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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.

Summary Background Food allergy is a common disease without effective treatment. Since strict elimination of food allergens may be difficult, strategies for effective intervention are urgently needed. Objective The aim was to investigate the prophylactic use of orally administrated FIP-fve, an immunomodulatory protein isolated from the edible mushroom Flammulina velutipes, in a murine model of food allergy. Methods BALB/c mice were immunized twice intraperitoneally with ovalbumin (OVA), at an interval of 2 weeks. Before and during each period of immunization, FIP-fve (200 μg per mouse) or phosphate-buffered saline was given orally every other day with a total of five doses. Then OVA-specific antibodies and cytokine profiles were determined. Subsequently, the mice were orally challenged with OVA. Symptoms of anaphylaxis, levels of plasma histamine, and histology of intestines were examined. Results Mice receiving oral FIP-fve treatment during sensitization to OVA had an impaired OVA-specific IgE response with a Th1-predominant cytokine profile. These mice were protected from systemic anaphylaxis-like symptoms induced by subsequent oral challenge with OVA. Conclusion Oral administration of FIP-fve has a Th1-skewing effect on the development of the allergen-specific immune response, and may serve the purpose of immunoprophylaxis for food allergy and other allergic diseases.

Summary Background The aetiology of food allergy remains unclear. Although failure to develop or breakdown in oral tolerance has been proposed, the existence of physiologic sensitization routes other than the gastrointestinal tract cannot be excluded. Objective The purpose of this study is to clarify whether or not exposure to allergen through the skin can promote food allergy. Methods BALB/c mice were shaved on the back, and a patch impregnated with 100 μg of ovalbumin (OVA) was applied to the dorsal skin for a 1-week period and then removed. After three courses of sensitization, OVA-specific antibodies in sera were measured, and then mice were orally challenged with 50 mg of OVA. Anaphylactic symptoms, plasma histamine levels, and histology of intestines and lungs after oral challenge were examined. Results Epicutaneous (EC) sensitization of mice to OVA induced a high level of OVA-specific IgE. Subsequent oral challenge with OVA resulted in symptoms of systemic anaphylaxis with elevated levels of plasma histamine as well as histological changes in both intestines and lungs. In the presence of anti-IL-4 antibodies, EC sensitization failed to provoke an IgE response, but still induced a Th2-predominant cellular immune response in lungs after oral challenge. Conclusion We demonstrated for the first time that food allergy can be induced by allergen exposure through the skin. Our results identify a novel role of EC sensitization in the pathogenesis of food allergy.

Induction of apoptosis of virus-infected cells is an important host cell defense mechanism. It is well documented that T cells may undergo apoptosis due to interactions between Fas and Fas ligand (FasL). In addition, signals that induce apoptosis in T cells can result from interaction of tumor necrosis factor (TNF)-alpha with TNF receptors (TNFRs). It has been shown that human T cell lines expressing HTLV-I have decreased sensitivity to Fas-mediated apoptosis. The susceptibility of HTLV-I-infected cells to TNF-alpha-induced apoptosis remains to be elucidated. In the present study, we examined the expression of TNFRs on HTLV-I-infected T cell lines that expressed T-cell activation markers and thus phenotypically resemble activated T cells. Different from primary activated T cells that expressed both TNFRs, none of the five HTLV-I-infected T cell lines studied had detectable TNFR1 and only three had TNFR2 on their cell surfaces, although, the RNA transcripts of both TNFR genes could be detected via reverse transcription-polymerase chain reaction in these cell lines. The T cell blasts, which we activated in vitro, were sensitive to apoptosis induced by TNF-alpha and by antibodies to TNFR1 and/or TNFR2. However, all of the HTLV-I-infected cell lines expressing TNFR2 were resistant to TNF-alpha-mediated apoptosis. These findings suggest that HTLV-I infection may interfere with the autonomous suicide programs of T cells, not only Fas/FasL but also TNFRs/TNF-alpha pathways, to prolong the life of the infected cells. This may contribute to viral persistence and favor survival and subsequent expansion of dysregulated infected T cells with the potential to produce HTLV-I-associated autoimmune-like diseases or malignancies.

Cord blood T cells are much more likely to be induced to apoptosis in vitro than adult T cells. Nevertheless, the expression of Fas is markedly lower on cord blood lymphocytes than on peripheral blood lymphocytes. In the current investigation, we determined the capacity of tumour necrosis factor-α (TNF-α) to induce apoptosis in human naive T cells in cord blood, and assessed the roles of two distinct TNF receptors (TNFRs) in mediating death signals. After activation, cord blood T cells were sensitive to TNF-α-induced apoptosis, and interleukin 2 (IL-2) could prevent this apoptotic response. Both TNFR1 (p55) and TNFR2 (p75) expressed on activated cord blood T cells were able to transmit apoptotic signals. Moreover, a synergistic effect was observed by a combination of TNFR1- and TNFR2-signals. Additionally, CD4+ T cells showed higher sensitivity to TNFR-mediated apoptosis than CD8+ T cells. These data suggest that TNF-α probably is a mediator of apoptosis in cord blood T cells in vivo and may contribute to the low incidence of graft-versus-host disease in cord blood transplantation.

Background House dust mite (HDM) Dermatophagoides pteronyssinus is the most important source of indoor allergens that cause allergic diseases in Taiwan. We prepared purified HDM allergens (Der p 1, Der p 2 and Der p 5) to detect allergen-specific immunoglobulin (Ig) E responsiveness among a large number of test subjects. The robust genetic typing system for HLA class II genes also facilitated the study on association of HLA and allergic response toward HDM. Objective This study intended to investigate the association between HLA class II alleles and the IgE responsiveness to the major allergens from HDM, D. pteronyssinus. Methods Two hundred and forty-eight subjects were selected for HLA association study. Plasma HDM allergen (Der p 1, Der p 2, Der p 5) -specific IgE and Der p 2-specific IgG antibodies were detected by ELISA, while HLA class II -DRB1, -DQA1, -DQB1, -DPB1 genetic polymorphism was determined by polymerase chain reaction/sequence-specific oligonucleotide probe hybridization (PCR/SSOPH). Statistical comparison of the allelic distribution of each HLA class II genes among the individuals with/without HDM allergen-specific IgE and IgG antibodies were performed. Results There was no significant association between HLA DRB1, DQB1, DQA1 alleles and HDM-specific IgE responsiveness noted. Only DRB1*0803 and the linked DQA1*0103 alleles showed positive association with Der p 5-specific IgE responsiveness. However, we found that HLA-DPB1*1301 predisposed subjects to IgE responsiveness to HDM Der p 5. HLA DPB1*0501 was weakly associated with the IgE responsiveness to HDM Der p 1 and Der p 5. There was a strong negative association between the HLA-DPB1*0201 allele with IgE responsiveness to Der p 1 (OR: 0.30, P ≤ 0.0001, P ≤ 0.0007, Pc ≤ 0.010). Conclusion We clearly observed the association between HLA DPB1 alleles and specific IgE responsiveness to HDM major allergens. The molecular mechanism of HLA-DPB1*0201 involvement in protecting subjects from HDM-specific IgE responsiveness awaits further investigation.

Background and objective Repeated exposure of aerosolized antigen via respiratory tract can induce immunoglobulin (Ig) E isotype-specific tolerance to this antigen. However, the atopic individuals often produce a higher titre of IgE in response to airborne environmental allergens. The mechanisms of this differential regulation of airway allergen-specific immune responses are not fully understood. This study investigated the role of airborne endotoxin on the initiation of antigen-specific airway allergic responses. Methods ELISA methods for detection of isotypes of antigen-specific antibodies and competitive reverse transcription polymerase chain reaction for detection mRNA of cytokines were used. In addition, Liu stain method was used to analyse the amounts of eosinophils in bronchoalveolar lavage fluid. Results Mice pre-exposed with airborne endotoxin mounted significantly higher amounts of OVA-specific IgE antibody responses to inhaled OVA than those OVA-only sensitized mice. Inhaled endotoxin could downregulate repeated airway antigen exposure-induced IgE isotype-specific tolerance and increase antigen-induced lung eosinophils infiltration. Conclusions These data show that airborne endotoxin exposure could potentiate allergen-specific airway inflammation. The results should have potential implications for understanding the development of allergen-induced airway allergic responses.

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 oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Two distinct oleosins are present in the oil bodies of diverse angiosperms, and classified as high and low Mr isoforms according to their relative molecular masses in each species. In sesame oil bodies, besides the two ubiquitous oleosin isoforms (17 and 15 kDa), an additional minor oleosin (15.5 kDa) was revealed on Tricine SDS-PAGE. A full-length cDNA fragment was cloned, sequenced and deduced to be a putative oleosin of 15,446 Da. The gene was constructed in a fusion or non-fusion vector and then over-expressed with different efficiency in Escherichia coli. All three oleosins purified from sesame oil bodies were subjected to immunoassaying using antibodies raised against the over-expressed oleosin. The results confirmed that this gene encodes the sesame 15.5 kDa oleosin. Sequence comparisons with other known oleosins revealed that sesame 15.5 kDa oleosin does not represent a new oleosin isoform class but may have been derived through gene duplication and truncation of sesame 17 kDa oleosin, and possesses the minimal structure of the high Mr oleosin isoform. A conserved amphipathic alpha-helix is predicted in sesame 15.5 kDa oleosin, which may imply a potential biological function associated with this isoform.

To investigate the activation status of tumor-infiltrating lymphocytes (TILs) within the tumor milieu of human cervical carcinoma, we quantitatively measured and compared the activation markers on lymphocyte subpopulations which infiltrating normal and neoplastic cervix. A total of 20 patients with stage IA to IIA cervical cancer (cancer group) and 10 women with normal cervix (control group) were enrolled in this study. Mononuclear cells were isolated from tissue specimens by mechanical dispersal technique and three-color flow cytometry was utilized for the quantification of activation markers on lymphocyte subsets. Compared with control group, lymphocytes isolated from cancer tissue consisted of higher proportions of B cells (7.23% +/- 4.49% vs. 3.67% +/- 3.19%, P = 0.016) and T cells (72.33% +/- 8.70% vs. 53.15% +/- 17.36%, P = 0.004), but an inverted CD4:CD8 ratio (0.74 +/- 0.27 vs. 1.14 +/- 0.28, P = 0.002) and decreased NK cells (7.53% +/- 4.33% vs. 16.00% +/- 11.82%, P = 0.035). Low expression of CD25, but not CD69 and HLA-DR was observed on both CD4+CD3+ and CD8+CD3+ T cells derived from cervical cancer (P < 0.0001). Further dual activation marker analysis demonstrated that the expression of CD25 was dissociated from CD69 and HLA-DR on the same TILs in cancer tissue (P < 0.001). TILs in the tumor microenvironment can be functionally inhibited and lose the ability of clonal proliferation due to depressed expression of CD25.

Human peritoneal mesothelial cells lie on a basement membrane-like material consisting of fibronectin (FN), type I collagen (CI), type III collagen (CIII) and laminin (LA). To understand how these extracellular matrix (ECM) proteins affect mesothelial cell behavior, we investigated their effect on the adhesion and proliferation of mesothelial cells. A modified methyltetrazolium dye method was used to assess cell number. The results showed that FN, CI, CIII and LA, all increased adhesion of mesothelial cells. The adhesive effect was blocked dose-dependently by a synthetic Arg-Gly-Asp-containing (RGD) peptide. When coated as a substratum (immobilized form), FN, CI, CIII and LA, all enhanced serum-stimulated and epidermal-growth-factor-stimulated cellular proliferation as compared with bovine-serum-albumin-blocked plastic surfaces. When added in a soluble form, all matrix proteins except FN inhibited serum-stimulated and epidermal-growth-factor-stimulated cellular proliferation at high concentrations (CI and CIII: 1-10 micrograms/ml, LA: 3-10 micrograms/ml). We conclude that peritoneal mesothelial cells possess an RGD-sensitive receptor and that the ECM can modulate adhesion and proliferation of peritoneal mesothelial cells. The growth-modulating effect depends on the form and concentration of the ECM proteins.

8-Methoxypsoralen (8-MOP) plus long-wavelength UV radiation (UVA, 320-400 nm) have been used to treat various diseases such as cutaneous T-cell lymphoma, systemic scleroderma, rheumatoid arthritis and rejection of heart transplants. However, the immunological mechanism of this treatment remains unknown. In this report, we investigated the effect of 8-MOP/UVA on the modulation of the immunogenicity of a T-cell leukemia cell line (RL male 1 cells). The results demonstrated that the stimulator function of the in vitro 8-MOP/UVA-treated RL male 1 cells was enhanced in both RL male 1-specific allogeneic and syngeneic immune responses. Furthermore, the enhancement of the immunogenicity of the 8-MOP/UVA-treated RL male 1 cells was found to be strongly associated with the increase of intercellular adhesion molecule-1 expression on these 8-MOP/UVA-treated tumor cells. Therefore, our findings suggested that the alteration of the expression of the immune-related cell surface molecules might be an important effect of 8-MOP/UVA treatment on the elevation of the immunogenicity of the 8-MOP/UVA-treated tumor cells.

Peritonitis is one of the most frequent complications of continuous ambulatory peritoneal dialysis (CAPD). Necrosis and exfoliation of the mesothelial cell layer of the peritoneum develop during the acute phase of peritonitis. Agents that hamper regeneration of mesothelial cells will cause delayed recovery of the peritoneal surface, which results in continuous exposure of underlying stem cells to the stimulation of growth factors and possibly leads to peritoneal fibrosis syndrome. The aim of the present study is to determine the effects of several intraperitoneal antibiotics on human peritoneal mesothelial cell (HPMC) growth at their usual loading and maintenance doses. HPMCs were isolated from human omenta. Proliferation of HPMC was evaluated by modified methyltetrazolium assay and cell membrane integrity was assessed by lactate dehydrogenase method. The results showed that most cephalosporins exert an inhibitory, even toxic, effect on HPMCs at their loading doses. Cephalothin, cephradine, cefamandole, cefoxitin, cefuroxime and cefoperazone inhibited HPMC proliferation at their maintenance doses. Vancomycin, clindamycin, aztreonam, piperacillin, imipenem, tobramycin and ceftriaxone have no effect in their usual intraperitoneal doses. From the viewpoint of peritoneal protection, not only drug sensitivity of the causative microorganisms but also effects of antibiotics on HPMC regeneration should be considered when selecting antibiotics for CAPD peritonitis.

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.

Mesangial cell activation and extracellular matrix accumulation are hallmarks of many forms of glomerulonephropathy. We investigated the effect of several agents possessing vasodilating activities on rat mesangial cell growth and collagen synthesis. Using cell counting and a modified MTT assay, it was shown that dipyridamole, pentoxifylline, dicentrine, prazosin and doxazosin all caused a dose-dependent inhibition of serum-stimulated rat mesangial cell proliferation. Platelet-derived growth factor-induced cell proliferation was also inhibited by doxazosin and pentoxifylline. Dipyridamole and pentoxifylline inhibited collagen synthesis in confluent mesangial cells while dicentrine and doxazosin did not. The procollagen alpha 1 (I) mRNA expression was also decreased by dipyridamole and pentoxifylline. These results suggested that, in addition to dipyridamole, pentoxifylline and alpha 1-adrenoceptor blockers may have a potential to delay the progression of chronic glomerulopathy associated with mesangial proliferation.

The human leucocyte antigen DR4-associated immune responses to Mycobacterium tuberculosis 65 kDa heat shock protein were considered to be relevant to the pathogenesis of rheumatoid arthritis (RA). In the Chinese population, DR4-Dw15 was found to be the predominant DR4 subtype in RA. To further define the immune responses associated with DR4-Dw15 molecules, the proliferative responses of peripheral (PBMC) and synovial mononuclear cells (SFMC) to mycobacterial 65 kDa antigen were evaluated. The SFMC of all of our RA patients responded significantly to 65 kDa mycobacterial antigen. The responses of PBMC to this antigen in RA were much lower than those of SFMC. Our results further indicated that relatively low numbers of peripheral antigen-specific T-cells, but not incompetence of peripheral antigen presenting cells, might be related to the observed low responsiveness to 65 kDa antigen in PBMC of RA patients. Of utmost importance, DR4-Dw15 was proved to be one of the major restrictive molecules in mycobacterial 65 kDa antigen-specific immune responses in Chinese RA patients.

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.

Presentation of a soluble self antigen, the fifth component of complement (C5), is discussed with emphasis on the differential ability of presentation by subpopulations of APC (dendritic cells, macrophages, B cells, fibroblasts, B cell lines and bone marrow macrophages). Constitutive presentation of C5 in C5 sufficient mice is a prerequisite for tolerance induction in MHC class II restricted T cells and can be directly demonstrated by the ability of ex-vivo APC from C5 sufficient, but not C5 deficient mice, to activate C5 specific T cells in vitro in the absence of added antigen. C5 presentation and tolerance induction in MHC class II restricted T cells is strictly dependent on an exogenous source of self antigen. C5 biosynthesized, but not secreted by macrophages is ignored by MHC class II restricted cells and induces neither tolerance nor autoimmunity. C5 presentation for tolerance induction depends largely on the efficiency of antigen uptake by APC, a property which varies within different APC subpopul...

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.

The preva lence of a s thma in schoolch i ldren in Ta ipe i was obse rved to increase f rom 1.3% in 1974. to 5.1% in 1985. and to 10.8% in 1994 (unpubl ished data) . An t i -mi t e IgE sera f rom pa t ien t s a l lergic to mites in Ta iwan r eac t ed with a n u m b e r of c o m p o n e n t s as d e t e r m i n e d by W e s t e r n blot analysis? To date . at least seven groups of these a l lergens have b e e n c loned and charac te r ized . 2 Both in vi t ro IgE binding s tudies and skin tests r evea led tha t more than 90% of the al lergic child ren with a s thma and mite al lergy in Ta iwan r eac t ed to the two well cha rac te r i zed majo r a l lergens D e r p 1 and D e r p 2. and approx imate ly 60% of these ch i ld ren reac ted to a newly cha rac te r i zed low molecu la r weight al lergen, D e r p 5. 3 In teres t ingly, a l though D e r p 5 r eac t ed with only 60% of sera f rom pat ien ts al lergic to mites, the IgE reactivity a p p e a r e d to be s t ronger than that of bo th D e r p 1 and D e r p 2. In addi t ion , the g roup with a s thma had significantly m o r e react ivi ty to D e r p 5 than the group with rhini t is alone. The seasona l va r ia t ion of the ma jo r a l le rgen D e r p 1 concen t ra t ions in Ta iwan has b e e n previous ly eva lua ted . 4 S imi lar env i ronmen ta l s tudy on the concen t ra t ions and d is t r ibut ions of D e r p 5 has no t been r e p o r t e d before . In this invest igat ion, D e r p 5 concen t ra t ions in mat t resses and the f loors of b e d r o o m s and living rooms were eva lua ted in the h o m e s of 12 as thmat ic ch i ld ren al lergic to house dust mi tes in

Induction of apoptosis of virus-infected cells is an important host cell defense mechanism. It is well documented that T cells may undergo apoptosis due to interactions between Fas and Fas ligand (FasL). In addition, signals that induce apoptosis in T cells can result from interaction of tumor necrosis factor (TNF)-alpha with TNF receptors (TNFRs). It has been shown that human T cell lines expressing HTLV-I have decreased sensitivity to Fas-mediated apoptosis. The susceptibility of HTLV-I-infected cells to TNF-alpha-induced apoptosis remains to be elucidated. In the present study, we examined the expression of TNFRs on HTLV-I-infected T cell lines that expressed T-cell activation markers and thus phenotypically resemble activated T cells. Different from primary activated T cells that expressed both TNFRs, none of the five HTLV-I-infected T cell lines studied had detectable TNFR1 and only three had TNFR2 on their cell surfaces, although, the RNA transcripts of both TNFR genes could be detected via reverse transcription-polymerase chain reaction in these cell lines. The T cell blasts, which we activated in vitro, were sensitive to apoptosis induced by TNF-alpha and by antibodies to TNFR1 and/or TNFR2. However, all of the HTLV-I-infected cell lines expressing TNFR2 were resistant to TNF-alpha-mediated apoptosis. These findings suggest that HTLV-I infection may interfere with the autonomous suicide programs of T cells, not only Fas/FasL but also TNFRs/TNF-alpha pathways, to prolong the life of the infected cells. This may contribute to viral persistence and favor survival and subsequent expansion of dysregulated infected T cells with the potential to produce HTLV-I-associated autoimmune-like diseases or malignancies.

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.

We used allergen-specific ELISA to evaluate environmental distributions of house dust mite allergens (Der p I and Der p V) in the homes of 46 asthmatic, 20 atopic, and 26 nonatopic control children during the summer and winter. Geometric mean Der p I levels were approximately 10 times higher than geometric mean Der p V concentrations. The concentrations of Der p I and Der p V in winter were significantly higher than concentrations found in summer. In addition, we found no differences in Der p I concentrations among the three groups in summer; however, in winter, Der p I levels in the control group were significantly higher than levels in the homes of both asthmatic and atopic children. During the summer, concentrations of Der p V were lower in the control group, compared with the asthmatic group, but during the winter, concentrations in the control group were higher than levels found in either asthmatic or atopic children's homes. In this study, we did not establish an important relationship between mite allergen exposure and atopy/asthma.

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.

To characterize the role of tumor necrosis factor (TNF)-alpha in regulating synovial T cell growth, cell cycle progression associated with TNF-alpha in mitogen-activated synovial T cells of patients with rheumatoid arthritis (RA) were analyzed. After mitogen stimulation, the majority of synovial T cells in RA patients accumulated in S-phase. Anti-human TNF-alpha monoclonal antibody and soluble recombinant human TNF receptor (rhTNFR) can block S-phase accumulation. Furthermore, synovial fluid (SF) from RA patients was able to inhibit the proliferation of these S-phase-accumulated T cells. These data indicate that TNF-alpha could regulate activated synovial T cell growth by driving them into S-phase. Combined with the activities of other components of SF, TNF-alpha seems to play an important role in down-regulating activated synovial T cells in RA patients. In addition, the elevated level of soluble TNFR in the SFof disease-active RA patients is believed to be associated with the promotion of synovial T cell responses.

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.

Direct recognition of major histocompatibility complex (MHC) molecules may occur when T cells are positively selected in the thymus and also during recognition of non-self MHC molecules. Since peptide recognition and binding of particular monoclonal antibodies is strongly influenced by the invariant chain (Ii) of the class II molecule, we have asked whether Ii also affects recognition of non-self MHC molecules by T cells. We find that Ii binding alters MHC class II conformation as detected by a monoclonal antibody, and that this alteration is retained in cell surface MHC class II molecules after Ii dissociates. This altered conformation also affects recognition by allogeneic T cells. Normal T cells and T cell clones preferentially recognize MHC class II molecules that have been associated with Ii, suggesting that thymic selection may be influenced by MHC conformation independently of specific peptide binding.

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.