Phylogenetic Groups and Pathogenicity Island Markers in Fecal Escherichia coli Isolates from Asymptomatic Humans in China

The study of phylogenetic groups and pathogenicity island (PAI) markers in commensal Escherichia coli strains from asymptomatic Chinese people showed that group A strains are the most common and that nearly half of all fecal strains which were randomly selected harbor PAIs.Escherichia coli is a well-diversified commensal species in the intestine of healthy humans but also includes intestinal or extraintestinal pathogens. It has been reported that pathogenic E. coli may be derived from fecal strains by acquisition of virulence determinants (11). The relationship between the E. coli genetic background and the acquisition of virulence factors is now better understood (1, 5). Extraintestinal E. coli strains may harbor several virulence factors, such as adhesins, fimbriae, and hemolysin, which can contribute to bacterial pathogenesis. These traits are usually encoded on pathogenicity islands (PAIs), which have been studied in pathogenic E. coli previously (15). The E. coli population includes 4 major phylogroups (A, B₁, B₂, and D) (2). Pathogenic strains belong mainly to groups B₂ and D, while most fecal isolates belong to groups A and B₁. Strains of groups B₂ and D often carry virulence factors that are lacking in group A and B₁ strains (3, 9, 13).In this study, we examined the distribution of phylogroups and the prevalence of PAIs in commensal E. coli strains isolated from asymptomatic persons in one region of China.     

Selective proliferation of T cells in the mixed lymphocyte interaction

The contribution of avian thymus-derived (T) and bursa-derived (B₂) cells to the proliferating cell population in mixed lymphocyte cultures (MLC) was evaluated. When spleen cells of chickens containing chromosomally marked T and B₂ cells were stimulated in a one-way MLC by mitomycin C blocked allogeneic spleen cells, only T cells proliferated during a 4–9 day culture period. No evidence for significant recruitment of B₂ cells, expressed as proliferation of B₂ cells, was found. The initial viability and proliferative potential of B₂ cells was shown by a substantial and selective B₂ cell response to anti-immunoglobulin serum.     

Adherence of Escherichia coli O157:H7 Mutants In Vitro and in Ligated Pig Intestines

There are contradictory literature reports on the role of verotoxin (VT) in adherence of enterohemorrhagic Escherichia coli O157:H7 (O157 EHEC) to intestinal epithelium. There are reports that putative virulence genes of O island 7 (OI-7), OI-15, and OI-48 of this pathogen may also affect adherence in vitro. Therefore, mutants of vt2 and segments of OI-7 and genes aidA₁₅ (gene from OI-15) and aidA₄₈ (gene from OI-48) were generated and evaluated for adherence in vitro to cultured human HEp-2 and porcine jejunal epithelial (IPEC-J2) cells and in vivo to enterocytes in pig ileal loops. VT2-negative mutants showed significant decreases in adherence to both HEp-2 and IPEC-J2 cells and to enterocytes in pig ileal loops; complementation only partially restored VT2 production but fully restored the adherence to the wild-type level on cultured cells. Deletion of OI-7 and aidA₄₈ had no effect on adherence, whereas deletion of aidA₁₅ resulted in a significant decrease in adherence in pig ileal loops but not to the cultured cells. This investigation supports the findings that VT2 plays a role in adherence, shows that results obtained in adherence of E. coli O157:H7 in vivo may differ from those obtained in vitro, and identified AIDA-15 as having a role in adherence of E. coli O157:H7.Escherichia coli O157:H7 is the prototypical enterohemorrhagic E. coli (EHEC) strain and is the most common serotype associated with large outbreaks and sporadic cases of hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS) (25). It is well established that EHEC O157:H7 can colonize the intestine of humans and animals and that adherence to intestinal epithelial cells occurs through the formation of attaching-and-effacing (AE) lesions, which is a critical early step in infection. Some researchers have suggested that EHEC uses fimbriae to make the initial contact with epithelial cells, prior to intimate attachment mediated by locus of enterocyte effacemen-encoded proteins (15). Several potential adherence factors of EHEC O157:H7 have been described, but only the outer membrane protein intimin has been demonstrated to play a role in intestinal colonization in animal models (16). Intimin mediates the intimate adherence component of the AE lesion by binding to the translocated intimin receptor Tir, resulting in close attachment of the bacteria to the host cell membrane (17). Intimin can also bind to β1 integrins and nucleolin on host cells (9, 36). Severe damage due to infection with EHEC is attributable to the cytotoxic verotoxin (VT), which damages epithelial and endothelial cells, leading to bloody diarrhea and HUS (16). Several investigators have reported that VT does not play a role in colonization of the intestine (2, 4, 34). However, Robinson et al. (30) reported recently that VT enhances adherence to epithelial cells and colonization of the mouse intestine by E. coli O157:H7. Therefore, the present study examined the involvement of VT in adherence in vitro and in vivo.Several putative virulence genes have been identified in O islands (OIs) in EHEC O157:H7 strain EDL 933 (26), including those encoding Iha and AIDA-I in OI-43/48, AIDA-I in OI-15, and a ClpB chaperone protein and a putative macrophage toxin in OI-7 (26, 27). OI-7 also contains many unknown open reading frames (ORFs) whose function in the pathogenesis of EHEC O157:H7 has not been investigated. Iha, an adherence-conferring outer membrane protein similar to IrgA (the product of iron-regulated gene A) (38), is a virulence factor in uropathogenic E. coli strain CFT073 (14). AIDA-I, encoded by aidA, was first identified in EPEC and confers the capacity for diffuse adhesion of the bacteria to epithelial cells (1). AIDA-I-like adhesins from OI-15 and OI-43/48 show 55% and 68% homology, respectively, to the AIDA-I of EPEC (26, 27). All three AIDA proteins show characteristics of an autotransporter membrane protein with a β-barrel structure (20), which is exposed at the surface of the bacteria (13). These observations suggest that the two homologs of AIDA-I may also function as adhesins in EHEC O157:H7; however, the roles of the AIDA-I-like adhesins in EHEC have yet to be determined.EHEC O157:H7 has been isolated from pigs, and conventional pigs are a permissive host and therefore a potential reservoir for human infection with EHEC O157:H7 (8). One recent family outbreak was associated with pork salami (3). Pigs are highly relevant models for the study of virulence of EHEC O157:H7 in humans and have been extensively used to characterize putative virulence factors and to investigate the pathogenesis of EHEC O157:H7 and other verotoxigenic E. coli strains (6, 11, 21). The present study was designed to examine VT2-negative mutants, OI-7 deletions, and aidA knockouts from OI-15 and OI-48 of EHEC O157:H7 in vitro and in the pig intestines for their roles in adherence.     

Immunologic unresponsiveness of mouse spleen sensitized to allogenic tumors.

CS7BL/6 mice were sensitized with an ip injection of allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the tumor allosensitized mice were cultured and tested for their responsiveness to mitogens and alloantigens, and for their ability to generate cytotoxic cells in vitro. The results indicate that 15 day tumor-sensitized spleen cells are hypo-responsive in mixed lymphocyte culture (MLC) with DBA/2 or AKR as stimulating spleen cells. The cells which are hypo-responsive in MLC can proliferate in response to mitogens and they also can generate cytotoxic cells in vitro. MLC reactivity recovers in about 2–3 months which is $\text{1}\text{1}\text{2}\text{–2}\text{1}\text{2}$ months after the mice have rejected their tumors. The mechanism of MLC hypo-responsiveness was investigated. The results suggest the presence of a suppressor cell which does not appear to be a macrophage or a B-cell. The suppressor cell can be separated from the cytotoxic cell and therefore appears to be a noncytotoxic T-cell.     

Functional heterogeneity among the T-derived lymphocytes of the mouse: III. Helper and suppressor T-cells activated by concanavalin A

We have studied the properties of T-cells which when activated by concanavalin A (Con A) either suppress or help the in vitro humoral response of mouse spleen cells. Previously established criteria for the T-cell populations, T₁ and T₂ were applied. T₁ cells were defined by their short half-life (2–3 wk) after adult thymectomy (ATx) and their resistance to small doses of antithymocyte serum (ATS). T₂ cells were defined by their long half-life (~15 wk) and their high sensitivity to ATS. T-cells which could be activated by Con A to help the response to the thymus-dependent antigen, sheep red blood cells, were found mainly in the T₂ subpopulation. T-cells which could be activated by Con A to suppress the response to the thymus-independent antigen, trinitrophenyl-lipopolysaccharide (TNP-LPS), were found within both the T₁ and T₂ subpopulations.These results, our previous results, and those of others suggest that the T-cell responses to phytomitogens distinguish precursors committed to different functions, while the T₁ and T₂ classifications distinguish T-cells at different stages of maturation.     

Suppressor factor from tumor-allosensitized spleen cells--its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF)² which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.     

Isogeneic and allogeneic lymphocyte interactions may be controlled by cell surface immunoglobulin tropism.

A study of allogeneic MLC (mixed lymphocyte culture) and two types of isogeneic MLC has been conducted in which subsets of B-cells were serologically removed from pools prior to using the remainder as stimulator cells. Cellular division in the two types of ILC (isogeneic lymphocyte culture) was found to be triggered by lymphocytes with IgG₁ on their surfaces. In contrast, the stimulator cells in ALC (allogeneic lymphocyte culture) possessed membrane-bound IgG_2A and/or possibly IgG_2B. Splenic T-cells were incapable of stimulating replication of splenic or lymph nodal T-cells in the absence of B-cells. Splenic T-cell preparations served as weak stimulators of other allogeneic T-cells but only when B-cells, either isogeneic or allogeneic to the stimulator T-cells, were present. We propose that stimulation in the MLC occurs in two distinct steps. First, immunoglobulins on cell surfaces may function to bring appropriate subsets of cells together. Next, antigenic recognition occurs that results in blastogenesis. Furthermore, the tropism or attraction that certain immunoglobulins have for some cell types may determine which subsets of cells participate in allogeneic and which take part in isogeneic MLC.     

Direct evidence for the response of B and T cells to pokeweed mitogen

Chicken spleen cells containing chromosomally marked thymus derived (T) and bursa-derived (B₂) cells were evaluated for their ability to respond to pokeweed mitogen (PWM), concanavalin A (Con A), and to anti-immunoglobulin serum during a 4-day culture period. The results indicate that soluble PWM induces a proliferative response of B₂ cells in addition to a predominant T cell response. The PWM-induced B₂ cell proliferative response was clearly detected only at 4 days after culture initiation. Soluble Con A did not induce detectable proliferation of B₂ cells and stimulated T cells exclusively. In contrast, anti-immunoglobulin serum was a specific stimulant for B₂ cells under the culture conditions used.     

<Emphasis Type="Italic">Hymenobacter sedentarius</Emphasis> sp. nov., isolated from a soil

A novel Gram-negative and red-pinkish bacterium designated DG5B^T was isolated from a dry soil. Cells were rods that were catalase- and oxidase-positive, and non-motile. The strain was found to grow at temperatures from 10 to 30°C (optimum 25°C) and pH 6.0–8.0, (optimum pH 7) on R2A broth. 16S rRNA gene sequence (1,452 bp) analysis of this strain identified it as a member of the genus Hymenobacter that belongs to the class Cytophagia. The highest gene sequence similarities were with Hymenobacter arizonensis OR362-8^T (98.3%), Hymenobacter humi DG31A^T (97.6%), and Hymenobacter glaciei VUG-A130^T (96.6%). Strain DG5B^T exhibited <70% DNA-DNA relatedness with H. arizonensis (34.7 ± 7.0%; reciprocally, 29.7 ± 1.2%) and H. humi (39.4 ± 4.3%; reciprocally, 39.5 ± 3.3%) as a different genomic species, and its genomic DNA G+C content was 59.8%. Strain DG5B^T had the following chemotaxonomic characteristics: the major fatty acids are iso-C_15:0, anteiso-C_15:0, C_16:1ω5c, and summed feature 3 (C_16:1ω7c / C_16:1ω6c); polar lipid profile contained phosphatidylethanolamine (PE), unknown aminophospholipid (APL), unknown glycolipids (GL), unknown phospholipids (PL), and unknown polar lipids (L); the major quinone is MK-7. The absorbance peak of pigment is at 481.0 nm. Strain DG5B^T showed low-level resistance to gamma-ray irradiation. Phenotypic, chemotaxonomic, and genotypic properties indicated that isolate DG5B^T represents a novel species within the genus Hymenobacter for which the name Hymenobacter sedentarius sp. nov. is proposed. The type strain is DG5B^T (=KCTC 32524^T =JCM 19636^T).     

Modulation of allo-immune responsesin vivo andin vitro by sperm specific lactate dehydrogenase-C4

The role of sperm specific lactate dehydrogenase-C₄ (LDH-C₄) in allo-immune responses using mixed lymphocyte cultures (MLC) and cytotoxic T cell (CTL) generationin vitro and local graft versus host (LGVH) reaction and allograft enhancementin vivo has been ascertained. LDH was purified from testes (LDH-C₄) and kidney (LDH-B₄) of C57 Bl/Ks mice. MLC and CTL were performed using C57 Bl/Ks-anti A/J lymphocytes in presence of 10^–3-1 g LDH-B₄ or LDH-C₄ per culture. The MLC and CTL responses showed biphasic action depending on the dose of LDH-C₄. Early MLC culture gave significantly low stimulation index at 10^–2–10^–1 g LDH-C₄ as compared to non-treated control cultures. However, the MLC response in presence of LDH-C₄ was not different from the LDH-B₄ treated one which showed a similar biphasic trend. On the other hand,⁵¹Cr release from YAC-222 target cells was practically abolished by LDH-C₄ at 10^–3–1^–1 g, and this was strikingly different from LDH-B₄ or non-treated cultures. LGVH reactivity as performed by using C57 Bl/Ks lymphocytes along with LDH-C₄ in (C57 Bl/Ks x A/J) F₁ hybrids indicated a suppression of stimulation index in primary and secondary (i.e. preimmunized in presence of LDH-C₄ or LDH-B₄) LGVH. Allograft enhancement of Sa I (A/J) in C57 Bl/Ks mice in presence of LDH-C₄, was delayed slightly but significantly during primary or secondary transplantation reaction. The reaction of LDH-C₄ in the modulation of allo-immune responses was more specificin vivo thanin vitro since the B₄ isozyme did not modify LGVH and Sa1 allograft rejection. Resultsper se suggest that LDH-C₄ is immunosuppressive for cell mediated allo-immune responsesin vivo andin vitro.     

Analysis of the APX, PGD1 and R1G1B constitutive gene promoters in various organs over three homozygous generations of transgenic rice plants

We have previously characterized the constitutively active promoters of the APX, PGD1 and R1G1B genes in rice (Park et al. 2010 in J Exp Bot 61:2459–2467). To have potential crop biotechnology applications, gene promoters must be stably active over many generations. In our current study, we report our further detailed analysis of the APX, PGD1 and R1G1B gene promoters in various organs and tissues of transgenic rice plants for three (T_3–5) homozygous generations. The copy numbers in 37 transgenic lines that harbor promoter:gfp constructs were determined and promoter activities were measured by real-time qPCR. Analysis of the 37 lines revealed that 15 contained a single copy of one of the three promoter:gfp chimeric constructs. The promoter activity levels were generally higher in multi-copy lines, whereas variations in these levels over the T_3–5 generations studied were observed to be smaller in single-copy than in multi-copy lines. The three promoters were further found to be highly active in the whole plant body at both the vegetative and reproductive stages of plant growth, with the exception of the APX in the ovary and R1G1B in the pistil and filaments where zero or very low levels of activity were detected. Of note, the spatial activities of the PGD1 promoter were found to be strikingly similar to those of the ZmUbi1, a widely used constitutive promoter. Our comparison of promoter activities between T₃, T₄ and T₅ plants revealed that the APX, PGD1 and R1G1B promoters maintained their activities at comparable levels in leaves and roots over three homozygous generations and are therefore potentially viable alternative promoters for crop biotechnology applications.     

Induction of preferential cytotoxicity against allogeneic mouse lymphoma cells: in vitro and in vivo studies

The aim of this study was to activate, in mixed leukocyte/tumor cell cultures (MLTC), cytotoxic lymphocytes exhibiting preferential activity in vitro and in vivo towards allogeneic mouse lymphoma cells. Whereas the lymphoma target cells were readily lysed by the MLTC-derived lymphocytes, the cytotoxicity against the corresponding allogeneic concanavalin-A(ConA)-induced lymphoblasts was more than tenfold lower. Both activities were mediated by CD3⁺, TCR⁺, CD8⁺, CD4⁻ cytotoxic T cells (CTL). ConA-induced lymphoblasts were readily lysed by anti-Thy1.2 antibodies and complement, by CTL derived from mixed leukocyte cultures (MLC) and by the MLTC-derived CTL in the presence of ConA, indicating that the lymphoblasts are not merely less lysable than the lymphoma cells but that the latter are specifically recognized by the CTL. Lymphoblasts poorly competed with ⁵¹Cr-labeled lymphoma cells in a “cold”-target competition assay, suggesting that the MLTC-derived CTL largely recognize epitopes expressed only by the lymphoma cells. Furthermore, analysis of the cytotoxic activity of more than 500 MLTC-derived CTL oligoclones and over 30 clones revealed that one-third of them were cytotoxic only against the allogeneic lymphoma cells, one-third were reactive against both the lymphoma and the allogeneic lymphoblast target cells and the remainder were not cytotoxic at all. Upon injection into sublethally irradiated, lymphoma-bearing allogeneic mice, the MLTC-derived CTL cured 56% of the recipients and caused graft versus host disease (GVHD) is only 22%, whereas CTL activated in MLC against allogeneic splenocytes were therapeutically ineffective and caused lethal GVHD in 89% of the recipients. Although the therapeutic efficacy of the in vitro-generated antitumor CTL was demonstrated against experimental lymphoma lines, this strategy might prove effective in tumor immunotherapy in conjunction with other modalities. Received: 24 December 1998 / Accepted: 5 April 1999     

Synthesis, crystal structures, DNA-binding properties, cytotoxic and antioxidation activities of several new ternary copper(II) complexes of N,N′-(p-xylylene)di-alanine acid and 1,10-phenanthroline

Three novel ternary copper(II) complexes, [Cu₂(phen)₂(l-PDIAla)(H₂O)₂](ClO₄)₂·2.5H₂O (1), [Cu₄(phen)₆(d,l-PDIAla)(H₂O)₂](ClO₄)₆·3H₂O (2) and [Cu₂(phen)₂(d,l-PDIAla)(H₂O)](ClO₄)₂·0.5H₂O (3) (phen = 1,10-phenanthroline, H₂PDIAla = N,N’-(p-xylylene)di-alanine acid) have been synthesized and structurally characterized by single-crystal X-ray crystallography and other structural analysis. Spectrometric titrations, ethidium bromide displacement experiments, CD (circular dichroism) spectral analysis and viscosity measurements indicate that the three compounds, especially the complex 3, strongly bind to calf-thymus DNA (CT-DNA). The intrinsic binding constants of the ternary copper(II) complexes with CT-DNA are 0.89 × 10⁵, 1.14 × 10⁵ and 1.72 × 10⁵ M⁻¹, for 1, 2 and 3, respectively. Comparative cytotoxic activities of the copper(II) complexes are also determined by acid phosphatase assay. The results show that the ternary copper(II) complexes have significant cytotoxic activity against the HeLa (Cervical cancer), HepG2 (hepatocarcinoma), HL-60 cells (myeloid leukemia), A-549 cells (pulmonary carcinoma) and L02 (liver cells). Investigations of antioxidation properties show that all the copper(II) complexes have strong scavenging effects for hydroxyl radicals and superoxide radicals.     

Cytotoxicity of lymphoid cells induced by Maclura pomifera (MP) lectin.

The cytotoxic activity of lymphoid cells stimulated with Maclura pomifera (MP) lectin was investigated. Spleen cells of Lewis (LEW) or Brown Norway (BN) rats induced a cell-dependent release of ⁵¹Cr from syngeneic, allogeneic, and xenogeneic erythrocytes when incubated with MP for 4–16 hr. The activity of MP differed from that of concanavalin A (Con A). MP exhibited a greater activity with spleen cells while Con A was more active when bone marrow cells were tested. Activity induced by MP required the presence of the lectin for at least 4 hr and was inhibited by melibiose, an inhibitor of MP binding. MP also stimulated phagocytosis by peritoneal macrophages of LEW rats, but phagocytosis was not responsible for the cytotoxic effect measured by ⁵¹Cr release. The ability of aggressor cells to bind MP did not correlate with their cytotoxic activity. The cytotoxic activity of spleen cells from athymic nude mice was equivalent to that of cells from euthymic littermates when stimulated with MP.