Dexamethasone accelerates catabolism of leukotriene C4 in bronchial epithelial cells.

Leukotriene (LT)C4, a potent chemical mediator in bronchial asthma, is metabolised to the less active LTE4 via LTD4 in two consecutive reactions catalysed by enzymes of the glutamyl transpeptidase and dipeptidase families. The activities of these catabolic enzymes may be influenced by glucocorticosteroids. This study was conducted to examine whether this inactivation of LTC4 is affected by dexamethasone (DEX) in transformed human bronchial epithelial cells and normal human bronchial epithelial cells. After incubation with DEX for 0-5 days, cells were resuspended in the presence of exogenous LTC4, and conversion of LTC4 to LTE4 was measured using high-performance liquid chromatography. Gamma-glutamyl transpeptidase (GGT) and GGT-related enzyme (GGTRE) messenger ribonucleic acid (mRNA) expression were examined using reverse transcriptase-polymerase chain reaction analysis, and GGT activity by enzyme assay. Conversion to LTE4 was accelerated by DEX pretreatment. GGTRE but not GGT mRNA expression was enhanced after incubation with DEX. The results indicate that dexamethasone transcriptionally upregulates the activity of gamma-glutamyl transpeptidase-related enzyme in human bronchial epithelial cells, which accelerates inactivation of leukotriene C4 via conversion to leukotriene E4. This is a novel mechanism of glucocorticosteroids in human bronchial epithelial cells.

Lipopolysaccharide (LPS) and zymosan-resistant mutant isolated from a macrophage-like cell line, WEHI-3, with a defective response to LPS under serum-free conditions.

A LPS-resistant mutant, W3SF-1, was isolated from a murine macrophage-like cell line, WEHI-3. The W3SF-1 mutant did not produce a significant amount of nitric oxide (NO) or TNF-alpha even with high concentrations of LPS in the presence or absence of FCS, whereas the parental WEHI-3 cells produced them in response to LPS. The parental cells expressed a significant level of TNF-alpha mRNA after LPS stimulation, whereas the mutant cells did not. This defective response of the mutant cells to LPS was neither dependent on the concentration or chemical structure of LPS, nor on the time of LPS treatment. The mutant cells also showed a defective response to zymosan, suggesting that the defect in the mutant cells is common to LPS and zymosan in the signal transduction pathways. The parental and mutant cells showed similar levels of Mac1, F4/80 and CD14, suggesting that these surface markers of macrophages are not linked directly to the defective responses of the mutant to LPS. The treatment of mutant cells with IFN-gamma did not restore the defect of NO or TNF-alpha production on LPS treatment. Binding experiments with 125I-labelled LPS showed a similar binding affinity for LPS in the parental and the mutant cells. These results suggest that the defect in the W3SF-1 mutant cells may not reside in the LPS binding but rather in the early step of signal transduction pathways in the cells after LPS binding.

Humoral immune-mediated acute, antigen-induced arthritis in rats is suppressed by the inducing antigen administered orally before, but not after, immunization.

Acute ovalbumin-induced arthritis (OIA), which is mediated by Arthus reaction to ovalbumin (OVA) in the joint space, can be induced by immunization of rats with OVA followed by the intraarticular injection of OVA. Because oral administration of antigen induces immunological unresponsiveness, we studied for the first time the effects of oral administration of OVA on acute OIA. The oral administration of OVA before immunization significantly suppressed the development of acute OIA, in accordance with decreases in both the anti-OVA IgG antibody production and in vitro lymphocyte proliferative responses to OVA. However, the oral administration of OVA after immunization did not show any decrease in antibody production or in vitro lymphocyte proliferation to OVA, or in the severity of acute OIA. These results indicate that the induction of oral tolerance to OVA in OIA is possible by oral administration of OVA before, but not after, immunization with the antigen. This supports the concept of using antigen feeding as a treatment for certain humoral immune-mediated diseases.

Tumor necrosis factor-alpha cooperates with receptor activator of nuclear factor kappaB ligand in generation of osteoclasts in stromal cell-depleted rat bone marrow cell culture.

A member of the tumor necrosis factor (TNF) family, receptor activator of nuclear factor kappaB ligand (RANKL; also known as ODF, OPGL, and TRANCE), plays critical roles in osteoclast differentiation and activation in the presence of macrophage colony-stimulating factor (M-CSF). Recently, TNF-alpha has also been shown to induce the formation of multinucleated osteoclast-like cells (MNCs) in the presence of M-CSF from mouse macrophages. We demonstrated that mononuclear preosteoclast-like cells (POCs) were formed in the presence of conditioned medium of osteoblastic cells in a rat bone marrow culture depleted of stromal cells. Using this culture system, in this study we examined whether TNF-alpha affects differentiation into POCs from hematopoietic progenitor cells. Human TNF-alpha (hTNF-alpha) markedly stimulated the formation of POCs. Moreover, a concentration as low as 0.005 ng/mL of hTNF-alpha increased the level of mRNA for calcitonin receptor (CTR) and cathepsin-K of POCs. The POCs induced by hTNF-alpha formed MNCs, which showed dentine-resorbing activity after coculture with primary osteoblasts. Stimulation was observed after 24 h of treatment with hTNF-alpha only on day 1 or day 2 of the culture. After 24 h of hTNF-alpha treatment, expression of the receptor activator of nuclear factor kappaB (RANK) mRNA was markedly increased. The addition of soluble RANKL (sRANKL) to the preformed POCs efficiently induced MNCs. Interestingly, treatment of bone marrow cells with hTNF-alpha and sRANKL synergistically augmented the formation of MNCs. This formation was abolished by the addition of human osteoprotegerin (hOPG). These results suggest that cooperation of TNF-alpha and RANKL is important for osteoclastogenesis.

Balance of Th1/Th2 cytokines associated with the preventive effect of incomplete Freund's adjuvant on the development of adjuvant arthritis in LEW rats.

Complete Freund's adjuvant (CFA) could induce adjuvant arthritis (AA) in LEW rats and incomplete Freund's adjuvant (IFA) could induce oil induced arthritis (OIA) in DA but not in LEW rats. Lymph node cells (LNCs) from these AA and OIA rats showed increased mRNA expression of IFN-gamma, IL-2 and TNF-alpha but not IL-4. LNCs from IFA immunized LEW rats showed increased expression of IL-4, reduced expression of IFN-gamma and TNF-alpha and no IL-2, in contrast to IFA immunized DA rats. The pretreatment of IFA before CFA challenge could completely prevent AA in LEW rats and their LNCs showed increased expression of IL-4 and IFN-gamma but not IL-2 and TNF-alpha. In F1 (LEW x DA) rats, IFA could not induce OIA but the pretreatment of IFA before CFA challenge could induce very mild AA with 80% incidence, LNCs showing an elevated expression of all the above cytokines. These findings suggest that increased Th1 cytokine expression is associated with disease development and that increased IL-4 expression or the balance of Th2 over Th1 cytokine expression plays an important regulatory role in disease development.

Dimethyl dioctadecyl ammonium bromide (DDA)-induced arthritis in rats: a model of experimental arthritis.

A single intradermal injection of 2 mg of dimethyl dioctadecyl ammonium bromide (DDA) in phosphate buffered saline (PBS) could induce polyarthritis in both LEW and DA rats with low incidence and severity whereas 2 mg of DDA in incomplete Freund's adjuvant (IFA) could induce very severe polyarthritis with 100% incidence in LEW rats. Histology of DDA-induced arthritis (DIA) revealed cellular infiltration, synovial hypertrophy, development of granulation tissue, destruction of cartilage and bone deformation in the articular joints. Lymph node cells (LNCs) but not immunoglobulin fractions from the DIA rats successfully transferred the severe disease into the naive recipients. A challenge injection of DDA in IFA in the rats, which had recovered from the DIA, could reactivate the disease. It is thus concluded that DDA has arthritis-inducing ability in the rats which is potentiated by IFA and the DIA is a cell-mediated immune disease which might be a model of experimental arthritis.

New induction of leukotriene A(4) hydrolase by interleukin-4 and interleukin-13 in human polymorphonuclear leukocytes.

Interleukin (IL)-4, IL-10, and IL-13, Th2 cell-derived cytokines, play major roles in the pathophysiology of allergic diseases. These cytokines up-regulate or down-regulate the production of arachidonic acid metabolites. In this study, we have investigated the effect of IL-4, IL-10, IL-13, and other cytokines on A23187-stimulated synthesis of leukotriene (LT) B(4) in human polymorphonuclear leukocytes (PMNs). Production of LTB(4) was measured by specific radioimmunoassay and high performance liquid chromatography. Messenger RNA (mRNA) expression of cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), and LTA(4) hydrolase, which were involved in the synthesis of LTB(4), was determined by reverse transcription-polymerase chain reaction and Northern blot analysis. Protein synthesis of their enzymes was determined by Western blot analysis. IL-4 and IL-13 enhanced A23187-stimulated LTB(4) synthesis and increased mRNA expression and protein synthesis of LTA(4) hydrolase, but not those of cPLA(2) or 5-LO. These results indicate that IL-4 and IL-13 transcriptionally or post-transcriptionally up-regulate the synthesis of LTB(4), a potent chemotactic factor to PMNs, at the enzyme level of LTA(4) hydrolase, and this up-regulation mechanism may participate in the development of allergic inflammation. (Blood. 2000;96:601-609)

The preventive effects of incomplete Freund's adjuvant and other vehicles on the development of adjuvant-induced arthritis in Lewis rats.

The present study showed a novel finding that the development of adjuvant-induced arthritis (AA) in Lewis rats was completely prevented by incomplete Freund's adjuvant (IFA) injected 21 or 28 days before complete Freund's adjuvant (CFA) challenge. Hexadecane also completely prevented AA and squalane, methyl oleate and pristane moderately prevented AA, though pristane by itself induced mild arthritis in two out of five rats. Concanavalin A-stimulated lymph node cells (LNCs) isolated from AA rats were able to adoptively transfer the severe polyarthritis to all the naive recipients or even to the IFA pretreated recipients with earlier onset and more rapid progression than those of AA. The LNCs from the donors who had been pretreated with IFA and subsequently challenged with CFA could induce mild arthritis in only two out of eight naive recipients, whereas all the recipients who were challenged with CFA immediately after intravenous injection of these LNCs developed significantly less severe arthritis. However, the LNCs from IFA-pretreated donors failed to prevent AA. According to the T helper type 1 (Th1)/Th2 paradigm, it was suggested that the adjuvant-active vehicles such as IFA, hexadecane, squalane, methyl oleate and pristane, can affect and deviate the Th1/Th2 balance of immune responses in host. CFA could promote the propagation of Th2 cells rather than Th1 cells in these vehicle-pretreated rats through as yet undetermined mechanisms, eventually resulting in the prevention of AA. Finally, we discussed a regulatory role of adjuvant vehicles for induction and suppression of AA.

Osteoclast-derived zinc finger (OCZF) protein with POZ domain, a possible transcriptional repressor, is involved in osteoclastogenesis.

The differentiation of osteoclasts is regulated by transcription factors expressed in cells of osteoclast lineage. We isolated here a potential transcription factor from a cDNA library of an enriched population of preosteoclasts and osteoclasts. The cDNA encodes a protein with N-terminal POZ domain and C-terminal Krüppel-like zinc fingers. We designate this protein as osteoclast-derived zinc finger (OCZF). OCZF was found to be rat homologue of mouse leukemia/lymphoma-related factor (LRF). Northern blot and in situ hybridization analysis showed OCZF mRNA at a high level in osteoclasts and kidney cells. OCZF had a nuclear targeting sequence and was localized in the nucleus of transfected cells. In addition, OCZF specifically bound to the guanine-rich consensus sequences of Egr-1 and c-Krox. Transient transfection assays indicate that OCZF can repress transcription activity like other POZ domain proteins. Furthermore, antisense but not sense phosphorothioate oligodeoxynucleotides (ODNs) for OCZF cDNA suppressed the formation of osteoclast-like multinucleated cells (MNCs) in bone marrow culture, whereas the same ODNs did not significantly affect the formation of macrophage polykaryons and mononuclear preosteoclast-like cells (POCs). These results suggest that OCZF is a unique transcription factor that plays an important role in the late stage of osteoclastogenesis.

Suppressive effects of serum on the LPS-induced production of nitric oxide and TNF-alpha by a macrophage-like cell line, WEHI-3, are dependent on the structure of polysaccharide chains in LPS.

The effect of serum on LPS-induced activation of a murine macrophage-like cell line, WEHI-3, was examined. Foetal calf serum strongly inhibited the production of nitric oxide (NO) and TNF-alpha by LPS-stimulated WEHI-3 cells, while it enhanced the production of both by other macrophage-like cell lines, J774.1 and BAM3, on treatment with LPS. This suppressive effect of serum on WEHI-3 cells was most remarkable when the cells were stimulated with rough-chemotype LPS, Ra LPS, Rc LPS and Rd2 LPS. Foetal calf serum also inhibited TNF-alpha production by the same cells stimulated with high concentrations of smooth-form LPS (S LPS; > 1000 ng/mL). Serum-mediated suppression was also observed for expression of the TNF-alpha gene in Rc LPS-stimulated WEHI-3 cells. This suppressive effect of FCS was most remarkable during the 1-2 h before the addition of LPS, but it was not observed when FCS was added at 1 h after the addition of LPS, suggesting dependence on the time of FCS addition to LPS-stimulated cells. No significant difference was observed in the expression of CD14 on WEHI-3 cells cultured in the presence and absence of serum, suggesting that CD14 is not involved in the serum-mediated suppression of these LPS-responses. On the contrary, FCS showed enhancing effects on the production of NO and TNF-alpha by WEHI-3 cells stimulated with low concentrations (< 100 ng/mL) of S LPS and rough mutant Salmonella minnesota Re LPS. These results suggest that the ability of FCS to suppress LPS-induced activation of WEHI-3 cells in mainly dependent on the structure of polysaccharide chains and also on the concentration of LPS employed.

A novel role of IL-15 in the development of osteoclasts: inability to replace its activity with IL-2.

IL-15 shares many activities with IL-2 on stimulating lymphocytes, hematopoietic progenitor cells, and macrophages. However, the role of IL-15 in osteoclastogenesis has not been elucidated. The recent finding of abundant IL-15 in rheumatoid arthritis synovial fluids suggested a possible role for this cytokine in the pathological destruction of bone and prompted us to determine whether IL-15 stimulates osteoclast formation. IL-15 stimulated the formation of multinucleated osteoclast-like cells in rat bone marrow cultures. In stroma-free cultures, IL-15 increased the number of mononuclear preosteoclast-like cells in the early stage of osteoclast formation. The stimulation was observed even after treatment with IL-15 for only 24 or 48 h of culture. Moreover, low IL-15 concentration (0.1 ng/ml) strongly increased the level of calcitonin receptor mRNA of mononuclear preosteoclast-like cells. Although IL-15 is known as a potent stimulator of TNF-alpha, its activity was not abolished by addition of anti-TNF-alpha Ab. Interestingly, IL-2 and IL-7, which utilize some IL-15R components, had no effect on osteoclast differentiation, but pretreatment with IL-2 or IL-7 of bone marrow cells before the addition of IL-15 inhibited the enhancing activity of IL-15. In summary, IL-15 has a novel activity to stimulate the differentiation of osteoclast progenitors into preosteoclasts, which cannot be replaced by IL-2 but may use components in common with IL-2R to mediate its effects.

Autoreactive CD4- CD8- alpha beta T cells to vaccinate adjuvant arthritis.

Studies suggested that experimental autoimmune diseases can effectively be prevented and treated by application of normal autoreactive T cells or autoreactive T cells in an attenuated form. In this study, several autoreactive CD4- CD8- T-cell clones (A2, A6, and A13 cells) were isolated for the first time from the draining lymph nodes of Lewis rats with adjuvant arthritis (AA). Surprisingly, intraperitoneal inoculation with A13 cells, but not A2 or A6 cells protected rats from AA both clinically and histologically. It was demonstrated that A13 cells were CD4- CD8- alpha beta T cells, and showed proliferative responses to irradiated syngeneic spleen cells (antigen-presenting cells; APC). Interestingly, A13 cells proliferated against concanavalin A (Con A) and staphylococcal enterotoxin B (SEB), but did not show any proliferation to Mycobacterium tuberculosis (Mt), or its 65 000 MW heat-shock protein (HSP). Rats protected from AA by inoculation with A13 cells showed a specific anti-idiotypic delayed-type hypersensitivity reaction compared with other autoreactive T cells (A2 or A6 cells). These findings demonstrate that AA can be suppressed by autoreactive CD4- CD8- alpha beta T cells, and these cells may be used as therapeutic agents in experimental autoimmunity.

Marked inhibition of the intracellular multiplication of Legionella pneumophila in monocytes isolated from carriers of human T lymphotrophic virus type I.

Intracellular growth patterns of Legionella pneumophila were examined in monocytes obtained from carriers of human T-lymphotropic virus type I (HTLV-1) and controls who were HTLV-1 seronegative. All subjects were seronegative for antibodies against L. pneumophila. Bacterial growth was determined 0, 1, 2, and/or 3 days after infecting peripheral blood mononuclear cells (PBMCs) with the bacteria. The intracellular growth of L. pneumophila was markedly inhibited in HTLV-1 carriers compared with normal controls. When the lymphocytes were depleted from the HTLV-1 carrier PBMC cultures before infection, this inhibition was abolished. Inhibition reappeared, however, when the 72-h culture supernatants of PBMCs from HTLV-1 carriers were added to the lymphocyte-depleted cultures. Culture supernatants of infected and uninfected PBMCs from HTLV-1 carriers exhibited markedly increased levels of interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) compared with the HTLV-1 seronegative controls. In the HTLV-1 carriers, IFN-gamma was produced by the CD4+ lymphocytes, whereas TNF-alpha was secreted by the monocytes. Addition of anti-IFN-gamma or anti-TNF-alpha antibodies to the HTLV-1 carrier PBMC cultures diminished the inhibition of intracellular growth of L. pneumophila. Results suggest that the monocytes are activated in HTLV-1 carriers. These findings may explain why an opportunistic infection by certain intracellular pathogens is rarely seen among HTLV-1 carriers.

Clonal anergy is a potent mechanism of oral tolerance in the suppression of acute antigen-induced arthritis in rats by oral administration of the inducing antigen.

The effects of oral administration of ovalbumin (OVA) on acute OVA-induced arthritis (OIA) in rats, which is mediated by Arthus reaction to the antigen in the joint space, were investigated. The oral administration of OVA before immunization with OVA significantly suppressed the development of acute OIA in a dose-dependent manner, in accordance with decreases in both the in vivo anti-OVA IgG antibody production and in vitro lymphocyte proliferative responses to OVA. These results were shown in both the single high-dose (200 mg x 1) or the multiple low-dose (200 microg x 5) feeding protocols. In vitro study showed that rat IL-2 could reverse the reduced OVA-specific lymphocyte proliferative responses. The spleen cells obtained from OVA-feeding, unprimed rats neither adoptively transferred the suppression to naive recipient rats nor suppressed the in vitro lymphocyte proliferation. These results demonstrate that the acute OIA can be suppressed by the induction of oral tolerance (OT) to OVA, and strongly suggest that the OT was due to clonal anergy of antigen-reactive T lymphocytes, not the active suppression by OVA-specific regulatory cells.

Suppression of adjuvant arthritis in rats by induction of oral tolerance to mycobacterial 65-kDa heat shock protein.

Oral administration of mycobacterial 65-kDa heat shock protein (HSP) given daily for 5 days prior to immunization with Mycobacterium tuberculosis (Mt) suppressed the development of adjuvant arthritis (AA) in rats. AA was significantly suppressed by 30 and 300 micrograms HSP, and variably by 0.3, 3 micrograms or 1 mg. Histological analysis of joint samples obtained from control and test rats confirmed the suppression of AA in the fed group. Feeding Mt or hen egg lysozyme (HEL) failed to affect AA, indicating that the suppression was HSP specific. The oral administration of 30 micrograms HSP decreased both delayed-type hypersensitivity (DTH) reactions and proliferative responses to HSP and Mt. In addition, the proliferation of lymph node cells (LNC) from Mt-sensitized rats was inhibited by the addition of spleen cells (SPC) from HSP-fed animals, possibly by the secretion of transforming growth factor (TGF)-beta. Spleen cells obtained from tolerized donors were capable of transferring the tolerance to naive recipients. These results demonstrate that feeding HSP is an effective way to suppress AA and that the suppression of AA may be mediated by regulatory T cells generated following oral administration of mycobacterial 65-kDa HSP.

Differences in immune functions between human T-lymphotropic virus type I carriers and patients with adult T-cell leukemia/lymphoma.

A variety of immunologic tests were compared between human T-lymphotropic virus type I (HTLV-I) carriers and patients with adult T-cell leukemia/lymphoma (ATL). The mitogenic responses of the lymphocytes to concanavalin A and phytohemagglutinin were depressed in the ATL patients but not in the carriers, while the response to pokeweed mitogen in the carriers and ATL patients was depressed compared to the HTLV-I-seronegative controls. A positive tuberculin reaction in the carriers was as frequent as in the controls, but less frequent in the ATL patients. A marked inhibition of the intracellular multiplication of Legionella pneumophila was observed within the monocytes isolated from the carriers, but not in the ATL patients or the normal controls. These results indicate that the ATL patients have severe immunodeficiency, while the HTLV-I carriers have some activation of anti-microbial activity in monocytes although they are somewhat immunosuppressed.

Effects of interferon-alpha, beta, and gamma on the function of differentiated leukemic HL-60 cells induced by 1,25-dihydroxyvitamin D3.

The differentiation of HL-60, a human leukemic cell line, into monocyte-like cells (D3-HL-60 cells) is induced by 1,25-dihydroxyvitamin D3 (D3). We examined the effects of interferon (IFN) treatment of D3-HL-60 cells on the expression of cell surface antigens, the phagocytic activity for fluorescent beads, production of oxygen radicals, and intracellular growth of Legionella pneumophila. Activation of D3-HL-60 cells with IFN-gamma, Beta, and alpha for 24 h significantly increased expression of CD16, CD36, CD71, and HLA-DR antigens. IFN-gamma markedly enhanced the phagocytic activity of beads in D3-HL-60 cells. There was no significant difference in phagocytic activity between cells exposed to IFN-alpha or beta and untreated D3-HL-60 cells. IFN-alpha, beta, and gamma enhanced production of oxygen radicals, including superoxide, by D3-HL-60 cells. Superoxide production was enhanced to the greatest degree by IFN-gamma, followed by IFN-beta and then IFN-gamma. Intracellular growth of L. pneumophila in D3-HL-60 cells was inhibited by interferons (IFN-gamma > beta > gamma). Similar results were obtained in human mononuclear cells. These data indicate that interferons can act as biologic response modifiers not only in human mononuclear cells but also in differentiated leukemic cells. Our results may have implications for the development of differentiation therapy for treatment of leukemia.

Effect of growth condition on in-vitro susceptibility of Shigella dysenteriae type 1 killing by murine peritoneal macrophages.

The intracellular fate of Shigella dysenteriae type 1 strains grown in casamino acid-yeast extract (CYE) broth and nutrient broth (NB) was studied in casein-elicited mouse peritoneal macrophages. Virulent strains 14731 and W30864 cultured in NB and opsonised with normal mouse serum were susceptible to killing by peritoneal macrophages (66 SEM 1.7% killing by 2 h). In contrast, both strains grown in CYE broth and opsonised with normal mouse serum showed resistance to killing by peritoneal macrophages (76 SEM 1.4% survival by 2 h). Electronmicroscopy demonstrated that the bacteria escaped from the phagosome compartment by lysing the phagocytic vacuole and remained within the cytoplasm. Lipopolysaccharide (LPS) stimulated peritoneal macrophages to kill the opsonised strains 14731 and W30864 grown in CYE broth (85.4 SEM 1.6% killing by 2 h). Recombinant murine gamma interferon (rIFN-gamma) also stimulated macrophages to kill CYE-grown bacteria (52.1 SEM 1.3% killing by 2 h). However, an avirulent rough mutant strain W30864-22 grown in either NB or CYE broth showed marked susceptibility to killing by peritoneal macrophages, which was similar to that of NB-grown strain 14731 or W30864. The results of the present study suggest that in-vitro growth conditions may modulate the susceptibility of S. dysenteriae type 1 to killing by phagocytes.

Effect of culture conditions on the susceptibility of enteroaggregative Escherichia coli to human serum and polymorphonuclear leucocytes.

Recent work has indicated that the pathogenicity of enteroaggregative Escherichia coli (EAggEC) is strictly regulated by environmental conditions. The growth conditions, susceptibility to the bactericidal activity of serum, and polymorphonuclear leucocytes (PMN) using strains of EAggEC grown in two different culture conditions were examined. Strains of EAggEC grown in casamino acids-yeast extract (CYE) broth at 37 degrees C for 20 h showed resistance to killing by human serum (86.8 +/- 6.9% survival by 2 h) and PMN (67 +/- 5.1% survival by 2 h). In contrast, these strains grown in nutrient broth (NB) showed sensitivity to serum (98.9 +/- 0.5% killing by 2 h) and PMN (98.9 +/- 0.5% killing by 2 h). EAggEC strains cultured in NB became resistant to killing by serum when mixed with extracellular material extracted from the bacteria grown in CYE broth. These results indicate that the growth condition may modulate the sensitivity of EAggEC, and the promotion of extracellular material formation in CYE broth is responsible for the resistance to killing by serum and PMN.