Suppressive effect of asbestos on cytotoxicity of human NK cells.

Asbestos, a naturally occurring fibrous mineral, causes malignant mesothelioma (MM). However, it takes a very long time to develop MM, which suggests that effects other than tumorigenicity of asbestos might contribute to the development of MM, and one of the possible targets is anti-tumor immunity. Therefore, we examined the effect of asbestos exposure on human natural killer (NK) cells using the cell line of YT-A1, Peripheral blood mononuclear cells (PBMCs) cultures and specimens from patients with MM. In particular, we focused on expression of NK cell-activating receptors, including NKG2D, 2B4 and NKp46. Analysis of the YT-CB5 subline of YT-A1, cultured with CB for over 5 months, showed a decrease in cytotoxicity with low expressions of NKG2D and 2B4, although there were no decreases after about one month. YT-CB5 showed decreases in phosphorylation of extracellular signal-regulated kinase (ERK) and degranulation stimulated by antibodies to NKG2D. Peripheral blood (PB-) NK cells from MM patients also showed decreased cytotoxicity compared with healthy volunteers (HV), and was accompanied with low expression of NKp46 unlike YT-CB5. PBMCs cultured with CB resulted in decreased expression of NKp46 on NK cells, although this did not occur when using glass wool, an asbestos substitute. These results indicate that asbestos has the potential to suppress cytotoxicity of NK cells. In particular, it is noteworthy that both NK cells from MM patients and those from a culture of PBMCs derived from HVs with asbestos showed the same characteristic of decreased cytotoxicity with low expression of NKp46.

Dysregulation of autoimmunity caused by silica exposure and alteration of Fas-mediated apoptosis in T lymphocytes derived from silicosis patients.

Silicosis patients suffer from pulmonary fibrosis caused by silica inhalation, as well as autoimmune diseases known as the adjuvant effects of silica. Caplan syndrome complicated with rheumatoid arthritis (RA) is well known epidemiologically, and the incidence of complicated systemic sclerosis (SSc), systemic lupus erythematosus (SLE) and antineutrophilic cytoplasmic antibody (ANCA)-related nephritis have been reported frequently in silicosis patients. To explore the detailed mechanisms of silica-induced dysregulation of autoimmunity, we had focused on Fas/CD95 and Fas-mediated apoptosis because Fas is one of the most important molecules regarding apoptosis of lymphocytes and its alteration makes some T cells survive longer. Additionally, if the long-survived T cells include the self-recognizing T-cell clones, it is easily thought that autoimmune diseases will appear in this situation. Furthermore, regulatory T cells (Treg) showing CD4+25+ and forkhead box P3 (FoxP3)-positive have been a central player in regulating activation of self- and foreign-antigen recognizing T cells, and it has been reported that activation of Treg causes its higher expression of Fas/CD95. Thus, in this review, we introduce the alteration of Fas and related molecules as found in silicosis and also present the Treg function of the CD4+25+ fraction in peripheral blood mononuclear cells derived from silicosis patients.

Reductive alteration of the regulatory function of the CD4(+)CD25(+) T cell fraction in silicosis patients.

Causal links have been documented between silica and rheumatoid arthritis, lupus erythematosus, systemic sclerosis and glomerulonephritis. Two different effects of silica have been suggested, an enhanced inflammatory response in the pulmonary region (e.g. activation of alveolar macrophages) and dysregulation of autoimmunity. Based on our previous reports showing in vitro activation of peripheral T cells by silica and reduced regulatory function of the peripheral CD4(+)CD25(+) fraction in which FoxP(3)+ regulatory T cells (Treg) are located, reconstitution of the CD4(+)CD25(+) fraction in silicosis patients (SILs) was investigated. Since T cells in peripheral CD4(+)CD25(+) and CD4(+)CD25(-) (effector T cells; Teff) fractions from SILs showed higher expression of pd-1 (a marker gene for T cell activation) in comparison to that of healthy donors (HDs), chronic T cell activation was considered to have occurred in SILs. In this study, a higher expression of the CD95/Fas molecule in Treg was recorded from silicosis patients (SILs) compared to healthy donors (HDs), and excess loss of FoxP3(+) Treg in freshly isolated peripheral blood mononuclear cells (PBMCs) from SILs relative to HDs was demonstrated when these cells were cultured with silica ex vivo, whereas CD25(+) cells were not reduced due to contamination of activated Teff in the CD4(+)CD25(+) fraction. The activation of both Teff and Treg results in reconstitution of the peripheral CD4(+)CD25(+) fraction, loss of Treg and contamination of activated Teff, resulting in reduction of the number and function of Treg. These results contribute to our understanding of the development of autoimmune diseases found in SILs.

Impairment in cytotoxicity and expression of NK cell- activating receptors on human NK cells following exposure to asbestos fibers.

Asbestos is well-known for its tumorigenic activity, but its effect on anti-tumor immunity remains unclear. Therefore, we prepared a sub-line of YT-A1 human NK cells exposed to chrysotile B (CB) asbestos (YT-CB5) as an in vitro model to analyze the effect of asbestos exposure on NK cells, and examined cytotoxicity and expressions of its related molecules. The cytotoxicity of YT-CB5 against K562 cells decreased compared with the original line of YT-A1 (YT-Org). YT-CB5 exhibited significant decreases in expressions of cell surface NKG2D, 2B4 and intracellular granzyme A. YT-CB5 also exhibited a decrease in the 2B4-dependent cytotoxicity. In addition, the degranulations stimulated via cell surface NKG2D and 2B4 also decreased in YT-CB5. Therefore, peripheral blood NK cells in patients with malignant mesothelioma (MM) were examined and compared with healthy volunteers. NK cells in patients with MM also showed decreases in cytotoxicity against K562. Although the expressions of NKG2D and 2B4 did not decrease in NK cells of MM patients, the expression of cell surface NKp46 decreased. To confirm the effect of asbestos exposure on peripheral blood NK cells, PBMCs were cultured under exposure to CB. NK cells in PBMCs exposed to CB in vitro showed a significant decrease in the expression of NKp46, whereas NK cells and alter the expression of NK cell-activating receptors including NKG2D, 2B4 and NKp46 and intracellular perforin/granzymes.cells in PBMCs exposed to glass wool did not show such a decrease. These results indicate that exposure to asbestos has the potential to impair the cytotoxicity of NK cells and alter the expression of NK cell-activating receptors including NKG2D, 2B4 and NKp46 and intracellular perforin/granzymes.

Decrease in phosphorylation of ERK following decreased expression of NK cell-activating receptors in human NK cell line exposed to asbestos.

YT-CB5, which had been continuously cultured with chrysotile B (CB)asbestos, showed impaired cytotoxicity with decreased expression of NKG2D and 2B4 NK cell-activating receptors. In the present study, the phosphorylation of extracellular signal-regulated kinase (ERK), which is known to induce degranulation downstream of many NK cell-activating receptors, was examined in YT-CB5 by flow cytometry and compared with the control line YT-Org. YT-CB5 exhibited impaired phosphorylation of ERK1/2 induced by the recognition of K562 cells, downstream of a process mediated by Src family kinase and phosphoinositide 3-kinase. YT-CB5 also exhibited impaired phosphorylation of ERK1/2 following incubation with K562 cells in the presence of anti-2B4 antibodies, where co-stimulation by 2B4 augmented the phosphorylation of ERK1/2 in YT-CB5 to a similar degree as in YT-Org. The phosphorylation of ERK1/2 induced by an inhibitor against phosphatase (PP) 1 and PP2A was also lower in YT-CB5 compared with YT-Org. Moreover, bead-bound antibodies to NKG2D, which contribute to cytotoxicity against K562 cells, induced negligible phosphorylation of ERK1/2 in YT-CB5, although antibodies to 2B4 induced a comparatively greater level of phosphorylation. Additionally, peripheral blood (PB-) NK cells with low expression of NKG2D showed lower phosphorylation of ERK1/2 mediated by anti-NKG2D antibodies compared with PB-NK cells with high expression of NKG2D. These results indicate that signal transduction events leading to the phosphorylation of ERK is impaired in YT-CB5 due to decreased expression of NKG2D. Further studies are required to clarify whether this suppressive effect of asbestos exposure on NK cells might promote lung cancer and mesothelioma in people who have inhaled asbestos.

Microsatellite analysis of the GLC1B locus on chromosome 2 points to NCK2 as a new candidate gene for normal tension glaucoma.

AIMS: The aim of this study was to investigate the association between normal tension glaucoma and the candidate disease locus glaucoma 1, open angle, B (GLC1B) on chromosome 2. There are many reports describing the results of association or linkage studies for primary open angle glaucoma (POAG), with GLC1B as one of the loci associated with normal or moderately elevated intraocular pressure. However, there are few reports about the association of genes or defined genomic regions with normal tension glaucoma, which is the leading type of glaucoma in Japan. The GLC1B locus is hypothesized to be a causative region for normal tension glaucoma. METHODS: Genomic DNA was extracted from whole blood of normal tension glaucoma (n = 143) and healthy controls (n = 103) of Japanese origin. RESULTS: Fifteen microsatellite markers within and/or near to the GLC1B locus were genotyped, and their association with normal tension glaucoma was analysed. Two markers D2S2264 and D2S176 had significant positive associations. CONCLUSION: The D2S176 marker had the strongest significant association and it is located 24 kb from the nearest gene NCK2, which now becomes an important new candidate gene for future studies of its association with normal tension glaucoma.

Complexity of the HVR-1 quasispecies and disease activity in patients with hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) easily undergoes genomic changes, especially in the hypervariable region (HVR) in the N-terminus of the E2/NS1 region. The quasispecies nature of HCV may have important biological implications in relation to viral persistence; however, the relationship between disease activity of chronic HCV infection and development of the genomic complexity have yielded conflicting results. We explored the changes in the complexity of the HVR-1 in the natural course of chronic HCV infection with and without elevation of serum alanine transaminase (ALT) levels. MATERIALS AND METHODS: Ten patients with chronic hepatitis C proven by liver biopsy, who showed persistent elevation of the serum ALT levels, and 15 patients with chronic HCV infection and persistently normal serum ALT levels (PNAL) were enrolled in this study. The number of the HCV quasispecies was determined twice for each patient at an interval of mean 2.5 years by fluorescence single-strand conformation polymorphism and sequence analysis. RESULTS: There was no significant difference in the changes in the number of quasispecies during the follow-up period between chronic hepatitis C and PNAL. There was also no significant difference in the change in the number of variable nucleotides sites between the two groups. In these patients, the number of quasispecies and the diversity of HVR-1 were correlated with platelet counts and serum hyaluronic acid levels previously shown to be associated with disease progression. CONCLUSION: Our results suggested that the disease activity is not always related to the generation of the HVR-1 quasispecies complexity.

Interleukin 10 and transforming growth factor beta contribute to the development of experimentally induced allergic conjunctivitis in mice during the effector phase.

AIM: To investigate the involvement of interleukin (IL)10 and transforming growth factor (TGF) beta in the development of experimentally induced allergic conjunctivitis in mice. METHODS: Balb/c mice were actively sensitised with ragweed in alum, and then challenged with ragweed in eye drops after 10 days. 24 h later, the conjunctivas, spleens and blood were collected for histological and cytokine expression analyses, proliferation and cytokine production assays and measurement of immunoglobulin (Ig) levels. Mice developing experimentally induced allergic conjunctivitis were injected intraperitoneally with 200 microg of anti-IL10 or anti-TGF beta antibodies at 0, 2, 4, 6 and 8 days (induction phase treatment) or 500 microg of antibodies 2 h before ragweed challenge (effector phase treatment). Normal rat IgG was used for control injections. RESULTS: Treatment with either anti-IL10 or anti-TGF beta antibodies during the induction phase did not affect eosinophil infiltration into the conjunctiva. By contrast, treatment with either antibody during the effector phase suppressed infiltration. During the effector phase, treatment with anti-TGF beta antibody, but not the anti-IL10 antibody, markedly up regulated proliferation and Th2 cytokine production by splenocytes. IL1alpha levels in the conjunctiva were reduced after treatment with either antibody; in addition, eotaxin and tumour necrosis factor alpha levels were reduced after treatment with antibody to TGF beta. CONCLUSIONS: IL10 and TGF beta do not have immunosuppressive roles in the development of experimentally induced allergic conjunctivitis. Rather, they augment the infiltration of eosinophils into the conjunctiva during the effector phase of experimentally induced allergic conjunctivitis.

Tumour necrosis factor alpha signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice.

BACKGROUND: While tumour necrosis factor alpha (TNF-alpha) appears to be associated with the development of non-alcoholic steatohepatitis (NASH), its precise role in the pathogenesis of NASH is not well understood. METHODS: Male mice deficient in both TNF receptors 1 (TNFR1) and 2 (TNFR2) (TNFRDKO mice) and wild-type mice were fed a methionine and choline deficient (MCD) diet or a control diet for eight weeks, maintaining isoenergetic intake. RESULTS: MCD dietary feeding of TNFRDKO mice for eight weeks resulted in attenuated liver steatosis and fibrosis compared with control wild-type mice. In the liver, the number of activated hepatic Kupffer cells recruited was significantly decreased in TNFRDKO mice after MCD dietary feeding. In addition, hepatic induction of TNF-alpha, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 was significantly suppressed in TNFRDKO mice. While in control animals MCD dietary feeding dramatically increased mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) in both whole liver and hepatic stellate cells, concomitant with enhanced activation of hepatic stellate cells, both factors were significantly lower in TNFRDKO mice. In primary cultures, TNF-alpha administration enhanced TIMP-1 mRNA expression in activated hepatic stellate cells and suppressed apoptotic induction in activated hepatic stellate cells. Inhibition of TNF induced TIMP-1 upregulation by TIMP-1 specific siRNA reversed the apoptotic suppression seen in hepatic stellate cells. CONCLUSIONS: Enhancement of the TNF-alpha/TNFR mediated signalling pathway via activation of Kupffer cells in an autocrine or paracrine manner may be critically involved in the pathogenesis of liver fibrosis in this NASH animal model.

Fibulin-5 deposition in human skin: decrease with ageing and ultraviolet B exposure and increase in solar elastosis.

BACKGROUND: Fibulin-5 was recently found as a secreted extracellular matrix protein that functions as a scaffold for elastic fibres. However, the distribution of fibulin-5 in human skin and its changes during the ageing process are not known. OBJECTIVES: To explore the involvement of fibulin-5 in skin ageing, the age-dependent changes in fibulin-5 localization in human skin were examined compared with those of other elastic fibre components including elastin, fibrillin-1 and fibulin-2. Methods The distribution of elastin, fibrillin-1, fibrillin-2, fibulin-2 and fibulin-5 was investigated by means of immunohistochemistry using their specific antibodies. Skin samples were recovered from 12 healthy subjects undergoing plastic surgery. Ultraviolet (UV) B-irradiated or control nonirradiated buttock skin samples were obtained from two healthy volunteers at 2 days after the irradiation at 2 minimal erythemal doses. RESULTS: In the reticular dermis of young sun-protected skin from the upper arm, fibulin-5 colocalized with the other elastic fibre components, while in the papillary dermis fibulin-5 showed candelabra-like structures perpendicular to the epidermis with an unstained area just beneath the epidermis, which was similar to that of elastin but not fibrillin-1. Fibulin-5 in the reticular dermis decreased and disappeared with age even in sun-protected skin from the thigh, abdomen and upper arm. In sun-exposed skin, fibulin-5 was extremely reduced in the dermis of cheek skin even from a 20-year-old man. UVB irradiation reduced fibulin-5, fibulin-2 and elastin markedly, moderately and weakly, respectively, compared with levels in control nontreated skin. Interestingly, the deposition of fibulin-5 was increased in solar elastosis, like that of other elastic fibre components. CONCLUSIONS: These results suggest that fibulin-5 is a good marker of skin ageing and that the earlier loss of fibulin-5 may involve age-dependent changes in other elastic fibre components.

Evaluation of autologous cultured epithelium as replacement skin after tattoo excision: correlation between skin texture and histological features.

BACKGROUND: Cultured epidermal autographs (CEAs) are currently used as a coverage treatment for burn wounds, for disfiguring burn scars involving depigmentation and in restoring the elasticity of the skin. The advantage of CEAs is that epidermal sheets prepared from small skin pieces can be enlarged sufficiently to cover large burn areas. OBJECTIVES: We examined the correlation between recovery of skin texture, and elastic fibre formation and keratinocyte differentiation (assessed by immunohistochemistry) in CEAs used as replacement skin after tattoo excision in a Japanese patient. METHODS: The tattooed skin was excised down to the deep dermal layer and then CEA was transplanted onto the patient. The skin textures were evaluated by taking replicas of the skin surface, and histological changes of filaggrin, transglutaminase, involucrin, fibrillin and elastin in the autograft skin were examined by immunohistochemistry. RESULTS: The skin texture improved with time after grafting the CEA, and appeared similar to that of normal skin at 39 months. Among keratinocyte differentiation markers, filaggrin recovered to a normal pattern at around 6 months, and transglutaminase did so at 39 months, whereas involucrin expression remained abnormal at 39 months. Fibrillin expression appeared similar to that of normal skin by 39 months, except for sparse candelabra-like structures of short fibres. Elastin expression remained at a low level throughout. CONCLUSIONS: Our results show that the recovery of skin texture after application of CEAs following tattoo excision is associated with the normalization of epidermal differentiation markers, except involucrin, and with the regeneration of elastic fibres in the dermis.

Synergistic effect of TNF-alpha and IL-4 on the expression of thymus- and activation-regulated chemokine in human corneal fibroblasts.

Chemokine production by resident fibroblasts contributes to the recruitment of migratory leukocytes to sites of tissue injury. The effects of cytokines on the expression of thymus- and activation-regulated chemokine (TARC), a potent chemoattractant for Th2 cells, were examined in cultured human corneal fibroblasts. The culture supernatants of cells incubated for 24 h in the absence or presence of either TNF-alpha (10 ng/ml) or IL-4 (10 ng/ml) alone did not contain TARC detectable by ELISA. However, exposure of cells to both cytokines resulted in marked release of TARC into the culture medium in a time- and dose-dependent manner. Similarly, quantitative RT-PCR analysis revealed that the two cytokines induced a synergistic increase in the amount of TARC mRNA in the cultured fibroblasts. This synergistic effect of TNF-alpha and IL-4 on TARC production by fibroblasts may contribute to the Th2 cell infiltration and consequent tissue damage associated with allergic inflammation.

Mitogenic action of lysophosphatidic acid in proximal tubular epithelial cells obtained from voided human urine.

Focal tubular cell multiplication at sites on an injured nephron is a critical event in the recovery phase following acute tubular necrosis. During this process, numerous viable tubular cells exfoliate and are shed into the urine. Lysophosphatidic acid (LPA) is generated in the plasma membrane of injured cells and acts as an intercellular mediator of various biological processes, including inflammation, proliferation and repair. In the present study, exfoliated proximal tubule (PT) cells were isolated from human urine and the mitogenic effects of LPA were investigated as a model of repair and proliferation following renal injury. LPA stimulated a 23. 5% increase in DNA synthesis, a 29.4% increase in cell number and an 86.6% decrease in cAMP content. All of these responses were pertussis toxin sensitive, indicating the involvement of G(i)-type G-proteins in LPA signalling. Conversely, the LPA-induced DNA synthesis and the decrease in intracellular cAMP content were insensitive to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), suggesting a mitogenic response via PI3K-independent mechanisms. Furthermore, we detected specific mRNA transcripts for the recently cloned human LPA-receptors, endothelial differentiation gene (Edg)-2 and Edg-4 (Edg-2>>Edg-4) by reverse transcription-PCR in PT cells. Our data suggest that LPA may behave as a local growth factor in PT cells following tubular injury.

Up-regulation of E-cadherin and I-catenin in human hepatocellular carcinoma cell lines by sodium butyrate and interferon-alpha.

Human E-cadherin is a homophilic cell adhesion molecule and its expression is well preserved in normal human hepatocytes; a decrease in its expression has been observed in poorly differentiated hepatocellular carcinoma cells. We examined the alteration of E-cadherin and catenin expressions caused by differentiation inducers in human hepatocellular carcinoma cells. Hepatocellular carcinoma cell lines, HCC-T and HCC-M, were cultured with all-trans retinoic acid (ATRA), dexamethasone (DEX), sodium butyrate, and interferon-alpha. E-cadherin expression was only up-regulated by butyrate and interferon-alpha (IFN-alpha) in both cell lines, studied by means of fluorescence immunostaining and flow cytometry. The localization of E-cadherin staining was shown at their cell membrane. According to the increase in E-cadherin expression, beta-catenin expression appeared at the cell membrane of both cell lines when treated with butyrate and IFN-alpha. Such an appearance was not observed when cells were treated with ATRA and DEX. Western blotting showed that alpha- and y-catenin expression was not changed, while only the expression of beta-catenin increased. Beta-catenin oncogenic activation as a result of amino acid substitutions or interstitial deletions within or including parts of exon 3, which has been demonstrated recently, was not detected in these cell lines by direct deoxyribonucleic acid sequencing. These results suggest that the expression and interaction between E-cadherin and wild-type beta-catenin are potentially modulated by butyrate and IFN-alpha, and that these two agents are potent inhibitors of hepatocellular carcinoma cell invasion and metastasis.

Synergistic induction of eotaxin expression in human keratocytes by TNF-alpha and IL-4 or IL-13.

PURPOSE: To investigate the effects of tumor necrosis factor (TNF)-alpha, interleukin (IL)4, and IL-13 on expression of the chemokine eotaxin by cultured human keratocytes. METHODS: Cultured human keratocytes were incubated with various combinations and concentrations of TNF-alpha, IL-4, and IL-13. The concentration of eotaxin in the culture supernatant was subsequently measured by enzyme-linked immunosorbent assay, and the amount of eotaxin mRNA in cell lysates was determined by reverse transcription-polymerase chain reaction analysis. RESULTS: Keratocytes incubated in the absence of cytokines did not release detectable amounts of eotaxin into the culture medium. Whereas incubation of keratocytes with TNF-alpha, IL-4, or IL-13 alone or with the combination of IL-4 and IL-13 had only a small effect on eotaxin release, exposure of the cells to TNF-alpha in combination with either IL-4 or IL-13 resulted in a marked increase in eotaxin production that was both time and dose dependent. The abundance of eotaxin mRNA in keratocytes was also increased in a synergistic manner by incubation of cells with TNF-alpha together with either IL-4 or IL-13. CONCLUSIONS: Stimulation of human keratocytes with the combination of TNF-alpha and either IL-4 or IL-13 resulted in synergistic increases in both the abundance of eotaxin mRNA and the release of eotaxin protein. This cytokine-induced increase in eotaxin production by keratocytes may contribute to eosinophil infiltration in inflammatory ocular diseases such as vernal keratoconjunctivitis.

Induction of hepatic stellate cell proliferation by LPS-stimulated peripheral blood mononuclear cells from patients with liver cirrhosis.

We studied hepatic stellate cell proliferation in vitro. Peripheral blood mononuclear cells (PBMC) from patients with chronic active hepatitis C (CAH) and liver cirrhosis (LC) were cultured for 24h in the presence or absence of Escherichia coli lipopolysaccharides (LPS). Hepatic stellate cell proliferation induced by the culture supernatants was measured, and interleukin-1 (IL-1) and IL-6 levels in the culture supernatants were quantified. Culture supernatants of LPS-stimulated PBMC from LC patients induced rat hepatic stellate cell proliferation by almost 2.8-fold (stimulation index, 2.83 +/- 1.41) compared with when the cells were cultured without addition of PBMC culture supernatants. Production of IL-1beta was significantly higher in the culture supernatants of both CAH and LC patients than in those of ten healthy controls (P < 0.01 and P < 0.05, respectively). But there was no significant correlation between IL-1 production and the induction of hepatic stellate cell proliferation by the culture supernatants. Although there were no significant differences in IL-6 production by LPS-stimulated PBMC among healthy controls and CAH and LC patients, we observed a significant correlation between IL-6 production and the induction of hepatic stellate cell proliferation in the culture supernatants of LC patients. Rat hepatic stellate cells themselves produced IL-6, and treatment with IL-6 antisense oligodeoxynucleotides suppressed the cell proliferation, suggesting that IL-6 is an autocrine growth factor for hepatic stellate cells. The addition of human recombinant IL-6 (hrIL-6) augmented rat hepatic stellate cell proliferation, indicating that excessive IL-6 may further facilitate cell proliferation. These findings suggest that a cytokine cascade including IL-6 may participate in hepatic stellate cell proliferation in LC patients when they are exposed to endotoxin.