restoration of the maxilla and mandible using osseointegrated implants with four types of bone graft.

A case is reported of a 66-year-old woman who could not use a conventional, full upper denture because of a gag reflex. In the maxillary alveolar ridge, restoration was performed on a moderately atrophied, edentulous anterior area and a small defect in the right-side posterior area. In the mandibular alveolar ridge, restoration was performed on a moderate osseous defect in each molar area resulting from tooth extraction due to severe periodontal disease. Based on careful treatment planning, four types of bone graft were used with previously designed osseointegrated implants. The atrophied maxillary alveolar ridge was restored with veneer iliac bone grafts to avoid fenestration during implant placement, while alveolar process deficiency was restored using inlay and sinus bone grafts as placements for long implant fixtures. The defects in the mandibular alveolar bone were filled with corticocancellous bone chips at the implant placement sites. A combination of immediate and secondary placement of Brånemark fixtures was used. Bone-anchored bridge-type implant prostheses were fitted approximately twelve months after surgery. Three years later, there had been no failure of implant fixtures and satisfactory functional and cosmetic restoration had been maintained.

Normal donor bone marrow is superior to Flt3 ligand-mobilized bone marrow in prolonging heart allograft survival when combined with anti-CD40L (CD154).

Flt3 ligand (FL) administration markedly increases bone marrow (BM) stem cells and immature dendritic cells. We investigated the influence of CD40-CD40Ligand (CD154) pathway blockade on antidonor immunity, cytokine production, microchimerism and heart graft survival in BALB/c (H2d) recipients of fully allogeneic C57BL/10 (H2b) FL-mobilized BM (FL-BM) or normal BM. Anti-CD40L mAb strongly suppressed anti-donor T-cell proliferative responses in recipients of either normal or FL-BM, but was less efficient in inhibiting antidonor cytolytic T-cell (CTL) activity, especially in recipients of FL-BM. Interestingly, CD40L blockade was more effective in recipients of multiple compared with single donor BM infusions. Anti-donor cytokine responses revealed complete impairment of IFN-gamma, IL-4 and IL-10 production in recipients of normal BM and CD40L mAb. By contrast, and in agreement with the CTL data, mice given FL-BM retained ability to produce IFN-gamma CD40-CD40L blockade did not promote microchimerism, as evidenced by immunohistology and real time polymerase chain reaction. Nevertheless, anti-CD40L mAb enhanced heart allograft survival in recipients of FL-BM, but the effect was inferior to that achieved with normal BM. These data provide insight into the influence of growth factor-expanded donor BM and costimulation blockade on antidonor immune reactivity and transplant outcome. The comparatively poor outcome obtained using FL-BM plus anti-CD40L mAb in this model may be ascribed to the failure of effectively interdicting antidonor CTL activity.

Promotion of skin graft tolerance across MHC barriers by mobilization of dendritic cells in donor hemopoietic cell infusions.

Flt3 ligand (FL) dramatically increases the number of immunostimulatory dendritic cells (DC) and their precursors in bone marrow (BM) and secondary lymphoid tissues. Herein we tested the ability of FL-mobilized donor hemopoietic cells to promote induction of skin graft tolerance across full MHC barriers. C57BL/10 (B10; H2(b), IE(-)) mice were given 10(8) spleen cells (SC) from normal or FL-treated, H-2-mismatched B10.D2 (H2(d), IE(+)) donors i.v. on day 0, 200 mg/kg i.p. cyclophosphamide on day 2, and 10(7) T cell-depleted BM cells from B10.D2 mice on day 3. B10.D2 skin grafting was performed on day 14. Indefinite allograft survival (100 days) was induced in recipients of FL-SC, but not in mice given normal SC. Tolerance was associated with blood macrochimerism and was confirmed by second-set skin grafting with donor skin 100 days after the first graft. In tolerant mice, peripheral donor-reactive T cells expressing TCR Vbeta11 were deleted selectively. Immunocompetence of tolerant FL-SC-treated mice was proven by rapid rejection of third-party skin grafts. To our knowledge this is the first report that mobilization of DC in donor cell infusions can be used to induce skin graft tolerance across MHC barriers, accompanied by specific deletion of donor-reactive T cells.

Retroviral delivery of transforming growth factor-beta1 to myeloid dendritic cells: inhibition of T-cell priming ability and influence on allograft survival.

BACKGROUND: Transforming growth factor (TGF)-beta inhibits the maturation and function of antigen-presenting cells. Our purpose was to evaluate the impact of retroviral delivery of human TGF-beta1 to murine myeloid dendritic cell (DC) progenitors on (i) their in vitro properties, (ii) their in vivo function, and (iii) their influence on organ allograft survival. METHODS: C57BL10 (B10; H2b) bone marrow cells were lineage depleted and stimulated with granulocyte-macrophage colony-stimulating factor for 6 days. Replicating DC progenitors were transduced on days 2, 3, and 4 of culture by ecotropic retrovirus encoding human TGF-beta1 using centrifugal enhancement. Secretion of TGF-beta1 and other cytokines was quantified by enzyme immunoassay. Allogeneic C3H/HeJ (C3H; H2k) T-cell proliferative responses and generation of cytotoxic T lymphocytes in mixed leukocyte reaction were determined by [3H]thymidine incorporation and 51Cr release assays, respectively. DC migration was analyzed by immunohistochemistry, and their impact on survival of intra-abdominal heart transplants was determined. RESULTS: Maximal TGF-beta1 transduction efficiency was 60%. The TGF-beta-transduced DC showed pronounced impairment (>80%) of T-cell allostimulatory activity in vitro. After their IV injection, B10 TGF-beta-transduced DC (IAb+) were detected in T-cell areas of spleens of allogeneic C3H recipients. Splenic T-cell responses to donor alloantigens of mice that received TGF-beta-transduced DC were severely impaired. This was accompanied by marked inhibition of interleukin-2 and interferon-gamma production in response to restimulation with donor alloantigen. Survival of B10 cardiac allografts in C3H mice given B10 TGF-beta-transduced DC (2x106 IV, 7 days before transplantation), was extended modestly but significantly. CONCLUSION: Retroviral transduction of myeloid DC progenitors to overexpress TGF-beta is associated with marked impairment of their T-cell allostimulatory activity but with only modest prolongation of organ allograft survival.