PEPped up: induction of Candida albicans virulence by bacterial cell wall fragments

In this issue, Xu et al. (2008) report that Candida albicans, a prevalent fungal pathogen, expresses virulence-related attributes in response to bacterial peptidoglycan components, muramyl dipeptides, in human serum. Conserved elements in human and fungal detection of muramyl dipeptides highlight a common ability to perceive proximal bacterial populations.     

The induction of ethylene production from pear cell culture by cell wall fragments

Macerase, a pectinase-containing enzyme mixture, was used to digest cell walls isolated from cultured pear cells. Following digestion, the reaction mixture was boiled to inactivate enzymes. Addition of soluble aliquots of the mixture to suspension cultures of pear cells led to a rapid and transient production of ethylene by the cells.     

Extraction and properties of hemagglutinin from cell wall fragments of Fusobacterium nucleatum.

To study the hemagglutinin of Fusobacterium nucleatum, methods were sought to solubilize and purify this component. When cells of F. nucleatum were ruptured by passage through a French press, the fragments lost virtually all ability to agglutinate human erythrocytes. Extraction of the fragments with 2% Triton X-100 for 30 min at 22 degrees C restored hemagglutinating activity (HA). Hemagglutination by these fragments could be inhibited by arginine, as can hemagglutination by intact bacteria. Treatment of active cell wall fragments with pronase and 2% Triton X-100-EDTA at 37 degrees C or with pronase and 0.1% Triton X-100-EDTA at pH 10.0 allowed recovery of solubilized HA. The former HA was inhibited by arginine (arg+) whereas the latter was not (arg-). Fractionation of the arg+ extract by preparative isoelectric focusing showed that HA was recovered from the gel sections having a pH between 4.5 and 5.5. Hemagglutination by this preparation was still arg+. Chromatography of this hemagglutinin on DEAE-Sephadex increased the specific activity to high levels with a loss of inhibition by arginine. A fraction from the DEAE-Sephadex column containing 10,700 HA units per mg of protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Solubilization at 22 degrees C before electrophoresis revealed three Coomassie blue-staining bands which migrated with apparent molecular weights of about 21,000, 38,000 and 60,000. When the same DEAE fraction was boiled in sodium dodecyl sulfate, electrophoresis revealed only one band with an apparent molecular weight of 21,000.     

Purification of cell wall fragments by sucrose gradient centrifugation

Summary A procedure for purification of cell wall fragments was developed. The method utilizes sucrose density gradients to efficiently remove soluble enzyme and membrane contaminants from the cell wall. Purification at each stage was monitored biochemically by the removal of cytoplasmic associated markers and ultrastructurally by thorough electron microscopic examination of the isolated cell wall fractions. Cell walls purified by the procedure were compared to those purified by the more conventional multiple washing procedure.     

Observation of microvasculature in intestinal wall after burn in rats

OBJECTIVE: To investigate the relationship between intestinal mucosa injury and changes in stereologic parameters for its vessels. STUDY DESIGN: PAS stain was used to observe the basement membrane of intestinal epithelium. To measure the parameters of microvasculature by stereology, microvasculature was shown by alkaline phosphatase stain. Diamine oxidase (DAO) activity was determined by spectrophotometry. RESULTS: Red mucus became thinner than that of the control group; basement membrane was disrupted at 3 post burn hours (PBH). Changes became more serious at 6 and 12 PBH. Villus height and crypt depth appeared shortened at post burn. Microvasculature diameter was reduced. Mean section area at 12 PBH was significantly lower than that of the control group (p < 0.01). Mean circumference at 6 PBH was 19.43 +/- 1.59 microm and in the control group 23.84 +/- 4.17 microm. After burn injury, DAO activity in the intestine was reduced, falling to minimum at 12 PBH (p < 0.01), and in plasma was raised significantly, reaching peak at 12 PBH (p < 0.05). CONCLUSION: Our results suggest disruption of basement membrane, reduction of microvasculature diameter and DAO activity in intestine will appear at post burn, which induces decreased blood flow in intestine and damage to intestinal mucosa.     

The cell wall of Bacillus licheniformis N.C.T.C. 6346. Isolation of low-molecular-weight fragments from the soluble mucopeptide

1. Soluble mucopeptide was prepared by lysozyme treatment of acid-extracted walls of Bacillus licheniformis N.C.T.C. 6346 and separated into fractions differing in molecular size by chromatography on Sephadex G-25 and G-50. 2. About 16% of the weight of soluble mucopeptide has a weight-average molecular weight in excess of 20000. About one half has a weight-average molecular weight of less than 2000 and the balance of soluble mucopeptide is of intermediate size. 3. In the mucopeptide fractions isolated from Sephadex there is a correlation between the weight-average molecular weight, the number of non-reducing muramic acid residues and the proportion of diaminopimelic acid residues recovered after treatment with 1-fluoro-2,4-dinitrobenzene. 4. The extent of cross-linking between peptide side chains is relatively low, even in mucopeptide material of the large molecular size. 5. The small amount of residual phosphorus present in preparations of B. licheniformis soluble mucopeptide remains associated mainly with mucopeptide material of large molecular size. 6. The mucopeptide components of lowest molecular weight are not produced as artifacts during the preparation of soluble mucopeptide, but are apparently incorporated in the insoluble mucopeptide present in walls of exponentially growing cells. 7. Soluble mucopeptide isolated in a complex with acidic polymers after lysozyme treatment of walls of B. licheniformis N.C.T.C. 6346 and Bacillus subtilis W23 retains a high molecular weight when the covalent bonds between mucopeptide and the acidic polymers are broken. 8. Pure fragments were isolated from B. licheniformis soluble mucopeptide. A major component, C1, of the material of smallest size is made up of one residue each of N-acetylglucosamine, N-acetylmuramic acid, l-alanine, glutamic acid and diaminopimelic acid. The N-acetylglucosamine is in beta-glycosidic linkage with a reducing N-acetylmuramic acid residue. The peptide unit is probably amidated. A quantitatively minor component, C2, has amino acid and amino sugar composition identical with that of component C1, but probably lacks an amide group. Another fragment, B1, is made up of two molecules of component C1 or C2 that are joined together through a molecule of d-alanine.     

Secretion of T cell receptor fragments from recombinant Escherichia coli cells.

This study describes the secretion and purification of T cell receptor (TCR) V alpha, V beta domains and single chain V alpha-V beta fragments (scTCRs) from recombinant Escherichia coli cells. The TCR V alpha and V beta genes are derived from a T cell hybridoma that is associated with disease pathogenesis in murine experimental allergic encephalomyelitis (EAE). Circular dichroism (c.d.) analyses of the single domains and the scTCR indicate that they are folded into beta-pleated sheet structures similar to those of immunoglobulin variable domains. The secreted TCR fragments can be purified in milligram quantities, and could therefore be used in high-resolution structural studies, in immunization to generate anti-clonotypic antibodies or in vaccination.     

Spontaneous autoreactive memory B cell formation driven by a high frequency of autoreactive CD4+ T cells

Although somatically mutated autoantibodies are characteristic of many autoimmune diseases, the processes that can lead to their development remain poorly understood. We have examined the formation of autoreactive memory B cells in PevHA mice, which express the influenza virus PR8 hemagglutinin (HA) as a transgenic membrane bound neo-self-Ag. Using a virus immunization strategy, we show that PR8 HA-specific memory B cell formation can occur in PevHA mice, even though a major subset of PR8 HA-specific B cells is negatively selected from the primary repertoire. Moreover, PR8 HA-specific memory B cells develop spontaneously in TS1 x PevHA mice, which coexpress a transgenic PR8 HA-specific TCR and contain a high frequency of HA-specific CD4(+) T cells. Notably, autoreactive memory B cell formation occurred in TS1 x PevHA mice even though approximately half of the HA-specific CD4(+) T cells were CD25(+)Foxp3(+) cells that could significantly attenuate, but did not completely abolish HA-specific autoantibody production in an adoptive transfer setting. The findings provide evidence that a high frequency of autoreactive CD4(+) T cells can be sufficient to promote autoreactive memory B cell formation in the absence of signals provided by overt immunization or infection and despite the presence of abundant autoantigen-specific CD4(+)CD25(+)Foxp3(+) regulatory T cells.     

Conversion of monophasic to recurrent autoimmune disease by autoreactive T cell subsets

Autoimmune uveitis has been elicited in susceptible rodents by several ocular-specific Ags. In most of these animal models the induced uveitis is acute and monophasic. Because recurrent uveitis poses the highest risk for blinding ocular complications in human disease, a spontaneous relapsing animal model would be most helpful in understanding the disease pathogenesis. In our current study we have observed that the adoptive transfer of interphotoreceptor retinoid-binding protein residues 1177-1191-specific T cells to naive Lewis rats induced a chronic relapsing disease, in contrast to the monophasic disease induced by immunization with interphotoreceptor retinoid-binding protein residues 1177-1191 emulsified in CFA. The chronic relapsing uveitis induced by autoreactive T cell subsets is dependent on the number of autoreactive T cells generated as well as their activation status. Our study documented a spontaneous model of recurrent uveitis in the rat, which should assist us in the study of disease pathogenesis and the design of specific therapy.     

Functional modulation of hepatitis B core antigen-specific T lymphocytes by an autoreactive T cell clone

Hepatitis B core (HBc)Ag-specific T cells present in the peripheral blood of a patient with chronic active hepatitis B were expanded by co-cultivation for 7 days with rHBcAg. After cloning at 1 cell/well in the presence of PHA and IL-2, five HBcAg-specific CD4+ cloned lines were obtained. All five lines proliferated and produced IL-2, IFN-gamma, and TNF in a dose-dependent fashion in response to HBcAg, but not to HBV envelope Ag. The cloned lines and derivative clones were HLA class II (DR1) restricted. All T cell clones were able to induce anti-HBc production by autologous B cells in response to HBcAg (helper effect). The proliferative response and the helper effect of the HBcAg-specific T cell lines and clones were augmented by co-cultivation with an autologous, autoreactive (HLA-DQ1 specific) T cell clone, even in the absence of HBcAg, and the autoreactive T cells directly stimulated anti-HBc secretion by autologous B cells, presumably due to the release of Ag-nonspecific factors. These findings define a model immunoregulatory circuit the physiologic significance of which remains to be determined.     

Active fragments obtained by cyanogen bromide cleavage of ovomucoid.

Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant O1, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11 000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56% of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol.wts. of approx. 12000 (fragment L) and 4700 (fragment S). Each of these peptides contained carbohydrate. Marked heterogeneity was observed in the hexose and hexosamine contents of fragment L. This may account for much of the heterogeneity in neutral carbohydrate occurring in ovomucoid preparations. It was found that fragment M was located at the N-terminal end, fragment S was in the centre and fragment L made up the C-terminal portion of the molecule.     

Shaping of the autoreactive regulatory T cell repertoire by thymic cortical positive selection

The main function of regulatory T lymphocytes is to keep autoimmune responses at bay. Accordingly, it has been firmly established that the repertoire of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) is enriched in autospecific cells. Differences in thymic-positive and/or -negative selection may account for selection of the qualitatively distinct regulatory and conventional T cell (Tconv) repertoires. It has previously been shown that precursors for Tregs are less sensitive to negative selection than Tconv precursors. Studies with TCR/ligand doubly transgenic mice suggested that an agonist ligand might induce positive selection of Treg (but not Tconv) cells. However, massive deletion of Tconv (but not Treg) cell precursors observed in these mice renders interpretation of such data problematic and a potential role for positive selection in generation of the autospecific Treg repertoire has remained therefore incompletely understood. To study this important unresolved issue and circumvent use of TCR/ligand-transgenic mice, we have developed transgenic mice expressing a single MHC class II/peptide ligand on positively selecting thymic cortical epithelial cells. We found that functional Treg (but not Tconv) cells specific for the single ligand were preferentially selected from the naturally diverse repertoire of immature precursors. Our data therefore demonstrate that thymic cortical positive selection of regulatory and Tconv precursors is governed by distinct rules and that it plays an important role in shaping the autoreactive Treg repertoire.     

Studies on bacterial cell wall inhibitors III. 3-amino-3-deoxy-D-glucose,an inhibitor of bacterial cell wall synthesis

It was shown that 3-amino-3-deoxy-D-glucose, one of the constituents of the kanamycin molecule and a metabolite of Bacillus sp., inhibits the bacterial synthesis of cell wall. The antibiotic (100 μg/ml) significantly inhibits the growth of Straphylococcis aureus FDA 209P as well as the incorporation of DL-[¹⁴C]alanine into the acid-insoluble macromolecular fraction of its growing cells in the presence of chloramphenicol (100 μg/ml). In contrast, the antibiotic doed not affect the incorporation of [³H]thymidine, [³H]uridine and L-[¹⁴C]leucine. The other constituents of kanamycin, 6-amino-6-deoxy-D-glucose and deoxystreptamine do not inhibit the synthesis of bacterial cell wall peptidoglycan.     

Staphylococcal enterotoxin B stimulates expansion of autoreactive T cells that induce apoptosis in intestinal epithelial cells: regulation of autoreactive responses by IL-10

T cell responses to self Ags and normal microbial flora are carefully regulated to prevent autoreactivity. Because IL-10-deficient mice develop colitis, and this response is triggered by luminal flora, we investigated whether IL-10 regulates the ability of microbial Ags to induce autoreactive T cells that could contribute to intestinal inflammation. T cells from wild-type mice were primed with staphylococcal enterotoxin B (SEB) in vitro, which induced an autoreactive proliferative response to syngeneic feeder cells. The cells were predominately CD3+ and CD4+. T cells from IL-10-deficient mice were constitutively autoreactive, and SEB priming enhanced this further. The autoreactive, proliferative response of T cells from wild-type mice was suppressed by IL-10 in the primary or secondary culture, and this effect was inhibited by neutralizing Abs to the IL-10R. To confirm that an autoreactive repertoire was expanded after SEB priming, we used CBA/J mice (Mls-1a) in which autoreactive T cells recognizing the endogenous viral superantigen are depleted (Vbeta6, 7, 8.1 TCR-bearing cells). However, SEB rescued these autoreactive T cell repertoires. Adding anti-MHC class II Ab blocked the autoreactive response. SEB-primed splenic or colonic T cells also induced apoptosis in syngeneic intestinal epithelial cells that was blocked significantly by IL-10. Thus, microbial Ags have the potential to abrogate self tolerance by stimulating autoreactive T cells that become cytolytic to target cells. IL-10 plays a protective role in maintaining self tolerance after microbial stimulation by preventing the activation of T cells that contribute to epithelial cell damage.     

Disruption of T cell tolerance to self-immunoglobulin causes polyclonal B cell stimulation followed by inactivation of responding autoreactive T cells

Scavenger receptor (SR)-specific delivery by maleylation of a ubiquitous self-protein, Ig, to SR-bearing APCs results in self-limiting induction of autoimmune effects in vivo. Immunization with maleyl-Ig breaks T cell tolerance to self-Ig and causes hypergammaglobulinemia, with increases in spleen weight and cellularity. The majority of splenic B cells show an activated phenotype upon maleyl-Ig immunization, leading to large-scale conversion to a CD138+ phenotype and to significant increases in CD138-expressing splenic plasma cells. The polyclonal B cell activation, hypergammaglobulinemia, and autoreactive Ig-specific T cell responses decline over a 2-mo period postimmunization. Following adoptive transfer, T cells from maleyl-Ig-immune mice taken at 2 wk postimmunization can induce hypergammaglobulinemia in the recipients, but those taken at 10 wk postimmunization cannot. Hypergammaglobulinemia in the adoptive transfer recipients is also transient and is followed by an inability to respond to fresh maleyl-Ig immunization, suggesting that the autoreactive Ig-specific T cells are inactivated peripherally following disruption of tolerance. Thus, although autoreactive T cell responses to a ubiquitous self-Ag, Ig, are induced by SR-mediated delivery to professional APCs in vivo resulting in autoimmune pathophysiological effects, they are effectively and rapidly turned off by inactivation of these activated Ig-specific T cells in vivo.     

PCR amplification of large genomic fragments from human and simian immunodeficiency virus infected cell lines.

Polymerase chain reaction (PCR) has been used to amplify the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from Western Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from highly conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have amplified by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from 1-2.3 kb from gag pol and env genes have been successfully amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a highly conserved stretch of the viral genome.     

Establishment of intestinal epithelial cell lines from adult mouse small and large intestinal crypts

Small and large intestinal epithelial cell (IEC) lines were established from adult murine intestinal crypts. Both established small and large IECs line (named aMoS7 and aMoC1 respectively) expressed epithelial markers. Similarly to IECs isolated from adult mouse intestines, the expression of major histocompatibility complex (MHC) class II molecules was induced by interferon-γ-treatment in both established cell lines. This expression of MHC class II molecules was higher in small intestinal aMoS7 cells than in large intestinal aMoC1 cells. Treatment with lipopolysaccharide and with ligands of Toll-like receptors 1, 2, 3, and 7 induced secretion of interleukin-6 from both adult IEC lines. These results suggest that the aMoS7 and aMoC1 cell lines can serve as useful tools in analyzing the immunological functions of IECs, especially in studying the IEC response to microbial components and its antigen presenting ability.     

Abnormal thymocyte development and production of autoreactive T cells in T cell receptor transgenic autoimmune mice.

Development of a C57BL/6-+/+ TCR transgenic mouse containing the rearranged TCR alpha- and beta-chain specific for the Db + HY male Ag results in production of a nearly monoclonal population of early thymocytes expressing the Db + HY reactive TCR. These thymocytes are autoreactive in H-2Db male mice and undergo clonal deletion and down-regulation of CD8. To study the effect of the lpr gene on development of autoreactive T cells, these transgenic mice were backcrossed with C57BL/6-lpr/lpr mice. T cell populations in the thymus and spleen were analyzed by three-color flow cytometry for expression of CD4, CD8, and TCR. The thymus of TCR transgenic H-2b/b lpr/lpr male mice had an increase in percent and absolute number of CD8dull thymocytes compared to TCR transgenic H-2b/b +/+ male mice. However, there was not a complete defect in clonal deletion, because clonal deletion and down-regulation of CD8 was apparent in both +/+ and lpr/lpr H-2Db HY+ male mice compared to H-2Db HY- female mice. The phenotype of splenic T cells was almost identical in TCR transgenic +/+ and lpr/lpr males with about 50% CD4-CD8- T cells and 50% CD8+ T cells. However, there was a dramatic increase in the SMLR proliferative response of splenic T cells from TCR transgenic lpr/lpr males compared to TCR transgenic +/+ males. To determine the specificity of this response, spleen cells from TCR transgenic lpr/lpr and +/+ mice were cultured with irradiated H-2b/b and H-2k/k male and female spleen cells. T cells from TCR transgenic C57BL/6-lpr/lpr male mice had an increased proliferative response to H-2b/b male spleen cells compared to T cells from TCR transgenic C57BL/6(-)+/+ male mice, but both lpr/lpr and +/+ mice had a minimal response to irradiated H-2b/b female or H-2k/k male or female stimulator cells. The splenic T cells from TCR transgenic lpr/lpr mice also had an increased specific cytotoxic activity against H-2b/b male target cells compared to TCR transgenic +/+ mice. These results demonstrate that there is a defect in negative selection of self-reactive T cells in the thymus of lpr/lpr mice and a defect in induction or maintenance of clonal anergy of self-reactive T cells in the periphery of lpr/lpr mice.