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Summary Erwinia chrysanthemi (Ech) RA3B produces a large amount of blue indigoidine. Using Tn5-induced mutagenesis, three indigoidine-deficient mutants were generated. Followed by library screening, a 5.8 kb fragment complemented mutants for indigoidine synthesis was cloned. This fragment contains four complete open-reading frames (ORFs), pecS, pecM, idgA ,a ndidgB, and two partial ORFs, argG ,a ndidgC .T hese genes are nearly identical to those in strain Ech3937. Primer extension assays demonstrated a clear transcriptional start site prior to idgA, while no promoter preceding idgB and idgC was detected, suggesting that idgA, idgB ,a ndidgC are organized as one transcription unit. In contrast,indAB is separated from indC in Ech3937. Interestingly, an ERIC sequence was present between idgB and idgC in place of the promoter region of the homolog indC, which may contribute to the loss of promoter activity in RA3B. Futhermore, idgB mutant displayed much lighter blue color, while indB mutant appeared white on media. Overexpression of pecS in RA3B resulted in significantly reduced indigoidine production and idgC transcript. Moreover, gel shift and luxAB reporter assays revealed that PecS specifically binds to the sequence preceding idg Aa nd inhibits gene expression, which is consistent with the results observed in Ech3937. & 2009 Published by Elsevier GmbH.

We previously discovered a hamster monoclonal antibody, TAB4, against mouse PSGL-1/CD162 that can induce death of activated T cells. Here, we further investigated the potential of TAB4 in treating two murine models of T cell-mediated diseases. The results showed that administration of TAB4 suppressed incidence and severity of both GVHD and type I diabetes. Analyses of apoptotic T cells ex vivo shortly after antibody injection revealed a higher percentage of apoptosis among activated T cells in the TAB4-treated group than in the control group. Furthermore, restoration of functional donor T cells was observed in TAB4-treated mice. As TAB4 does not affect the binding of P-selectin to activated T cells, our data suggest that its long-lasting therapeutic effect on inhibiting disease progression is attained by specifically inducing apoptosis of activated T cells. These data hence extend our previous finding of the novel property of PSGL-1 and strongly indicate that the PSGL-1-specific apoptosis-inducing antibody is a new therapeutic agent possessing a great potential for controlling GVHD and other T cell-mediated autoimmune diseases.

Increasing evidence has shown that death signaling in T cells is regulated in a complicated way. Molecules other than death receptors can also trigger T-cell death. Here, we demonstrate for the first time that P-selectin glycoprotein ligand-1 (PSGL-1) or CD162 molecules cross-linked by an anti–PSGL-1 monoclonal antibody, TAB4, can trigger a death signal in activated T cells. In contrast to classic cell death, PSGL-1–mediated T-cell death is caspase independent. It involves translocation of apoptosis-inducing factor from mitochondria to nucleus and mitochondrial cytochrome c release. Ultrastructurally, both peripheral condensation of chromatin and apoptotic body were observed in PSGL-1–mediated T-cell death. Collectively, this study demonstrates a novel role for PSGL-1 in controlling activated T-cell death and, thus, advances our understanding of immune regulation.

BACKGROUND To investigate the clinical significance of tumor-infiltrating lymphocytes (TILs) within the tumor milieu of human cervical carcinoma, the authors quantitatively measured and compared the subpopulations of lymphocytes infiltrating the neoplastic cervix. METHODS A total of 30 patients with Stage Ia–IIa cervical carcinoma were enrolled. TILs were isolated from tissue specimens by means of a mechanical dispersal technique, and the immunocyte subsets were quantified with dual-color flow cytometry. Bulky tumor was defined as tumor size >4 cm in greatest dimension according to the 1995 staging of the International Federation of Gynecology and Obstetrics. RESULTS The CD4/CD8 ratios of TILs were reversed in both cervical squamous cell carcinoma (n = 20) and cervical adenocarcinoma (n = 10). The proportion of CD4+ T cells was significantly lower in tumors from patients with lymph node metastasis (n = 8) than in those from patients without lymph node metastasis (n = 22) (24.5 vs. 32.7, P = 0.001), as was the reversed CD4/CD8 ratio (0.50 vs. 0.81, P = 0.001). The proportion of CD4+ T cells was much lower in bulky tumors (n = 5) than in nonbulky tumors (n = 25) (21.4 vs. 32.5, P < 0.001), reflecting in a more strongly reversed CD4/CD8 ratio (0.41 vs. 0.81, P = 0.001). CONCLUSIONS Decreased proportions of tumor-infiltrating CD4+ T cells with reversed CD4/CD8 ratios are highly correlated with rapid tumor growth and lymph node metastasis in cervical carcinoma. The regional immune escape is of prognostic importance with regard to cancer progression. Cancer 1999;86:1537–43. © 1999 American Cancer Society.

Repeated inhalation of allergen leads to the down-regulation of allergen-specific IgE responses in non-atopic individuals as well as in mice. This phenomenon is named inhalation-induced IgE tolerance. In contrast, inhaled allergen causes significant IgE and allergic responses in atopic persons. The mechanisms involved in this differential regulation of airway allergen-specific immune responses remain unclear. Besides the allergen exposure of genetically susceptible individuals, environmental contamination is considered to play a role as an initiating factor for airway allergic responses. Using a murine model, we demonstrate here that airborne β-1,3-D-glucan, which exists frequently in our environment, particularly in highly humid areas, can abrogate inhalation-induced IgE isotype-specific down-regulation and promote airway eosinophil infiltration to inhaled antigen.

Sufficient CD4 F T cell help is very important in generating specific cytotoxic T cell responses. The inadequate activation of tumor-specific Th cells leads to failure of antitumor immunity. In general, each individual consists of some primed Th cells responding to certain antigens. If these tumor non-specific pre-primed Th cells can provide sufficient help, the generation of tumor-specific T cells may be enhanced. In the present study, we tested this hypothesis by cognating and reactivating pre-primed ovalbumin (OVA)-specific Th cells with OVA-pulsed tumor cells which could simultaneously present both OVA and tumor-associated antigen on the same cell. We clearly demonstrated that immunization of OVA-sensitized mice with OVA-pulsed P388 cells, but not unpulsed P388 cells, led to the induction of P388-specific cytotoxicity and tumor resistance. Both CD4 F and CD8 F tumor-specific cytotoxic T cells were detected in vitro, but only CD8 F T cells played the major effector role in preventing the growth of challenged tumor in vivo. Taken together, our study demonstrated that the immunogenicity of tumor cells can be enhanced effectively by cognating pre-primed foreign antigen-specific Th cells with tumor cells. These findings have potential implications in developing methods to control tumor growth.

The mechanisms involved in the differential regulation of airway immune responses in atopic versus non-atopic individuals are poorly understood. In this study, the association between non-specific immunity and the differential airway antigen-specific immune responses was examined in a murine model. The disparity in antigen-specific IgE and IgG2a productions between the two strains of mice was observed to be significant. C57BL/6J mice were much more efficient than BALB/cJ mice in making IgE antibody to inhaled ovalbumin (OVA) antigen. On the contrary, BALB/cJ mice did make more IgG2a antibodies than C57BL/6J mice to inhaled OVA. These findings suggest that in C57BL/6J mouse strain a predominant Th2 type of immune response develops in response to inhaled OVA antigen. In contrast, BALB/cJ mice mount a Th1 type of immune response to aerosolized OVA antigen. Furthermore, after lipopolysaccharide (LPS) stimulation, the IL-12 mRNA expression of lung-derived cells from BALB/cJ mice was higher than that from C57BL/6J cells. However, the lung-derived cells of C57BL/6J mice stimulated by LPS produced higher levels of IL-10 and prostaglandin E2 than BALB/cJ lung-derived cells did. Therefore, our study demonstrated that the difference of lung-derived cells in their ability to produce cytokine and prostaglandin between BALB/cJ and C57BL/6J mice correlates well with the type of the airway antigen-specific immune effector functions.

A new fungal immunomodulatory protein (FIP-fve) has been isolated and purified from the edible golden needle mushroom (Flammulina velutipes). The apparent molecular mass of FIP-fve determined by SDS/PAGE agrees well with the value of 12704 Da calculated from its amino acid composition and sequence. The complete amino acid sequence of FIP-fve was elucidated by protein sequencing techniques. FIP-fve consists of 114 amino acid residues with an acetylated amino end, and lacks methionine, half-cystine and histidine residues. FIP-fve was able to hemagglutinate human red blood cells. The immunomodulatory activity of FIP-fve was demonstrated by its stimulatory activity toward human peripheral blood lymphocytes, and its suppression of systemic anaphylaxis reactions and local swelling of mouse footpads. FIP-fve was found to enhance the transcriptional expression of interleukin-2 and interferon-gamma.

The size distributions of the submicrometer particles generated from ten metered dose inhalers (MDIs) were determined by a high resolution particle sizer, which could measure the particles in the size range of 0.01 μm to 1 μm. The particle sizer contains a differential mobility analyzer (TSI 3071) and a condensation particle counter (TSI 3022). The median diameters ranged from 0.06-0.10 μm with a geometric standard deviation of 2. In addition, the surface median diameters and volume median diameters were found to be 0.30 μm and 0.45 μm, respectively. Moreover, none of the size distributions of the generated submicrometer particles fits log-normal distributions. The deposition probabilities of the submicrometer particles in the airways were evaluated. It was observed that the average deposition percentages of the particles in the alveolar, tracheobronchial, and extrathoracic regions are 32%, 4%, and 4%, respectively. Understanding the deposition of submicrometer particles from MDIs should benefit the clinical practice in inhalation therapy.

Bacterial galU coding for a uridine diphosphate-glucose pyrophosphorylase plays an important role in carbohydrates biosynthesis, including synthesis of lipopolysaccharides (LPS), membrane-derived oligosaccharides, and capsular polysaccharides. In this study, we characterized the galU mutant of Pseudomonas syringae pv. syringae 61 (Psy61), a necrotizing plant pathogen whose pathogenicity depends on a functional type III secretion system (T3SS), and showed that the Psy61 galU mutant had reduced biofilm formation ability, was nonmotile, and had an assembled T3SS structure but failed to elicit hypersensitive response in resistant plants and necrotic lesions in susceptible plants. Moreover, the defective LPS and other pathogen-associated molecular patterns (PAMPs) on the surface of the Psy61 galU mutant were capable of inducing PAMP-triggered immunity, which severely compromised the ability of the Psy61 galU mutant to survive in planta. Our results demonstrated that the complete LPS protected plant-pathogenic bacteria from host innate immunity, similar to what was found in animal pathogens, prior to the translocation of T3S effectors and bacterial multiplication.

We have used cloned T cell receptor (TCR) genes from closely related CD4 T cell lines to probe the interaction of the TCR with several specific major histocompatibility complex (MHC) class II ligands. Complementarity determining region 3 (CDR3) equivalents of both alpha and beta TCR chains are required for antigen-MHC recognition. Our data provide novel information about the rotational orientation of TCR-MHC contacts in that exchange of the amino terminal portion of the TCR alpha chain containing the putative CDR1 and CDR2 regions results in both gain and loss of MHC class II specificity by the resulting receptor. These two TCRs differ primarily in recognition of polymorphisms in the second hypervariable region of the MHC class II alpha chain. These results document the involvement of CDR1 and/or CDR2 of the TCR alpha chain in MHC recognition and suggest a rotational orientation of this TCR to its MHC ligand.

Despite evidence that interleukin (IL)-1 promotes the proliferation of some T helper 2 (Th2) cell clones in vitro, the physiological role of IL-1 in the regulation of antigen-specific immune responses remains undefined. Using a liposome-DNA delivery system, we transiently expressed IL-1 receptor antagonist (IL-1Ra) to suppress IL-1 functions at the site of the antigen-specific primary immune response. Our data indicate, for the first time, that IL-1Ra downregulates antigen-specific IL-4 and IgE responses, with concomitant enhancement of interferon-gamma and IgG2a responses in vivo. In addition, IL-1 can promote Th2 development in an IL-4-independent manner in vitro. Thus, the balance between endogenous IL-1 and IL-1Ra during the primary immune response can be an important factor in determining the antigen-specific effector function of T cells.

Cytotoxic T lymphocytes (Tc) play a central role in cellular immunity against cancers. The cytotoxic potential of freshly isolated tumor-infiltrating lymphocytes (TILs) is usually not expressed. This suggests the possible existence of as yet unspecified and perhaps complex immunosuppressive factors or cytokines that affect the anti-tumor capacity of these TILs in the tumor milieu. In the present study, we demonstrated for the first time that TILs derived from human cervical cancer tissue consist mainly of Th2/Tc2 phenotypes. In vitro kinetic assays further revealed that cancer cells could direct the tumor-encountered T cells toward the Th2/Tc2 polarity. Cancer cells promote the production of IL-4 and down-regulate the production of IFN-γ in cancer-encountered T cells. The regulatory effects of cervical cancer cells are mediated mainly by IL-10, and TGF-β plays only a synergistic role. The cancer-derived effects can be reversed by neutralizing anti-IL-10 and anti-TGF-β Abs. IL-10 and TGF-β are present in cancer tissue and weakly expressed in precancerous tissue, but not in normal cervical epithelial cells. Our study strongly suggests important regulatory roles of IL-10 and TGF-β in cancer-mediated immunosuppression.

Aging is associated with a decline in immune function. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), two important immune deviation-related cytokines, are mainly produced by type 1 and type 2 T cells, respectively. To investigate the age-associated changes in the secretion of these two cytokines, 20 elderly and 20 young subjects fulfilling the SENIEUR protocol were enrolled. The ratios of CD4+ to CD8+ T cells were not different between the two age groups. The CD4+ and CD8+ T cells were purified by a magnetic cell sorting system, and then activated by concurrent anti-CD3 and anti-CD28 stimulation. The released cytokines were determined by ELISA. Both the CD4+ and the CD8+ T cells of the elderly individuals secreted a significantly larger amount of IFN-gamma after activation. Profound IL-4 production by CD8+ T cells was observed in the older subjects compared with that of the young subjects. These data suggested that age-associated decrease in immunity may be related to an imbalance in the secretion of immune deviation cytokines. The number of IL-4-secreting CD8+ T cells (T cytotoxic 2) rose significantly in the older individuals. Our design also provided a useful way to differentiate the T cell subsets secreting the same cytokine, such as IFN-gamma-producing T helper 1 and T cytotoxic 1 cells.

A new fungal immunomodulatory protein (Fip) has been purified from the edible mushroom, Volvariella volvacea, and designated Fip-vvo. Analysis of the purified protein by SDS/PAGE followed by Coomassie Blue staining demonstrated that Fip-vvo is a single polypeptide with an apparent molecular mass of 15 kDa. Periodic acid/Schiff staining showed that this single polypeptide lacks carbohydrates. Using an in vitro bioassay measuring blast-formation stimulatory activity, Fip-vvo was shown to stimulate the maximum proliferation of human peripheral blood lymphocytes at a concentration of 5 μg/ml. Fip-vvo was capable of agglutinating rat red blood cells. Neither haemagglutination nor mitogenic activities were inhibited by mono- or dimeric sugars. In vivo, repeat administration of Fip-vvo greatly reduced the production of BSA-induced Arthus reaction in mice, whereas little effect was observed on the prevention of systemic anaphylaxis reactions. The selectively enhanced transcriptional expression of interleukin (IL)-2, IL-4, interferon-γ, tumour necrosis factor-α, lymphotoxin and IL-2 receptor by Fip-vvo was also demonstrated by reverse transcriptase-PCR. This finding suggests that Fip-vvo exerts its immunomodulatory effects via cytokine regulation. In addition, the complete amino acid sequence of Fip-vvo was obtained by direct protein sequencing. This protein consists of 112 amino acid residues with a blocked N-terminal end and has a calculated molecular mass of 12667 Da not including the N-terminal blocking group. By gel filtration analysis, Fip-vvo exhibited a molecular mass of 26 kDa for the native molecules in PBS. This result indicates that native Fip-vvo is most likely a non-covalently associated homodimeric molecule.

Pseudomonas syringae pv. syringae 61 contains a 25-kb hrp cluster that is sufficient to elicit the hypersensitive response (HR) in nonhost plants. Previous studies have shown that mutations in complementation groups VIII, IX, and XI in the hrp cluster abolished the ability of the bacterium to cause the HR. The sequence of a 3.7-kb SmaI-SstI fragment covering groups VIII and IX now reveals five open reading frames (ORFs) in the same transcript, designated as hrpU, hrpW, hrpO, hrpX, and hrpY, and predicted to encode proteins of 14,795, 23,211, 9,381, 28,489, and 39,957 Da, respectively. The hrpU, hrpW, hrpO, hrpX, and hrpY genes are homologous and arranged colinearly with the yscQ/spa33/spaO, yscR/spa24/spaP, yscS/spa9/spaQ, yscT/spa29/spaR, and yscU/spa40/spaS genes of Yersinia spp., Shigella flexneri, and Salmonella typhimurium, respectively. These proteins also show similarity to Fli/Flh proteins of Bacillus and enteric bacteria. The Ysc and Spa proteins are involved in the secretion of virulence factors, like the Yop and Ipa proteins. Fli/Flh proteins are involved in flagellar biogenesis. The sequence of a 2.9-kb EcoRV-EcoRI DNA fragment containing mainly group XI revealed five ORFs, designated hrpC, hrpD, hrpE, hrpF, and hrpG, predicted to encode proteins of 29,096, 15,184, 21,525, 7,959, and 13,919 Da, respectively. The first three genes belong to an operon containing hrpZ, which encodes an extracellular protein that elicits the HR. hrpF and hrpG are two potential ORFs upstream of hrpH in the hrpH operon. HrpC is homologous to Yersinia YscJ, Pseudomonas solanacearum HrpI, Xanthomonas compestris pv. vesicatoria HrpB3, and Rhizobium fredii NolT. HrpE is similar to YscL of Yersinia spp. P. s. pv. syringae 61 Hrp proteins are most similar to Ysc proteins among those homologs. TnphoA insertions in hrpC, hrpE, hrpW, hrpX, and hrpY abolished the ability of P. s.pv. syringae 61 to secrete HrpZ (harpinPss), as determined by immunoblot analysis of cell-bound and culture supernatant fractions. Thus, many of the proteins required for flagellar biogenesis and virulence protein secretion in plant and animal pathogens may have a common ancestry.

OBJECTIVE--To clarify the significance of the humoral immune response triggered by the Mycobacterium tuberculosis (M.tb) 65 kDa heat shock protein (hsp) in the pathogenesis of rheumatoid arthritis (RA). METHODS--M.tb 65 kDa hsp-specific IgG, IgA, IgM, and IgG subclass antibodies in serum or synovial fluid (SF) of RA and other disease patients were determined by enzyme linked immunosorbent assay (ELISA). RESULTS--RA patients did not show any characteristic increase in mycobacterial 65 kDa hsp-specific antibodies compared with healthy individuals. In contrast, antigen-specific IgG and IgG2 antibody titres in the serum of RA patients were significantly lower than those of patients with tuberculosis and normal controls. In addition, there was also no significant difference in antibody titre between the serum and SF of RA patients, nor was any significant difference found between the SF of RA and Reiter's patients. CONCLUSION--The failure to detect a significant increase in IgG anti-M.tb 65 kDa hsp antibodies in RA patients does not exclude the possibility of microbial immunity in the aetiology of RA. Nevertheless, anti-M.tb 65 kDa hsp antibodies clearly do not appear to be the disease specific markers for RA and their relatively reduced concentrations may argue against their playing a major role in the disease pathogenesis.

Preliminary analysis indicated that a 19-amino-acid peptide sequence (435 to 453 of GtfC) within a highly conserved region of the glucosyltransferases of the cariogenic streptococci might be functionally important (J.-S. Chia, S.-W. Lin, T.-Y. Hsu, J.-Y. Chen, H.-W. Kwan, and C.-S. Yang, Infect. Immun. 61:1563-1566, 1993). To obtain antipeptide monoclonal antibodies (MAbs), the 19-amino-acid peptide was conjugated to bovine serum albumin and used as an antigen in BALB/c mice. Six immunoglobulin G-secreting hybridoma clones, CJSm18-S1 to -S6, specifically reacted with this peptide and with purified GtfC and GtfD but not with bovine serum albumin in an enzyme-linked immunosorbent assay. The concentrated hybridoma supernatant of all six MAbs inhibited GtfC enzymatic activity but failed to inhibit GtfD, although GtfD contains the same peptide sequence. Further analysis of a purified immunoglobulin G2b MAb from one of the clones, CJSm18-S3, confirmed that this MAb specifically inhibited GtfC enzymatic activity for insoluble-glucan synthesis in a dose-dependent manner. CJSm18-S3, even at high concentrations, had no effect on GtfD, which synthesizes water-soluble glucan exclusively. Furthermore, the in vitro sucrose-dependent adherence of Streptococcus mutans was also inhibited by CJSm18-S3 in a dose-dependent manner. Our results indicate that the peptide containing the N-terminal conserved region of glucosyltransferases is functionally important for both enzymatic activity and bacterial adherence.

The Pseudomonas syringae pv. syringae 61 hrpJ and hrpI genes were sequenced and found to encode predicted proteins of 37,710 Da and 76,490 Da, respectively. The products of these genes were confirmed by T7 polymerase-dependent expression and sodium dodecyl sulfate-polyacrylamide gel analysis. HrpI belongs to a superfamily of proteins represented by Yersinia pestis LcrD.

A novel mechanism for breaking T cell self tolerance is described. B cells induced to make autoantibody by immunization of mice with the non-self protein human cytochrome c can present the self protein mouse cytochrome c to autoreactive T cells in immunogenic form. This mechanism of breaking T cell self tolerance could account for the role of foreign antigens in breaking not only B cell but also T cell self tolerance, leading to sustained autoantibody production in the absence of the foreign antigen.

Mice deficient for the fifth component of murine complement (C5), unlike normal mice, do not possess the secreted form of C5 in their body fluids and can be readily immunized to serum-derived normal C5. Although macrophages from C5-deficient mice do not secrete C5, they synthesize the precursor form (pro-C5). Therefore contact of T cells with autologous pro-C5 presented by macrophages is theoretically possible. We show that macrophages from C5-deficient mice can indeed stimulate a class II restricted C5-specific T cell clone without addition of exogenous C5. Immunization of C5-deficient mice with autologous pro-C5 induces vigorous C5-specific T cell proliferation and pro-C5 is recognized by C5-specific T cells in vitro, demonstrating that this protein fails to induce tolerance under physiological conditions. Thus, intracellular pro-C5 is processed and presented by C5-deficient macrophages and can activate T cell clones in vitro, yet is neither immunogenic nor tolerogenic for T cells in vivo.