Flagellar filament structural protein induces Sjögren's syndrome-like sialadenitis in mice.

OBJECTIVE: Sjögren's syndrome (SS) is a systemic autoimmune disease that primarily affects lacrimal and salivary glands. We previously reported that FliC derived from Escherichia coli could induce autoimmune pancreatitis-like lesions. From these results, we speculated that FliC could also induce SS-like exocrinopathy. In this study, we investigated the effects of chronic exposure to FliC on lacrimal and salivary glands and the possibility that it might lead to an autoimmune response. METHODS: C57BL/6 mice were repeatedly injected with FliC and histological changes, serum levels of cytokine/chemokines and autoantibodies were evaluated at different time points after the final injection. The presence of sialadenitis was diagnosed by histological methods. RESULTS: In FliC-treated groups, 57% of subjects developed inflammatory cell infiltrates around ducts in mandibular salivary glands, but not lacrimal glands. In addition, serum levels of total IgG, IgG1, and IgG2a were significantly higher in FliC-treated groups. Intriguingly, serum anti-SSA/Ro levels were also significantly higher in FliC-treated groups. Cytokine analysis revealed that serum levels of IL-1ß, IL-12p70, IL-13, IFN-γ, IL-15, and IL-23 seemed to be higher in FliC-treated mice. CONCLUSIONS: Our data suggest that FliC-treated mice develop an SS-like phenotype. Our model may elucidate the relationship between commensal bacteria and SS.

Effect of Lipopolysaccharide on the Progression of Non-Alcoholic Fatty Liver Disease in High Caloric Diet-Fed Mice.

The incidence of non-alcoholic steatohepatitis (NASH) is increasing. Because gut microbiota have been highlighted as one of the key factors in the pathogenesis of metabolic syndrome, we investigated the involvement of the bacterial component in the progression of non-alcoholic fatty liver (NAFL) to NASH. C57BL/6 mice were fed with maintenance food (MF, groups A and B) or a high caloric diet (HCD, groups C and D) for 1 month. Mice were then divided into four groups: Groups A and C were inoculated with PBS, while groups B and D were inoculated with lipopolysaccharide (LPS) plus complete Freund's adjuvant (CFA). The inoculations were performed a total of 3 times over 3 months. At 6 months, while hepatic steatosis was observed in groups C and D, cellular infiltration and fibrosis were less evident in group C than in group D. Inflammatory cytokines were upregulated in groups B and D. 16S rRNA pyrosequencing of whole colon homogenates containing faeces showed that certain bacterial groups, such as Bacteroidaceae, Peptostreptococcaceae and Erysipelotrichaceae, were increased in groups C and D. Although loading of bacterial components (LPS) resulted in hepatic inflammation in both MF- and HCD-fed mice, HCD feeding was more crucial in the progression of NAFL during the triggering phase.

Epitopes associated with MHC restriction site of T cells. III. I-J epitope on MHC-restricted T helper cells.

I-J epitopes were found to be associated with the functional site of the class II MHC-restricted helper T (Th) cells: Virtually all of the H-2k-restricted Th cell function of H-2kxbF1 T cells was inhibited by the anti-I-Jk mAb, leaving the H-2b-restricted function unaffected. The I-Jk epitope was inducible in Th cells of different genotype origin according to the environmental class II antigens present in the early ontogeny of T cells. Although above results suggested that I-J is the structure reflecting the inducible MHC restriction specificity, further studies revealed some interesting controversies: First, the I-J phenotype did not always correlate with the class II restriction specificity, e.g., I-Ab-restricted Th from 5R was I-Jk-positive, whereas I-Ak-restricted Th of 4R was not. Second, there was no trans expression of parental I-J phenotypes and restriction specificities in F1 Th, e.g., the I-J phenotype was detected only on I-Ab-restricted Th of (4R X 5R)F1, whereas it was absent on I-Ak-restricted Th. This strict linkage between the restriction specificity and I-J phenotype was also found on Th cells developed in bone marrow chimera constructed with intra-H-2-recombinant mice. The expression of I-Jk was always associated with the restriction specificity of the relevant host. Thus, the restriction specificity of Th cells followed the host type, and the I-J expression on Th was exactly the same as that expressed by the host haplotype. These results indicate that I-J is an isomorphic structure adaptively expressed on Th cells that is involved in the unidirectional regulatory cell interactions, and that the polymorphism cannot be explained merely by the restriction specificity of the conventional T cell receptor heterodimer.

Mutagenicity and cytotoxicity of benzo(a)pyrene benzo-ring epoxides.

Four benzo-ring epoxides of the environmental carcinogen benzo(a)pyrene (BP) were tested for mutagenic and cytotoxic activity in 3 strains of Salmonella typhimurium (TA1538, TA98, and TA100) and in Chinese hamster V79 cells. Although very unstable in aqueous solution, 7beta,8alpha-dihydroxy-0beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol epoxide 1), with the 7-hydroxyl group on the same face of the molecule as the epoxide oxygen, was 1.5 to 4 times as mutagenic in the bacterial strains as was its more stable stereoisomer 7beta,8alpha-dihydroxy-9alpha,10beta-epoxy-7,8,9.10-tetrahydrobenzo(a)pyrene (diol epoxide 2). In V79 cells, diol epoxide 1 had one-third the mutagenic activity of diol epoxide 2 but was at least 10 times more labile than diol epoxide 2 in the tissue culture medium. The half-life of diol epoxide 1 in tissue culture medium was about 30 sec, whereas the half-life of diol epoxide 2 was between 6 and 12 min. 9,10-Epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, which is saturated in the benzo ring, is also very unstable and has mutagenic activity equal to or greater than diol epoxide 1 in the bacterial and mammalian cells. 7,8-Epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was more stable in aqueous solution than any of the 9,10-epoxides of BP but was much less mutagenic in both the bacterial and mammalian cells. In v79 cells, diol epoxides 1 and 2 and 9,10-opoxy-7,8,9,10-tetrahydrobenzo(a)pyrene were more than 40 times more cytotoxic than 7,8-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene. The mutagenicity of the 2 tetrahydro epoxides toward strain TA98 of S. typhimurium was readily abolished by purified epoxide hydrase, whereas the mutagenic activity of the 2 diol epoxides was relatively unaffected by coincubation with the enzyme.

Prevention of diabetes in NOD mice by injection of autoreactive T-lymphocytes.

The nonobese diabetic (NOD) mouse develops a high incidence of autoimmune diabetes and is believed to be a good model for insulin-dependent diabetes mellitus (IDDM) in humans. We isolated T-lymphocyte lines from islets of newly diabetic NOD mice, some of which are autoreactive to NOD spleen cells. Because autoreactive T-lymphocytes have been implicated in immune suppression, we injected NOD mice with an autoreactive T-lymphocyte line. The injected mice had a marked decrease in incidence of IDDM compared with control mice. Moreover, their islets showed no insulitis at 1 yr of age. We conclude that autoreactive T-lymphocytes can prevent the development of IDDM in NOD mice. This result suggests that 1) islets contain both effector cells capable of damaging pancreatic beta-cells and cells able to regulate this autoimmune response, and 2) development of IDDM depends on the balance between these opposing forces.

Superantigenic staphylococcal exotoxins induce T-cell proliferation in the presence of Langerhans cells or class II-bearing keratinocytes and stimulate keratinocytes to produce T-cell-activating cytokines.

Several staphylococcal toxins are among a growing number of immunostimulatory molecules called "superantigens" because of their ability, when presented by appropriate major histocompatibility complex class II+ accessory cells, to activate essentially all T cells bearing particular T-cell receptor V beta gene segments. We have examined the ability of murine epidermal Langerhans cells and/or keratinocytes to act as accessory cells in the T-cell response to the superantigens staphylococcal enterotoxin B and exfoliative toxin, also known as epidermolysin. Purified murine splenic T cells were stimulated with staphylococcal enterotoxin B or exfoliative toxin in the presence of Langerhans cells--enriched epidermal cells from normal mice or epidermal cells isolated from mice pretreated with recombinant interferon-gamma, a procedure that induces the expression of major histocompatibility complex class II molecules on keratinocytes. The data show that both Langerhans cells and class II-bearing keratinocytes can act as accessory cells in the T-cell response to staphylococcal enterotoxin B and exfoliative toxin. We also observed that both human and murine keratinocytes cultured in the presence of staphylococcal enterotoxin B or exfoliative toxin produce increased amounts of cytokine(s) capable of stimulating thymocytes and D10 cells, and that this toxin activity is independent of the level of expression of class II on keratinocytes. Studies by enzyme-linked immunosorbent assay showed that staphylococcal enterotoxin B stimulates keratinocytes to produce tumor necrosis factor-alpha but not interleukin-1, suggesting tumor necrosis factor-alpha and perhaps other cytokines are responsible for the T-cell proliferative activity. These results demonstrate that two distinct epidermal constituents (i.e. Langerhans cells and keratinocytes) can serve as accessory cells in the responses of T cells to superantigenic bacterial toxins. It is possible that such toxins contribute to the pathogenesis of a variety of skin diseases by either locally activating T cells bearing particular V beta genes and/or enhancing keratinocyte production of immunomodulatory cytokines.

Enhanced hepatocyte growth factor expression associated with prolonged rat hepatic allograft survival in recipients pretreated with donor-specific blood.

BACKGROUND: Pretransplantation injection of freshly heparinized donor blood (donor-specific blood transfusion, or DST) significantly prolongs the survival of hepatic allografts from ACI(RT1a) to LEW(RT1l) rats. We investigated hepatocyte growth factor (HGF) expression in rat hepatic allografts of recipients pretreated with or without DST. METHODS: The levels of HGF mRNA and protein in hepatic allografts were determined after transplantation. The localization of HGF+ cells was identified with a rat anti-HGF monoclonal antibody. RESULTS: Plasma HGF concentrations in transplanted rats treated with DST were significantly and persistently increased compared to untreated rats with hepatic allografts. The number of HGF+ cells in hepatic allografts of recipients pretreated with DST on day 14 was significantly greater than that in allografts of untreated recipients on day 7. HGF+ cells were also found in the marginal zone and red pulp of recipient spleens. Northern blot analysis revealed the presence of three HGF+ cell phenotypes: HGF+ED1+, HGF+ED2+, and HGF+ED1-ED2-. Most HGF+ cells were ED1-ED2-. In situ hybridization demonstrated HGF mRNA in the mononuclear cells in the portal and sinusoidal areas as well as the marginal zone and red pulp in both DST-treated and untreated recipient spleens. CONCLUSIONS: Enhanced HGF expression in rat hepatic allografts is associated with immunologic unresponsiveness induced by DST.

Synaptic organization of the vestibulo-collic pathways from six semicircular canals to motoneurons of different neck muscles.

The pattern of inputs from six semicircular canals to neck motoneurons was investigated by stimulating six ampullary nerves electrically and recording intracellular potentials from motoneurons of the rectus capitis dorsalis (RD), the complexus (COMP) and the obliquus capitis caudalis (OCA) muscles at the upper cervical cord of the cat. RD and COMP motoneurons received disynaptic excitation from bilateral anterior and contralateral horizontal ampullary nerves and disynaptic inhibition from bilateral posterior and ipsilateral horizontal ampullary nerves. OCA motoneurons received excitation from ipsilateral vertical and contralateral horizontal ampullary nerves and inhibition from contralateral vertical and ipsilateral horizontal ampullary nerves. Ipsilateral disynaptic inhibitory postsynaptic potentials and contralateral disynaptic excitatory postsynaptic potentials to these motoneurons were mediated by the medial longitudinal fasciculus (MLF) and the other postsynaptic potentials by the extra-MLF pathways. The results indicated that motoneurons of a neck muscle have its own characteristic pattern of inputs from six semicircular canals.

Effect of interstimulus interval on perceived sensation and intradental nerve activity during thermal tooth stimulation in man.

The effect of interstimulus interval on nerve responses and subjective sensory ratings evoked by thermal stimulation of the teeth were studied in man. A total of 30 unitary discharges during heat stimulation of the lower incisor teeth were recorded from the inferior alveolar nerves using microneurographic technique. Fifteen of them had threshold sensory ratings above 3 (pain related-units) and 15 had ratings of less than 2 (non-pain-related units). Repetitive heat stimulation was applied to the teeth with interstimulus intervals of 180 s (ISI 180) and 30 s (ISI 30). Repetitive (ISI 180 and ISI 30) non-painful heat stimulation of the teeth did not alter either the intensity ratings or unitary discharge activities of non-pain-related units (P > 0.05). Repetitive painful heat stimulation of the teeth with ISI 180 did not alter the intensity ratings of pain-related units (P > 0.05), whereas that with ISI 30 significantly reduced the intensity ratings of pain-related units during a second session of heat trials (P < 0.05). On the other hand, repetitive heat stimulation of the teeth with ISI 180 caused a slight reduction in the firing frequency of pain-related units (P > 0.05). Repetitive painful heat stimulation with ISI 30 significantly reduced the firing frequency of pain-related units (P < 0.05).

Response properties of primary somatosensory cortical neurons responsive to cold stimulation of the facial skin and oral mucous membrane.

The distribution and response characteristics of the primary somatosensory cortical (SI) neurons activated by cold stimulation of the facial skin and oral mucous membrane were studied in cats. The discharge activities of 53 cold-sensitive SI neurons that responded to a decrease in temperature of the facial skin and/or oral mucous membrane were recorded. Each of these neurons was classified according to its responsiveness to mechanical stimulation as follows: LTM (low-threshold mechanoreceptive, 14/53), WDR (wide dynamic range, 39/53) and NS (nociceptive-specific, none identified). Encoding and non-encoding SI cold-sensitive neurons were identified, according to their responsiveness to decremental thermal stimulation. Only 14 cold-sensitive SI neurons demonstrated increased firing frequencies when subjected to incremental stimulus intensity increases and were classified as the encoding-type, whereas 39 non-encoding-type neurons did not.

Inhibitory effect of 8-methoxypsoralen plus ultraviolet-A on interleukin-1 production by murine keratinocytes.

We report the effects of 8-methoxypsoralen (8-MOP) plus ultraviolet-A (UV-A) irradiation on interleukin-1 (IL-1) production by murine epidermal keratinocytes, correlating its effect on IL-1 with cell viability, DNA synthesis, and 8-MOP-DNA photoadduct formation. Freshly isolated murine keratinocytes were treated with various doses of 8-MOP (5-100 ng/mL; incubation time, 30 min) plus 1 J/cm2 UV-A and cultured for 1-3 days. The IL-1/epidermal cell-derived thymocyte-activating factor (ETAF) activity in both supernatant and cell extract was reduced proportionately with increasing doses of 8-MOP/UV-A. Interleukin-1 inhibitors induced by 8-MOP plus UV-A were not detected in either supernatant or cell extract. A clear reduction of the IL-1 production was induced by the treatment as low as 15 ng/mL 8-MOP plus 1 J/cm2 UV-A, which led to the formation of 0.52 8-MOP photoadducts per million DNa bases and affected neither cell viability nor DNA synthesis of the treated cells. Cells treated with 100 ng/mL 8-MOP and 1 J/cm2 UV-A exhibited 57% suppression of IL-1 production in both 2- and 3-day culture samples. This treatment resulted in the formation of 3.8 photoadducts per million bases as well as significant abrogation of DNA synthesis although cell viability was unchanged. These observations provide some insights into the phototoxicity mechanisms of 8-MOP and the effect of PUVA therapy on the cytokine regulation in keratinocytes.

Accessory cell ability of Langerhans cells for superantigen is resistant to ultraviolet-B light.

We examined the effects of ultraviolet-B (UVB) irradiation on the accessory cell ability of Langerhans cells (LC) to induce a T-cell response to a superantigen, staphylococcal enterotoxin B (SEB). The ability of LC-enriched epidermal cells (LC-EC) to evoke a T-cell response to SEB was retained at the doses of UVB (up to 40 mJ/cm2) that profoundly affected the antigen-presenting function of LC-EC for a hapten, trinitrophenyl (TNP), and a protein antigen, conalbumin. Thus, the LC accessory function for superantigens is more resistant to UVB irradiation than that for ordinary antigens. This UVB resistance is presumably due to no requirement of antigen processing for superantigens as chemically fixed or chloroquine-treated LC-EC still retained their ability to induce T-cell responses to SEB. Higher doses of UVB (more than 60 mJ/cm2) reduced the accessory cell ability of LC-EC for SEB up to 50% of control. The addition of monoclonal antibodies against adhesion molecules between LC and T cells to the culture resulted in a substantial suppression of the T-cell response to SEB induced by nonirradiated LC-EC, while the UVB-irradiated LC-EC-induced T-cell response was not significantly blocked with these monoclonal antibodies. This suggested that the reduction of LC ability for superantigen by high doses of UVB is at least partly due to the impairment of adhesion molecules on LC by UVB irradiation.

Spontaneous persistent epithelial defects after cataract surgery.

The natural course of corneal epithelial erosions was studied retrospectively in patients who had undergone cataract extraction. Postsurgical epithelial defects occurred in 41 of 796 eyes (5.2%). No difference was noted in the incidence of corneal epithelial defects in eyes after intracapsular and extracapsular cataract extraction. Of the 41 eyes with postsurgical corneal erosion, 26 (63.4%) showed corneal epithelial defects by the fourth postoperative day. Two types of healing patterns were noticed: rapid healing, in which there was complete epithelial resurfacing within 4 days after the onset of corneal erosion, and prolonged healing, in which the period for complete epithelial resurfacing ranged from 6 days to 34 days. Since fibronectin has been reported to facilitate corneal epithelial migration and adhesion, we administered autologous fibronectin eyedrops in five cases in which corneal epithelial erosion developed after cataract surgery and epithelial defects persisted more than 7 days (except case 2). In all cases receiving fibronectin treatment, complete epithelial resurfacing occurred. In four of five eyes, fibronectin eye drops were effective in epithelial resurfacing within 3-9 days; in one eye, complete epithelial resurfacing took 23 days. The present results indicate that there are two types of epithelial erosion and that fibronectin eye drops might be one of the possible therapeutic approaches for the treatment of persistent epithelial defects. The weak adhesion of epithelial cells underlying basement membrane may be one of the causes of persistent epithelial defects after cataract surgery.

Localization of fibronectin and actin in cultured rabbit keratocytes.

Migration of activated keratocytes toward the corneal stromal wound is one of the most important processes of successful healing. To understand the motility of keratocytes and the interaction of fibronectin and intracellular actin filaments, we cultured rabbit corneal keratocytes and studied dynamic movements of the cells by time-lapse cinematography. We also examined the changes in the localization of fibronectin and actin using double staining immunofluorescent microscopy. The cultured keratocytes first attached to the substratum in round globular shape and then spread with many extending processes. In the early stage of the cultivation, fibronectin was observed inside the cells. Later, fibronectin was observed outside the cells, suggesting formation of the extracellular matrix. When keratocytes spread, actin was observed as a stress fiber inside the cells. At the edge of the cellular processes, a close interaction between fibronectin and actin was observed. The present results demonstrated that cultured keratocytes had active motility and that there were close interactions between the extracellular fibronectin and intracellular actin filaments. The organization of fibrillar actin filaments (F-actin) might be affected by the binding of extracellular fibronectin to the cell surface receptor for fibronectin.

Ecology of Increasing Diseases: Population Growth and Environmental Degradation.

The World Health Organization (WHO) and other organizations report that the prevalence of human diseases during the past decade is rapidly increasing. Population growth and the pollution of water, air, and soil are contributing to the increasing number of human diseases worldwide. Currently an estimated 40% of world deaths are due to environmental degradation. The ecology of increasing diseases has complex factors of environmental degradation, population growth, and the current malnutrition of about 3.7 billion people in the world.

A prospective randomized comparative study on the safety and tolerability of transnasal esophagogastroduodenoscopy.

BACKGROUND AND STUDY AIM: Transnasal esophagogastroduodenoscopy (EGD) with a small-caliber endoscope is well tolerated by patients. However, the effect of this procedure on cardiopulmonary function has not been fully investigated. The aim of this prospective, randomized study was to investigate the effect of transnasal EGD in comparison with transoral EGD on cardiopulmonary function. PATIENTS AND METHODS: The study involved 450 patients referred for diagnostic EGD. Patients were randomly assigned to one of three types of unsedated EGD (150 patients per group): transnasal EGD using a small-caliber endoscope (the "XP-N" group), transoral EGD using the same small-caliber endoscope ("XP-O" group), and transoral EGD using a conventional endoscope ("XQ" group). Systolic and diastolic blood pressure, pulse rate, and arterial oxygen saturation were monitored before, and 2, 4 and 6 minutes after intubation, and just after endoscope extubation. Gagging episodes were also counted, to determine tolerance. RESULTS: It was not possible to perform transnasal EGD in 12 patients (8.0%). A small amount of epistaxis was observed in eight (5.8%) of 138 patients who were examined successfully by transnasal EGD. Systolic and diastolic blood pressure, pulse rate, rate-pressure product (pulse rate x systolic blood pressure/100), and the drop in arterial oxygen saturation in the XQ group were significantly greater than in the XP-N and XP-O groups at each time point. In the XP-N group, these parameters were significantly lower than those in the XP-O group at 2 minutes after intubation. Of the tree groups the number of gagging episodes was significantly lower in the XP-N group. CONCLUSION: Transnasal EGD is safer than transoral EGD as it is associated with fewer adverse effects on cardiopulmonary function and is better tolerated by patients.