ECA-immunogenicity of Proteus mirabilis strains

Introduction  Bacteria of the genus Proteus are opportunistic pathogens and cause mainly urinary tract infections. They also play a role in the pathogenesis of reactive arthritis (RA). Patients suffering from Yersinia-triggered RA often carry high titers of antibodies specific to enterobacterial common antigen (ECA). The immunogenicity of ECA has not received much attention thus far and studies have focused mainly on the ECA of Escherichia coli and Yersinia enterocolitica. In this paper the ECA-immunogenicity of Proteus mirabilis is elucidated using two wild-type strains (S1959 and O28) as well as their rough (R) derivative strains R110/1959, which expresses lipopolysaccharide (LPS) with a full core, and R4/O28, which expresses LPS with only an inner core. Materials and Methods  Rabbit polyclonal antisera were produced by immunization with boiled suspensions of the four P. mirabilis strains. The antisera were tested for the presence of antibodies specific to ECA by Western blotting using glycerophospholipid- linked ECA (ECA _PG ) of Salmonella montevideo as antigen. Lipopolysaccharide (LPS) was isolated from the four strains by the hot phenol/water procedure in which ECA _PG is co-extracted with LPS and by the phenol/chloroform/petroleum ether extraction that results in the isolation of LPS and/or LPS-linked ECA (ECA _LPS ) free of ECA _PG . The LPS preparations were tested for the presence of ECA by Western blotting using ECA-specific antibodies. Results  The results demonstrated that all four P. mirabilis strains were ECA immunogenic. The rabbit antisera immunized by the four strains all contained ECA-specific antibodies. Analysis of the LPS preparations demonstrated that the P. mirabilis wild-type strains O28 and S1959 and the Ra mutant strain R110/1959 expressed ECA _LPS , suggesting that it induced the anti-ECA antibody responses. Only the presence of ECA _PG could be demonstrated in the Rc mutant strain R4/O28. Conclusions  These results therefore suggest that, similar to E. coli, LPS with a full core is also required as the acceptor of ECA for P. mirabilis strains to produce ECA _LPS . Since ECA _PG is not immunogenic unless combined with some proteins, it is likely that ECA _PG -protein complexes formed during the intravenous immunization with the Rc mutant strain R4/O28.     

Acinetobacter and E. coli lipopolysaccharide preparations comparative mitogenicity and induction in vitro of immunoglobulin synthesis in adult and neonatal pig lymphocytes.

Lipopolysaccharide (LPS) was prepared by phenol/water extraction of bacterial membranes prepared from Acinetobacter and Escherichia coli. The mitogenicity of laboratory-prepared LPS was significantly greater than that of commercial E. coli LPS for pig, sheep, calf and rat lymphocytes, assayed as [3H]-thymidine incorporation. Mouse lymphocytes responded well to commercial LPS and no greater response was obtained with other LPS preparations. A small proportion (14%) of the Acinetobacter LPS preparations was soluble in aqueous medium, the remainder comprising membraneous fragments of variable form and size. It is suggested that the insoluble presentation of LPS to cells may contribute to the improved mitogenicity compared with wholly soluble LPS. Acinetobacter LPS preparations were used to induce synthesis and secretion in vitro of immunoglobulin by adult blood lymphocytes and pre-suckled, neonatal spleen cells of the pig. IgM was the dominant class of immunoglobulin secreted. This work thus demonstrated that virgin, unprimed B cells could be induced into immunoglobulin secretion by mitogen stimulation.     

Histopathology and cell-mediated immune reactivity in halothane-associated lymphomagenesis and autoimmunity in BXSBMp and MRLMp mice

The present study examined whether halothane anesthesia could alter immunoregulation in autoimmune disease-prone $\text{BXSB}\text{Mp}$ and $\text{MRL}\text{Mp}$ mice. This was judged by histopathological examination of lymph nodes and by studies on mitogen-induced transformation of splenic lymphocytes. The development of lymphoma-like disease in BXSB males and the appearance of lymphoproliferation and systemic lupus erythematosus-like disease in $\text{MRL}\text{Mp}\text{lpr}\text{lpr}$ mice were revealed by histologic examination of lymph nodes and other tissues and by identification of immune complexes in kidney sections by immunofluorescence. Splenic lymphocytes from $\text{MRL}\text{Mp}$ +/+ mice exposed to halothane showed a 44% reduction in mean stimulation indices (MSI) compared to exposure to O₂. Likewise, the MSI of splenic lymphocytes from $\text{MRL}\text{Mp}\text{lpr}\text{lpr}$ mice exposed to halothane were reduced to 42% when compared with those from $\text{MRL}\text{Mp}\text{lpr}\text{lpr}$ mice subjected to O₂. In accord with the MRL results, halothane induced a 38% reduction in the stimulation indices of autoimmune and lymphoma-like disease-prone BXSB mice and a 27% decline in the lymphocyte proliferative responsiveness of autoimmune disease-insusceptible control $\text{C57B1}\text{6}$ mouse spleen cells, compared to their respective O₂ control values. Results demonstrate that halothane facilitated an earlier onset in loss of immunoregulation associated with development of autoimmunity and lymphomagenesis in genetically disease-prone mice receiving halothane than in those receiving oxygen.     

Identification and characterization of a novel intelectin in the digestive tract of Xenopus laevis

The intelectin (Intl) family is a group of secretory lectins in chordates that serve multiple functions, including innate immunity, through Ca²⁺-dependent recognition of carbohydrate chains. Although six Intl family lectins have so far been reported in Xenopus laevis, none have been identified in the intestine. Using a monoclonal antibody to the Xenopus embryonic epidermal lectin (XEEL or Intl-1), I identified cross-reactive proteins in the intestines. The proteins were purified by affinity chromatography on a galactose-Sepharose column and found to be oligomers consisting of N-glycosylated 39 kDa and 40.5 kDa subunit peptides. N-terminal amino acid sequencing of these peptides, followed by cDNA cloning, identified two novel Intls (designated Intl-3 and Intl-4) that showed 59–79% amino acid identities with known Xenopus Intl family proteins. From the amino acid sequence, immunoreactivity, and properties of the recombinant protein, Intl-3 was considered the intestinal lectin identified by the anti-XEEL antibody. The purified Intl-3 protein could potentially bind to Escherichia coli and its lipopolysaccharides (LPS), and to Staphylococcus aureus and its peptidoglycans, depending on Ca²⁺. In addition, the Intl-3 protein agglutinated E. coli cells in the presence of Ca²⁺. Intraperitoneal injection of LPS increased the intestinal and rectal contents of Intl-3 and XCL-1 (or 35K serum lectin) proteins within three days; however, unlike XCL-1, Intl-3 was detectable in neither the sera nor the other tissues regardless of LPS stimulation. Immunohistochemical analyses revealed accumulation of the Intl-3 protein in mucus secretory granules of intestinal goblet cells. The results of this study suggest that Xenopus Intl-3 is involved in the innate immune protection of the digestive tract against bacterial infections.     

Blastogenesis of Human Lymphocytes by Endotoxin

Lipopolysaccharides of S. typhimurium, S. enteritidis and E. coli at wide range of concentrations were used to induce blastogenesis in human and mouse (nude) lymphocytes. Human lymphocytes from peripheral blood showed positive response to at least one source of LPS (stimulation index of 2-9). The optimum concentration resulting in maximum stimulation varied with different individual, sources and concentrations of LPS used. Lymphocytes from cord blood failed to respond to LPS, but had positive response to PHA. All three LPS produced about equally strong mitogenic effects on mouse spleen cells.     

Effect of hydrocortisone on immune system of the lizard,Chalcidesocellatus II. Differential action on T and B lymphocytes

Lymphocytes of thymus, spleen, peripheral blood (PB) and bone marrow (BM) collected from adult lizards, $\text{Chalcides}$$\text{ocellatus}$ were cultured for 24 hr in the presence of 10^?3M hydrocortisone acetate (HC) in order to assess the effect of in vitro HC on lizard T and B cell viability. The results indicated that HC induced stepwise, time-dependent mortality of the majority of thymocytes carrying T cell specific antigen(s) (TSA), 30–50% of T cells of spleen, PB and BM, and of a proportion of splenic B lymphocytes. Administration of 1 mg/g body weight HC to adult $\text{Ch}$. $\text{ocellatus}$ lead to depletion of all TSA⁺ thymocytes. In contrast, T lymphocytes in the peripheral lymphoid compartments revealed both sensitivity and resistance to HC; similarly, B lymphocytes constituted susceptible and resistant subpopulations.     

Further Characterization of an Interleukin-2-1Ike Cytokine Produced by Xenopus Laevis T Lymphocytes

A T-cell growth factor (TCGF) is produced by antigen- or mitogen-stimulated T lymphocytes from the South African clawed frog Xenopus laevis. This study further defines the physical and biological properties of this cytokine and demonstrates that TCGF is biochemically similar to mammalian interleukin-2 (IL-2). Biologically active TCGF eluted from SDS-PAGE displays a M_r of 16 kD and lectin-affinity chromatography indicates that the three-dimensionmal configuration of carbohydrates on TCGF and human IL-2 is similar. Secretion of TCGF is detectable 1 day after stimulation of splenocytes with the T-cell mitogen phytohemagglutinin (PHA) and peaks following 2 to 3 days of stimulation. Finally, despite the biological and physical similarities between Xenopus TCGF and mammalian IL-2, anti-human IL-2 monoclonal antibodies do not recognize Xenopus TCGF.     

Purification and some properties of beet yellow virus

Beet yellows virus was purified by a method, based on ultracentrifuging at 35,000 g, that preserved the normal length of the virus particle and eliminated a uv-absorbing contaminant retained by previously described methods. The purified particles had a modal length in shadowed preparations of 1250 nm and sedimented in the analytical centrifuge usually as one component at 130 S. Purified preparations had an $\text{A}{}_{260}\text{A}{}_{280}$ ratio of 1.44 and an extinction coefficient at 260 nm of 2.9. In Cs₂SO₄ isopycnic banding, the virus had a density of 1.285 g/cm³, while in CsCl two bands were produced at 1.307 and 1.312 g/cm³.     

The invitro generation of an antibacterial activity from the fat body and hemolymph of non-immunized larvae of Galleriamellonella

A state of immunity in $\text{Galleria}$$\text{mellonella}$ against the pathogen $\text{Pseudomonas}$$\text{aeruginosa}$ is known to be induced by the injection of lipopolysaccharide (LPS), isolated from the homologous organism. An $\text{in}$$\text{vitro}$ mixture of the LPS and whole or cell-free hemolymph from non-immunized larvae is not antibacterial. $\text{In}$$\text{vitro}$ mixtures of fat body and cell-free hemolymph from non-immunized larvae, incubated at 25°C for 20 hours generated a proteinaceous antibacterial activity. The generation of this activity was enhanced by the presence in the incubation mixture of LPS and/or hemocytes from non-immunized larvae. It is suggested that LPS causes the release of a hemocyte factor(s) which acts in conjunction with or directly on the fat body resulting in an enhanced production of antibacterial factors.     

Thymus-dependent immune functions in chickens bursectomized with colchicine applied to the anal lips

Thymus-dependent immune functions were investigated in chickens bursectomized neonatally with colchicine solution given $\text{per anum}$. Antibody responses to thymus-dependent antigens sheep red blood cells (SRBC) and human gammaglobulin (HGG) were delayed, reaching the normal level after the third antigen stimulation. Also the mitogenic responses of peripheral blood lymphocytes were preserved, and no changes in the thymic morphology were found. In contrast, antibody responses to bursa-dependent antigen $\text{Brucella abortus}$ were low and the switch of immunoglobulin isotypes from IgM to IgA and IgG was disturbed. It can be concluded that neonatal bursectomy with cloacal administration of colchicine does not significantly affect T celi functions, whereas B cell functions are partially deficient.     

Antigen trapping cells in xenopus laevis: Tissue distribution

In the spleen of $\text{Xenopus laevis}$, antigen trapping (XL) cells are located at the periphery of the B-lymphocyte follicles and extend pseudopods through the boundary cell layer into the T-lymphocyte rich marginal zone to provide an anatomical bridge between the B-and T-cell compartments. The relationship between XL cells and nonsplenic antigen trapping cells was probed with a T-“independent” antigen, $\text{Aeromonas salmonicida}$. ¹²⁵I-$\text{Aeromonas}$ were trapped in high concentrations in marrow, spleen and liver. Splenic trapping was most efficient at low doses and liver trapping at high doses of antigen. Immunofluorescence stains demonstrated large cells with trapped $\text{Aeromonas}$ in spleen, marrow, liver, thymus, gut, kidney and blood. An antisera to XL cells stained large cells in all of these organs as well as vascular endothelium of the spleen, thymus, gut and lung. Thus, $\text{Xenopus}$ has a widely distributed system of antigen trapping cells that may be related to some vascular endothelial cells.     

Immunoglobulin bearing blood leucocytes in the Pacific hagfish

The occurrence of immunoglobulin (Ig) bearing leucocytes in the blood of the Pacific hagfish, $\text{Eptatretus}$$\text{stoutii}$, was examined using a murine monoclonal antibody (45.3) and a rabbit antiserum specific for hagfish serum Ig. Binding of antibody 45.3 to hagfish leucocytes assessed by radioimmunoassay was inhibited by preincubation of antibody with purified serum Ig thus verifying the presence of cell surface Ig cross reactive with serum Ig. The monoclonal antibody identified approximately 65% of blood leucocytes as Ig+ve while the rabbit antiserum indicated 81% Ig+ve cells. Both antibody preparations failed to react specifically with cells from mouse, horned shark, tunicate or sea star; this indicates the distinctive nature of hagfish Ig. The high percentage of blood cells bearing surface Ig in the hagfish raises the possibility that lymphocyte divergence to separate B and T pathways may not have occurred in this most primitive vertebrate. Alternatively, an Ig-like specificity characteristic of both “T” and “B” lymphocytes may have been detected. In any event, a subset of Ig negative leucocytes is evident in hagfish.     

Specific activity of carp antisera against β2-microglobulin (β2m) and evidence for a β2m homologue in carp (Cyprinuscarpio)

Antisera against rabbit and human β₂-microglobulin were produced in carp ($\text{Cyprinus}$$\text{carpio}$). These antisera were found to be specific for β₂m, and detected β₂m epitopes in various vertebrates including guinea-pig, cow, mouse, rat, dog, horse, cat, sheep, goat, parrot, chicken and frog. In addition, these antisera were also inhibited by an extract from oyster. The differences between the reactivity of these two antisera and the ability of the anti-rabbit β₂m to distinguish between mouse and rat β₂m's suggested that the carp itself carries β₂m epitopes. This hypothesis was corroborated by the ability of a variety of mammalian anti-β₂m antisera to induce a mitogenic response in carp leukocytes. Finally, a rabbit antiserum against dog β₂m was shown to precipitate a low molecular weight molecule from carp leukocyte extract. This molecule is likely to represent the carp homologue of mammalian β₂m.     

RNA contents of abnormally long particles of certain strains of alfalfa mosaic virus

A nucleoprotein particle longer than bottom component occurring in small amounts in preparations of the alfalfa mosaic virus (AMV) strain $\text{15}\text{64}$ has been partially purified by gradient centrifugation and characterized. The particle has the same shape, width, and RNA percentage as the major components of the virus. Its length and sedimentation coefficient are 87 nm and 113 S, respectively. It does not contain a single abnormally long RNA molecule, but two molecules of middle component RNA. Electrophoresis in polyacrylamide gel revealed that in addition to this type of particle other nucleoproteins containing different sets of RNA molecules occur in trace amounts in preparations of strain $\text{15}\text{64}$.Particles with a length of up to about 400 nm of strain VRU have also been studied after they were freed from normal length particles by gradient centrifugation. In accordance with the results of others it has been found that the abnormally long particles did not contain any RNA molecules longer than bottom component RNA.     

Endotoxin Tolerance Induced by Lipopolysaccharides Derived from Porphyromonas gingivalis and Escherichia coli: Alternations in Toll-Like Receptor 2 and 4 Signaling Pathway

Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a hyporesponsive state to subsequent challenge, which is termed endotoxin tolerance. In this experiment, we studied the cytokine production in THP-1 cells upon single or repeated Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) or Escherichia coli (E. coli) LPS stimulation by ELISA. In addition, the protein expression profiles of Toll-like receptor 2 (TLR2), TLR4, IL-1 receptor-associated kinase 4 (IRAK4) and IRAK-M and the gene expression changes of Toll-interacting protein (Tollip) and suppressor of cytokine-signaling-1 (SOCS1) were explored to identify possible mechanisms for changes in cytokine secretion. After repeated stimulation with P. gingivalis LPS or E. coli LPS, secretions of TNF-α and IL-1β were decreased significantly compared with those following single challenge, while the levels of IL-10 were increased (p?<?0.05). Only comparable levels of IL-8 were confirmed in P. gingivalis LPS-tolerized cells (p?>?0.05). In addition, severe downregulation of TLR2 was detected in THP-1 cells retreated with P. gingivalis LPS, and the reduction of TLR4 expression was observed in cells restimulated with E. coli LPS (p?<?0.05). Precondition with P. gingivalis LPS or E. coli LPS also led to an enhancement of IRAK-M and SOCS1, while maintaining the expressions of IRAK4 and Tollip. This pattern of cytokine production indicates the different effects of endotoxin tolerance triggered by P. gingivalis LPS and E. coli LPS, which might contribute to limiting inflammatory damage. Moreover, TLR2, TLR4, IRAK-M, and SOCS1 might play important roles in developing tolerance.     

Studies of immunogenicity of Westphal lipopolysaccharides from Pasteurella multocida in mice.

The immunogenicity of Westphal lipopolysaccharides (LPS) from 2 strains of Pasteurella multocida was tested in mice. When lipopolysaccharide from P. multocida strain P-1234 (bovine source) was inoculated into CF1 or $\text{C}\text{3}\text{H}\text{HeJ}$ mice, less than 20 per cent of the mice survived homologous challenge infection 4 weeks later. However, 90 per cent, or more, of the CF1 or $\text{C}\text{3}\text{H}\text{HeJ}$ mice survived homologous challenge infection after immunization with formolized P-1234 bacteria. Sera from the CF1 mice, but not $\text{C3H}\text{HeJ}$ mice, formed precipitin lines against P-1234 LPS in an agar gel diffusion test.Hyperimmune sera from rabbits inoculated with formolized P-1234 bacteria passively protected more than 80 per cent of the CF1 mice against challenge infection; however, sera from rabbits inoculated with P-1234 LPS or LPS adsorbed to sheep red blood cells protected less than 20 per cent of the challenged mice.Absorption of rabbit anti-whole cell sera with LPS slightly reduced the capacity of serum to passively protect mice against challenge exposure.     

Exogenous estradiol and sexual receptivity in photoinhibited female Lemur catta.

Nine Lemur catta females whose ovarian cycles had been inhibited by exposure to long daylength (18L:6D) for 220 days were implanted with Silastic tubules (4.0 cm×0.132 in. i.d.) containing estradiol-17β (E₂) while caged continuously with a male. Radioimmunoassay demonstrated a transitory peak (261.8 pg/ml ± 2.42 SEM) in mean plasma E₂ concentration $\text{4-}\text{1}\text{2}$ hr after insertion of implants that was followed by relatively constant hormone titers (approximately 100 pg/ml) until removal of implants. Five females received an ejaculation after a decline in plasma E₂ concentration. These data demonstrate that a consistent, predictable and reproducible plasma estradiol hormone profile follows insertion of E₂ implants. These data also lend support to the hypothesis that declining estradiol titers activate sexual receptivity in L. catta.     

Maturation of transplantation antigens in Ambystomamexicanum

Both juvenile (14–16 week) and adult (18 month) $\text{Ambystoma}$$\text{mexicanum}$ reject skin allografts from adult $\text{Ambystoma}$ more speedily than they reject such grafts from juvenile axolotls. Donor-specific histocompatibility antigen, prepared from splenocytes, is more effective in inhibiting adult host splenocyte migration when the antigen is prepared from spleen cells from adult, rather than from juvenile $\text{Ambystoma}$. The thymus is fully developed in juvenile $\text{Ambystoma}$, suggesting that the delayed kinetics of rejection of juvenile allografts reflects immaturity in the expression of histocompatibility antigens to donor skin cells.     

Comparative electron microscopic studies on Pseudomonas aeruginosa endotoxin

The endotoxin from Pseudomonas aeruginosa C₉ was obtained by five standard methods of extraction for comparative electron microscopic studies. Observation of the five preparations demonstrated the presence of two major types of structures. The first of these was seen in endotoxins prepared by trichloroacetic acid, ethyl ether, hot water, and ethylenediamineteraacetate-lysozyme extraction, and consisted of discrete spherules containing smaller spherules within and having a homogeneous staining centre and rod-like border. The other morphologic type was seen only in preparations obtained by the aqueous phenol technique and consisted of pleomorphic staining material and rodlets. Preparations isolated by trichloroacetic acid extraction by phenol extraction. Loss in structural integrity was encountered upon exposure to polymxin B, colistin sulfate and carbenicillin, but not with other antibiotics tested.