A progressive decline in the integrity of the immune system is one of the physiologic changes during aging. The frequency of autoimmune diseases or immune disorders increases in the aging population, but the state of regulatory T (Treg) cells in aged individuals has not been well determined. In the present study, we investigated the levels, phenotypes, and function of CD4(+)CD25(+) Treg cells in Balb/c mice, which were older than <em>20</em> months. Significantly enhanced percentages of CD4(+)CD25(+) Treg cells in the periphery (blood, spleen, and lymph nodes) of the aged mice were observed. These Treg cells showed modified Vbeta family distribution, reduced levels of CD45 receptor B and CD62 ligand molecules, as well as normal levels of forkhead box p3. However, when the inhibiting function of Treg cells was assayed in the in vitro assays and in a delayed-type hypersensitivity (DTH) model, CD4(+)CD25(+) Treg cells of aged mice displayed significantly lower inhibiting ability on alloantigen-induced DTH reaction or cytokine productions (<em>IL</em>-2 and IFN-gamma) but not cell proliferation of effector T cells, as compared with CD4(+)CD25(+) Treg cells of young mice. In addition, the percentages of CD4(+)CD8(-)CD25(+) Treg cells in the thymi of aged mice increased significantly, but their total cell numbers decreased markedly in these mice. Our present studies indicated collectively that the percentages, phenotypes, the size of TCR repertoire, and function of CD4(+)CD25(+) Treg cells were altered significantly with aging in mice. The functional defects of CD4(+)CD25(+) Treg cells may shed light on the role of CD4(+)CD25(+) Treg cells in the increased sensitivity to autoimmune diseases of aged populations.

<strong class="sub-title"> Background: </strong> Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic in <em>20</em><em>20</em>. Testing is crucial for mitigating public health and economic effects. Serology is considered key to population-level surveillance and potentially individual-level risk assessment. However, immunoassay performance has not been compared on large, identical sample sets. We aimed to investigate the performance of four high-throughput commercial SARS-CoV-2 antibody immunoassays and a novel 384-well ELISA.

<strong class="sub-title"> Methods: </strong> We did a head-to-head assessment of SARS-CoV-2 IgG assay (Abbott, Chicago, <em>IL</em>, USA), LIAISON SARS-CoV-2 S1/S2 IgG assay (DiaSorin, Saluggia, Italy), Elecsys Anti-SARS-CoV-2 assay (Roche, Basel, Switzerland), SARS-CoV-2 Total assay (Siemens, Munich, Germany), and a novel 384-well ELISA (the Oxford immunoassay). We derived sensitivity and specificity from 976 pre-pandemic blood samples (collected between Sept 4, <em>20</em>14, and Oct 4, <em>20</em>16) and 536 blood samples from patients with laboratory-confirmed SARS-CoV-2 infection, collected at least <em>20</em> days post symptom onset (collected between Feb 1, <em>20</em><em>20</em>, and May 31, <em>20</em><em>20</em>). Receiver operating characteristic (ROC) curves were used to assess assay thresholds.

Findings: At the manufacturers' thresholds, for the Abbott assay sensitivity was 92·7% (95% CI 90·2-94·8) and specificity was 99·9% (99·4-100%); for the DiaSorin assay sensitivity was 95·0% (92·8-96·7) and specificity was 98·7% (97·7-99·3); for the Oxford immunoassay sensitivity was 99·1% (97·8-99·7) and specificity was 99·0% (98·1-99·5); for the Roche assay sensitivity was 97·2% (95·4-98·4) and specificity was 99·8% (99·3-100); and for the Siemens assay sensitivity was 98·1% (96·6-99·1) and specificity was 99·9% (99·4-100%). All assays achieved a sensitivity of at least 98% with thresholds optimised to achieve a specificity of at least 98% on samples taken 30 days or more post symptom onset.

Interpretation: Four commercial, widely available assays and a scalable 384-well ELISA can be used for SARS-CoV-2 serological testing to achieve sensitivity and specificity of at least 98%. The Siemens assay and Oxford immunoassay achieved these metrics without further optimisation. This benchmark study in immunoassay assessment should enable refinements of testing strategies and the best use of serological testing resource to benefit individuals and population health.

Funding: Public Health England and UK National Institute for Health Research.

Delayed neutrophil apoptosis is a feature of persistent acute inflammation. Neutrophil-mediated damage has been shown to be associated with the development of inflammatory bowel disease (IBD). Persistence of these cells both at the colonic site and circulation may further contribute to IBD. The aims of this study were to determine whether neutrophils isolated from IBD patients delay apoptosis and to investigate possible mechanisms involved in this delay. We studied <em>20</em> patients with IBD, 13 with Crohn's disease, and 7 with ulcerative colitis, all of whom were undergoing intestinal resection for symptomatic disease. Seventeen patients undergoing elective resection of colon cancer acted as operative controls. Systemic, mesenteric arterial, and mesenteric venous blood was harvested. Neutrophils isolated from patients with IBD showed decreased spontaneous apoptosis compared to cancer patients. Mesenteric venous serum of IBD patients contributed to this delay, which contained higher concentrations of interleukin-8 (<em>IL</em>-8). Pro-caspase 3 expression was also reduced in IBD neutrophils, which may contribute to decreased spontaneous and Fas antibody-induced apoptosis. Neutrophil apoptosis may be altered in Crohn's disease and ulcerative colitis through release of anti-apoptotic cytokines and altered caspase expression. The alterations in cell death mechanisms may lead to persistence of the inflammatory response associated with IBD.

Progressive disease caused by pathogenic SIV/HIV infections is marked by systemic hyperimmune activation, immune dysregulation, and profound depletion of CD4(+) T cells in lymphoid and gastrointestinal mucosal tissues. <em>IL</em>-17 is important for protective immunity against extracellular bacterial infections at mucosa and for maintenance of mucosal barrier. Although <em>IL</em>-17-secreting CD4 (Th17) and CD8 (Tc17) T cells have been reported, very little is known about the latter subset for any infectious disease. In this study, we characterized the anatomical distribution, phenotype, and functional quality of Tc17 and Th17 cells in healthy (SIV-) and SIV+ rhesus macaques. In healthy macaques, Tc17 and Th17 cells were present in all lymphoid and gastrointestinal tissues studied with predominance in small intestine. About 50% of these cells coexpressed TNF-α and <em>IL</em>-2. Notably, ∼50% of Tc17 cells also expressed the co-inhibitory molecule CTLA-4, and only a minority ((<em>20</em>%) expressed granzyme B suggesting that these cells possess more of a regulatory than cytotoxic phenotype. After SIV infection, unlike Th17 cells, Tc17 cells were not depleted during the acute phase of infection. However, the frequency of Tc17 cells in SIV-infected macaques with AIDS was lower compared with that in healthy macaques demonstrating the loss of these cells during end-stage disease. Antiretroviral therapy partially restored the frequency of Tc17 and Th17 cells in the colorectal mucosa. Depletion of Tc17 cells was not observed in colorectal mucosa of chronically infected SIV+ sooty mangabeys. In conclusion, our results suggest a role for Tc17 cells in regulating disease progression during pathogenic SIV infection.

The influence of an exercise training program and age on inflammatory cytokine production and CD14+cell-surface expression of toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) was examined in 60 younger and older subjects. Subjects were assigned to: young physically active (YPA, n = 15; 25.2 +/- 5.0 years), young physically inactive (YPI, n = 14; 24.9 +/- 4.7 years), older physically active (OPA, n = 14; 71.2 +/- 4.4 years) or older physically inactive (OPI, n = 17; 71.0 +/- 4.3 years) groups. YPI and OPI completed 12 weeks (3 days/week) of endurance (<em>20</em> min) and resistance exercise (eight exercises, two sets). YPA and OPA groups were instructed to continue their normal activity for 12 weeks. Blood was collected at rest, before and after the 12-week training and control period. A whole blood method was used to determine lipopolysaccharide-(LPS) and peptidoglycan-(PGN) stimulated <em>IL</em>-6, <em>IL</em>-1beta, and TNF-alpha production with supernatants analyzed using ELISA. CD14+ cell-surface expression of TLR2 and TLR4 were measured using flow cytometry. Training increased estimated VO(2max) by 10.4% and increased strength by an average of 38.1%. YPI and OPI had a post-training reduction in LPS-stimulated <em>IL</em>-6 production (P < .01), but LPS-stimulated <em>IL</em>-1beta and TNF-alpha and PGN-stimulated cytokines were not changed. CD14+ cell TLR4 was significantly reduced (P < .05) in YPI and OPI groups after training, but TLR2 was not significantly changed. An exercise training program reduced LPS-stimulated <em>IL</em>-6, concomitant with lower TLR4. These results provide further support for a training- or physical activity-induced lowering of TLR4 and inflammation.

T cells oxidized with sodium periodate proliferate polyclonally in response to accessory cells. We confirmed previous work showing that DC are potent stimulators of this response. In addition, the accessory function of unfractionated mouse spleen and spleen adherent cells was markedly reduced after elimination of DC with a specific monoclonal antibody and complement. Therefore oxidative mitogenesis was used as a model to study the mechanism by which DC stimulate T cell proliferative responses. A two-stage mechanism was identified. The first stage occurred during the first <em>20</em> h of culture, required live DC, and involved the progressive release of interleukin 2 (<em>IL</em>-2) into the medium and acquisition of responsiveness to this growth factor. The second stage occurred between <em>20</em> and 40 h, did not require live DC, and involved DNA synthesis in response to <em>IL</em>-2. Similar events occurred during culture of DC with unmodified T cells (syngeneic MLR) but were quantitatively reduced. The experimental approach was to co-culture DC and T cells for up to <em>20</em> h and then kill the DC with specific antibody, or anti-Ia antibody, and complement. Subsequent proliferation was inhibited if the T cells were cultured in fresh medium. However, proliferation was restored when the lymphocytes were cultured in the original DC-T cell medium, or with a crude or a purified preparation of <em>IL</em>-2. <em>IL</em>-2 did not induce the proliferation of T cells that had been cultured in the absence of DC, and did not synergize with viable DC. We conclude that DC induce proliferation by tightly coordinating the release of, and responsiveness to, T cell growth factor or <em>IL</em>-2.

Inflammation and proinflammatory cytokines suppress the expression of several hepatic transporters and metabolic enzymes, often resulting in cholestatic liver disease. However, mechanism(s) of this down-regulation have not been fully elucidated. As the pregnane X receptor (PXR) is involved in inducing many of these hepatic proteins, it is possible that PXR is also involved in their down-regulation during inflammation. Thus, we compared the effect of inflammation on hepatic gene regulation in wild-type (PXR(+/+)) versus PXR-null (PXR(-/-)) mice. Treatment of PXR(+/+) but not PXR(-/-) mice with the PXR activators 5-pregnen-3beta-ol-<em>20</em>-one-16alpha-carbonitrile (PCN) or 17beta-hydroxy-11beta-[4-dimethylamino phenyl]-17alpha-[1-propynyl] estra-4,9-dien-3-one (RU486) resulted in increased mRNA levels of bsep, mdr1a, mrp2, mrp3, oatp2, and cyp3a11, indicating involvement of PXR in their regulation. Significantly lower mRNA levels of bsep, mdr2, mrp2, mrp3, ntcp, oatp2, and cyp3a11 were found in endotoxin-treated PXR(+/+) mice. In endotoxin-treated PXR(-/-) mice, the extent of mrp2 suppression was significantly diminished. Changes in MRP2 expression were supported by Western blot analysis. Although interleukin (<em>IL</em>)-6 imposed significant decreases in the expression of bsep, mrp2, and cyp3a11 in PXR(+/+) mice, this was not observed in PXR(-/-) mice. Of note, significantly lower levels of PXR mRNA and protein were detected in endotoxin- and <em>IL</em>-6-treated PXR(+/+) mice. In addition, endotoxin and <em>IL</em>-6 were also able to suppress PCN-mediated induction of bsep, mrp2, cyp3a11, and PXR. Taken together, our results suggest that PXR plays a role in the down-regulation of several hepatic proteins during inflammation.

Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (<em>IL</em>-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of <em>IL</em>-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of <em>IL</em>-8 and ICAM-1 expression following brief challenge were such that (i) secretion of <em>IL</em>-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) <em>IL</em>-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to <em>20</em> h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of <em>IL</em>-8 secretion was facilitated by adherent P. gingivalis strains. The <em>IL</em>-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalis or its culture supernatant. Although these results suggested that <em>IL</em>-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for <em>IL</em>-8 attenuation. In addition, while P. gingivalis up-regulated <em>IL</em>-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The <em>IL</em>-8 mRNA levels in epithelial cells following sequential challenge with P. gingivalis and F. nucleatum and vice versa were approximately identical and were lower than those following F. nucleatum challenge alone and higher than control levels or those following P. gingivalis challenge alone. Thus, together with the protease effect, P. gingivalis possesses a powerful strategy to ensure the down-regulation of <em>IL</em>-8 and ICAM-1.

Although several sensitive and specific assays have been developed to quantify murine cytokines, these assays do not allow individual cells to be correlated with the specific cytokines they produce. The purpose of this study was to develop a sensitive and reproducible method for the detection of individual T cells which secrete either interferon-gamma (IFN-gamma) or interleukin-5 (<em>IL</em>-5). We have used an adaptation of the enzyme-linked immunospot (ELISPOT) assay in which monoclonal antibodies to IFN-gamma (R4-6A2) and to <em>IL</em>-5 (TRFK-5) were used to coat 96-well plates with a nitrocellulose base. Mouse splenic T cells, either nonstimulated or activated with concanavalin A (ConA) or phytohemagglutinin (PHA), were cultured in individual wells. Following incubation, the cells were removed, and the bound cytokines probed with either biotinylated mAb anti-IFN-gamma (XMG 1.2) or anti-<em>IL</em>-5 (TRFK-4) followed by avidin-peroxidase. The spots which developed with 3-amino-9-ethylcarbazole were discrete and enumerated with a dissecting microscope. Although unstimulated splenic T cells contained low numbers of cytokine-specific spot-forming cells (SFC), 24-72 h activation with mitogen was required to induce significant numbers of cytokine producing cells. When mitogen-stimulated splenic CD4+ T cells were assessed, approximately equal numbers of IFN-gamma and <em>IL</em>-5 SFC were seen. Approximately <em>20</em>-30% of all mitogen-activated splenic T cells produced at least one of these two cytokines. Pre-incubation of biotinylated anti-IFN-gamma with recombinant IFN-gamma (rIFN-gamma) or anti-<em>IL</em>-5 mAbs with r<em>IL</em>-5 completely inhibited cytokine-specific SFC. Further, use of nonrelevant antibodies did not result in spot formation, and treatment of mitogen-activated T cells with cycloheximide inhibited both IFN-gamma- and <em>IL</em>-5-specific SFC. A sensitive method has been developed which allows detection of individual T cells that produce either IFN-gamm or <em>IL</em>-5, and should be useful for detection of cytokine secretion at the single cell level.

To clarify the mechanisms that cause elevation of plasma fibrinogen levels in diabetes, we first examined the effect of hyperglycemia on the production of interleukin 6 (<em>IL</em>-6) and tumor necrosis factor (TNF) by cultured human peripheral blood monocytes. Monocyte-enriched fractions isolated from <em>20</em> healthy volunteers were incubated with 11 mmol/l glucose, 33 mmol/l glucose, or mannitol as an osmolar control for 6 or 24 h. After 6 h of incubation, <em>IL</em>-6 and TNF-alpha mRNA levels were analyzed by reverse transcription and polymerase chain reaction. In addition, after 24 h of incubation, <em>IL</em>-6 and TNF-alpha immunoreactivity in the culture medium was measured by enzyme-linked immunoassay. Both <em>IL</em>-6 and TNF-alpha mRNA levels and immunoreactivity were significantly increased by treatment with 33 mmol/l glucose compared with treatment with 11 mmol/l glucose or 11 mmol/l glucose with 22 mmol/l mannitol. In addition, preincubation of the cells with an anti-TNF monoclonal antibody (mAb) blocked the stimulatory effect of 33 mmol/l glucose on <em>IL</em>-6 synthesis and secretion. Second, we examined the ability of conditioned media from human peripheral blood monocytes to stimulate beta-fibrinogen mRNA synthesis in HepG2 cells. The conditioned medium from monocytes treated with 33 mmol/l glucose increased beta-fibrinogen mRNA levels. The results of this study demonstrate that hyperglycemia stimulated <em>IL</em>-6 and TNF synthesis and secretion by human peripheral monocytes in vitro and that the <em>IL</em>-6 response to hyperglycemia may be mediated by TNF. Furthermore, hyperglycemia may increase fibrinogen levels through stimulation of peripheral monocytes. These results suggest that hyperglycemia may cause hyperfibrinogenemia in diabetic patients through an <em>IL</em>-6-dependent and TNF-dependent mechanism.

OBJECTIVE

Caspase recruitment domain 9 (CARD9) is an adaptor protein that integrates signals downstream of pattern recognition receptors. CARD9 has been associated with autoinflammatory disorders, and loss-of-function mutations have been associated with chronic mucocutaneous candidiasis, but the role of CARD9 in intestinal inflammation is unknown. We characterized the role of Card9 in mucosal immune responses to intestinal epithelial injury and infection.

METHODS

We induced intestinal inflammation in Card9-null mice by administration of dextran sulfate sodium (DSS) or Citrobacter rodentium. We analyzed body weight, assessed inflammation by histology, and measured levels of cytokines and chemokines using quantitative reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Cell populations were compared between wild-type and Card9-null mice by flow cytometry analysis.

RESULTS

Colon tissues and mesenteric lymph nodes of Card9-null mice had reduced levels of interleukin (<em>IL</em>)-6, interferon-γ, and T-helper (Th)17 cytokines after administration of DSS, compared with wild-type mice. <em>IL</em>-17A and <em>IL</em>-22 expression were reduced in the recovery phase after DSS administration, coincident with decreased expression of antimicrobial peptides and the chemokine (C-C motif) ligand <em>20</em> (Ccl<em>20</em>). Although Card9-null mice had more intestinal fungi based on 18S analysis, their Th17 responses remained defective even when an antifungal agent was administered throughout DSS exposure. Moreover, Card9-null mice had impaired immune responses to C rodentium, characterized by decreased levels of colonic <em>IL</em>-6, <em>IL</em>-17A, <em>IL</em>-22, and regenerating islet-derived 3 gamma (RegIIIγ), as well as fewer <em>IL</em>-22-producing innate lymphoid cells (<em>IL</em>Cs) in colon lamina propria.

CONCLUSIONS

The adaptor protein CARD9 coordinates Th17- and innate lymphoid cell-mediated intestinal immune responses after epithelial injury in mice.

Differentiating eosinophilic esophagitis from gastroesophageal reflux disease is important given their pathogenetic differences and responses to therapy. Eotaxins are a family of chemokines important for activation and recruitment of eosinophils mediated by their receptor, chemokine receptor-3 (CCR-3). Interleukin 5 (<em>IL</em>-5) is a key cytokine involved in many steps of eosinophil production and recruitment. The aim of this study was to compare the messenger RNA expression of the eotaxins, CCR-3, and <em>IL</em>-5 between well-characterized groups of patients with eosinophilic esophagitis, patients with gastroesophageal reflux disease, and healthy individuals. This was a retrospective study using esophageal biopsies from 33 patients with eosinophilic esophagitis, <em>20</em> patients with gastroesophageal reflux disease, and 17 healthy controls. Parameters studied included demographic features, presenting symptoms, endoscopic findings, histopathologic features, and messenger RNA levels of eotaxins 1, 2, and 3, CCR-3, and <em>IL</em>-5 by quantitative real-time polymerase chain reaction using formalin-fixed, paraffin-embedded tissue. Patients with eosinophilic esophagitis were predominantly males (M/F=3:1), with a mean age of 15.9 years and a mean eosinophil count of 55 per x400 high-power field. Patients with gastroesophageal reflux disease had a mean age of 31.5 years and a mean eosinophil count of 5.8 per high-power field. Total intraepithelial eosinophil and lymphocyte counts, the presence of superficial eosinophil clusters, microabscesses, and basal cell hyperplasia were all significantly associated with eosinophilic esophagitis as opposed to gastroesophageal reflux disease (P<.0001). The mean expression levels of eotaxin-3 were markedly elevated in patients with eosinophilic esophagitis as compared with the gastroesophageal reflux disease and healthy control groups (731+/-276, 31+/-12, and 1.5+/-0.4 pg/ng beta-actin, respectively; P<.001). Mean expression levels of eotaxins 1 and 2, <em>IL</em>-5, and CCR-3 were also significantly increased in the patients with eosinophilic esophagitis, albeit at lower levels than eotaxin-3. In conclusion, our results highlight the important contribution of eotaxin-3 in the pathogenesis of eosinophilic esophagitis. Determination of eotaxin-3 levels by real-time polymerase chain reaction on paraffinized, formalin-fixed tissue may be a useful test in the differentiation of eosinophilic esophagitis from gastroesophageal reflux disease.

Bacterial endotoxin-lipopolysaccharide (LPS) rapidly induced hepatic metallothionein (MT) mRNA levels in the LPS-sensitive CD-1 strain of mice. This LPS effect was severely attenuated in the LPS-resistant C3H/HeJ strain of mice, but could be mimicked by injection of human recombinant interleukin-1 alpha (<em>IL</em>-1 alpha) or human recombinant tumor necrosis factor (TNF-alpha). In the CD-1 strain, LPS induction of MT gene expression occurred in each of 10 organs examined (liver, kidney, pancreas, intestine, lung, heart, brain, ovary, uterus, and spleen). Solution hybridization with probes specific for MT-I or MT-II mRNA established that these genes were co-induced in each of the organs and that the liver and kidney contained the highest absolute levels of these mRNAs, whereas in the intestine and spleen they were 10-<em>20</em>-fold lower. LPS and cytokine induction of hepatic MT gene expression occurred in hypophysectomized mice, which suggests a lack of significant involvement of glucocorticoids. Several recombinant cytokines (TNF-alpha, <em>IL</em>-1 alpha, <em>IL</em>-1 beta, <em>IL</em>-6, interferon-gamma (IFN-gamma), as well as poly(rI.rC) were effective inducers of hepatic MT-I and MT-II genes. As an attempt to determine which of these cytokines may mediate LPS effects on MT gene expression in vivo, CD-1 mice were injected with LPS or various cytokines, and RNA from liver, ovary, and uterus was extracted at various times postinjection and analyzed by Northern blotting using probes specific for <em>IL</em>-1 alpha, <em>IL</em>-1 beta, TNF-alpha, <em>IL</em>-6, and MT mRNA. In each organ examined, LPS, <em>IL</em>-1 alpha, or <em>IL</em>-1 beta injection caused a rapid, coordinate, transient increase in the levels of each of the cytokine mRNAs which peaked by 1 h and declined to low levels by 4 h. In contrast, levels of MT mRNA did not reach a peak until 4-6 h postinjection. TNF-alpha had minimal effects on expression of cytokine and MT genes in organs other than liver. <em>IL</em>-6 had no effect on hepatic cytokine mRNA levels, and induced MT mRNA only in the liver which suggests a direct effect of <em>IL</em>-6 on hepatic MT gene expression. These data suggest that the acute effects of LPS on MT gene expression may include complex paracrine interactions between a variety of cytokines and the cells expressing MT genes in each organ, and tissue-specific cytokine effects on the MT genes.

The Her2/neu (c-erbB-2) oncogene encodes a 185-kDa protein tyrosine kinase which is overexpressed in <em>20</em>% of breast adenocarcinomas and is recognized by a humanized anti-Her2/neu monoclonal antibody (mAb) (rhu4D5 or Herceptin). Natural killer (NK) cells are capable of mediating antibody-dependent cell cytotoxicity (ADCC) against antibody-coated targets via their expression of a low-affinity receptor for IgG (FcgammaRIII or CD16). NK cells can be expanded in cancer patients via the administration of low-dose interleukin-2 (<em>IL</em>-2) and become potent cytotoxic effectors following exposure to high doses of <em>IL</em>-2. We tested <em>IL</em>-2-activated NK cells against Her2/neu+ (MCF-7Her2/neu) and Her2/neu- (MDA-468) breast cancer cell lines in a 4-h 51Cr-release cytotoxicity assay in the presence or absence of rhu4D5 mAb (effector : target ratio = 10 : 1). Specific lysis of rhu4D5-coated MCF-7Her2/neu and MDA-468 target cells by <em>IL</em>-2-activated NK cells was 35% and 3%, respectively (p < 0.05). Lysis was less than 5% when targets were treated with either the non-humanized mu4D5 mAb or control huIgG. Lysis of rhu4D5-coated MCF-7Her2/neu cells was inhibited by 80 % when NK cells were pre-treated with an anti-Fc receptor antibody prior to use in the cytotoxicity assay. Enhanced ADCC of MCF-7Her2/neu target cells was seen when the effector cells consisted of mononuclear cells obtained from a patient demonstrating significant expansion of NK cells secondary to therapy with low-dose <em>IL</em>-2. Serum from patients receiving infusions of rhu4D5 mAb could substitute for exogenous antibody in the ADCC assay. NK cells activated by rhu4D5-coated tumor cells in the presence of <em>IL</em>-2 also produced large amounts of IFN-gamma with concomitant up-regulation of cell-surface activation markers CD25 and CD69. These results lend support to the concurrent use of rhu4D5 mAb and <em>IL</em>-2 therapy in patients with cancers that express the Her2/neu oncogene.

The aim of the present study was to determine if the human erythroid (E) and megakaryocytic (MK) lineages were closely linked to the existence of a bipotent burst-forming unit (BFU) E/MK progenitor. In methylcellulose cultures, BFU-E/MK colonies were observed at day 12 and closely resembled mature BFU-E with the exception that the erythroid component was surrounded by MK. These colonies were quite different from the colony forming unit (CFU)-GEMM-derived colonies, which were composed of a larger number of erythroblasts and which developed later in culture. The existence of these bilineage colonies composed of 100 to 1,000 erythroblasts intermingled with a few MK and without granulocytic cells was confirmed by the plasma clot technique and immunoalkaline phosphatase labeling of the MK. To investigate if this bipotent progenitor belonged to the compartment of primitive progenitors, CD34+ marrow cells were subfractionated according to expression of the CD38 antigen. The bipotent BFU-E/MK progenitor as well as a large fraction of MK progenitors were found in the CD34+ CD38+/- or in the CD34+ CD38- cell fractions. Growth of this bipotent BFU-E/MK progenitor required the combination of stem cell factor (SCF), Interleukin-3 (<em>IL</em>-3), and Epo in serum free conditions. Addition of <em>IL</em>-6 had only a marginal effect, whereas megakaryocyte growth and development factor (MGDF) was not an absolute requirement, but slightly increased the plating efficiency of CFU-MK and of BFU-E/MK progenitors when combined with SCF, <em>IL</em>-3, and Epo. In contrast, when these cultures were performed in the presence of 30% fetal calf serum, no BFU-E/MK colonies were observed irrespective of the combination of growth factors used, including the presence of MGDF; however, inclusion of the MS-5 cell line restored the growth of this bipotent progenitor. In contrast, in cultures performed in the presence of human normal or aplastic plasma, MS-5 had only a slight effect on the cloning efficiency but improved MK cytoplasmic maturation and MK size, suggesting that the main effect of MS-5 is to diminish the inhibitory effect of the fetal calf serum on the MK differentiation. The clonal origin of bipotent BFU-E/MK colonies was demonstrated in liquid culture of single CD34+ CD38low cells by immunophenotyping individual clones. At day 12, 30% of the clones contained erythroblasts (glycophorin A+) and some MK (CD41+) without granulocytes (G) or macrophages (M) (CD14+ and CD15+). At day <em>20</em>, clones containing erythroblasts and MK were rare (5%). In contrast multilineage clones could be frequently detected at this time without passage from BFU-E/MK clones at day 12 to GEMM at day <em>20</em>. These results suggest that a bipotent BFU-E/MK progenitor may be a nonrandom step in the hierarchical development of stem cells.

Large cytomegalovirus (CMV)-specific CD8 T-cell responses are observed in both young and, somewhat more often, old people. Frequent CMV reactivation is thought to exhaust these cells and render them dysfunctional so that larger numbers of them are needed to control CMV. Expansions of CMV-specific CD4 T cells are also seen but are less well studied. In this study, we examined the T-cell response to the dominant CMV pp65 and IE-1 antigens in healthy CMV-infected people across a wide age range (<em>20</em> to 84 years) by using multicolor flow cytometry. CMV-specific T cells were characterized by the activation markers CD40 ligand (CD40L), interleukin-2 (<em>IL</em>-2), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) and the memory markers CD27 and CD45RA. The proportions of effector memory T cells increased in large responses, as did the proportions of polyfunctional CD8 (IFN-γ(+) <em>IL</em>-2(+/-) TNF-α(+)) and CD4 (CD40L(+/-) IFN-γ(+) <em>IL</em>-2(+) TNF-α(+)) T-cell subsets, while the proportion of naïve T cells decreased. The bigger the CD4 or CD8 T-cell response to pp65, the larger was the proportion of T cells with an advanced memory phenotype in the entire (including non-CMV-specific) T-cell compartment. In addition, the number of activation markers per cell correlated with the degree of T-cell receptor downregulation, suggesting increased antigen sensitivity in polyfunctional cells. In summary, our findings show that polyfunctional CMV-specific T cells were not superseded by dysfunctional cells, even in very large responses. At the same time, however, the memory subset composition of the entire T-cell compartment correlated with the size of the T-cell response to CMV pp65, confirming a strong effect of CMV infection on the immune systems of some, but not all, infected people.

Preeclampsia is associated with autoimmune cells T(H)17, secreting interleukin-17, autoantibodies activating the angiotensin II type I receptor (AT1-AA), and placental oxidative stress (ROS). The objective of our study was to determine whether chronic <em>IL</em>-17 increases blood pressure by stimulating ROS and AT1-AAs during pregnancy. To answer this question four groups of rats were examined: normal pregnant (NP, n = <em>20</em>), NP+<em>IL</em>-17 (n = 12), NP+tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) (n = 7) (a superoxide dismutase mimetic that scavenges ROS), and NP+<em>IL</em>-17+tempol (n = 11). <em>IL</em>-17 (150 pg/day) was infused into NP rats while tempol was administered via the drinking water ad libitum. On day 19 blood pressure (MAP) was recorded, and plasma, urine, and tissue were collected for isolation of ROS detected by chemilluminescent technique. Urinary isoprostane was measured by ELISA. AT1-AAs were determined via cardiomyocyte assay and expressed as beats per minute. MAP increased from 98 ± 3 mmHg in NP to 123 ± 3 mmHg in <em>IL</em>-17-infused NP rats. Urinary isoprostane increased from 1,029 ± 1 in NP to 3,526 ± 2 pg·mg(-1)·day(-1) in <em>IL</em>-17-infused rats (P < 0.05). Placental ROS was 436 ± 4 RLU·ml(-1)·min(-1) (n = 4) in NP and 702 ± 5 (n = 5) RLU·ml(-1)·min(-1) in <em>IL</em>-17-treated rats. Importantly, AT1-AA increased from 0.41 ± 0.05 beats/min in NP rats (n = 8) to 18.4 ± 1 beats/min in <em>IL</em>-17 rats (n = 12). Administration of tempol attenuated the hypertension (101 ± 3 mmHg) ROS (459 ± 5 RLU·ml(-1)·min(-1)) and blunted AT1-AAs (7.3 ± 0.6 beats/min) in NP+<em>IL</em>-17+tempol-treated rats. Additionally, AT1 receptor blockade inhibited <em>IL</em>-17-induced hypertension and placental oxidative stress. MAP was 105 ± 5 mmHg and ROS was 418 ± 5 RLU·ml(-1)·min(-1) in NP+<em>IL</em> 17-treated with losartan. These data indicate that <em>IL</em>-17 causes placental oxidative stress, which serves as stimulus modulating AT1-AAs that may play an important role in mediating <em>IL</em>-17-induced hypertension during pregnancy.

Asthma affects almost <em>20</em> million people in the United States and more than 300 million people worldwide. Of these, 10-15% have severe asthma, which is refractory to commonly available drugs. New drugs are needed because those that are currently available cannot control symptoms and exacerbations in all patients and can cause adverse reactions. In the past 10 years, there have been substantial advances in the understanding of asthma genetics, airway biology, and immune cell signaling. These advances have led to the development of small molecule therapeutics and biologic agents that may improve asthma care in the future. Several new classes of asthma drugs--including ultra long acting β agonists and modulators of the interleukin 4 (<em>IL</em>-4), <em>IL</em>-5, <em>IL</em>-13, and <em>IL</em>-17 pathways--have been evaluated in randomized controlled trials. Other new drug classes--including dissociated corticosteroids, CXC chemokine receptor 2 antagonists, toll-like receptor 9 agonists, and tyrosine kinase inhibitors--remain in earlier phases of development. Despite some preliminary efficacy data, there is insufficient evidence to make strong recommendations about the use of these newer agents. Future research on the clinical efficacy of these biologic agents, the effect of newer agents on severe asthma in pediatric patients, and the biology of non-eosinophilic and corticosteroid resistant asthma is needed to reduce the morbidity of asthma worldwide.

We investigated the effects of an Ironman triathlon race on markers of muscle damage, inflammation and heat shock protein 70 (HSP70). Nine well-trained male triathletes (mean +/- SD age 34 +/- 5 years; VO(2peak) 66.4 ml kg(-1) min(-1)) participated in the <em>20</em>04 Western Australia Ironman triathlon race (3.8 km swim, 180 km cycle, 42.2 km run). We assessed jump height, muscle strength and soreness, and collected venous blood samples 2 days before the race, within 30 min and 14-<em>20</em> h after the race. Plasma samples were analysed for muscle proteins, acute phase proteins, cytokines, heat shock protein 70 (HSP70), and clinical biochemical variables related to dehydration, haemolysis, liver and renal functions. Muscular strength and jump height decreased significantly (P < 0.05) after the race, whereas muscle soreness and the plasma concentrations of muscle proteins increased. The cytokines interleukin (<em>IL</em>)-1 receptor antagonist, <em>IL</em>-6 and <em>IL</em>-10, and HSP70 increased markedly after the race, while <em>IL</em>-12p40 and granulocyte colony-stimulating factor (G-CSF) were also elevated. <em>IL</em>-4, <em>IL</em>-1beta and tumour necrosis factor-alpha did not change significantly, despite elevated C-reactive protein and serum amyloid protein A on the day after the race. Plasma creatinine, uric acid and total bilirubin concentrations and gamma-glutamyl transferase activity also changed after the race. In conclusion, despite evidence of muscle damage and an acute phase response after the race, the pro-inflammatory cytokine response was minimal and anti-inflammatory cytokines were induced. HSP70 is released into the circulation as a function of exercise duration.

BACKGROUND

Recent studies have shown that cytokines are pivotal to the pathogenesis of periodontal diseases and may be used as markers in diagnosis.

OBJECTIVE

The aim of the present study was to determine the levels of interleukin (IL)-1beta, IL-4, IL-6 and IL-8 in gingival crevicular fluid of periodontally healthy and diseased individuals and to study their association to smoking, stress and clinical periodontal parameters.

METHODS

A total of 80 patients were included in the study : 20 patients with early onset or aggressive periodontitis (EOP), 20 with chronic adult periodontitis (AP), 20 with gingivitis (G) and 20 patients with healthy periodontium (H). GCF was collected by means of Durapore strips, from four sites per patient, randomly selected in each quadrant. The contents of IL- 1beta, IL-4, IL-6 and IL-8 were measured in 320 samples by use of commercially available sandwich enzyme-linked immunoadsorbent assays.

RESULTS

In periodontally diseased subjects the total amounts of IL-1beta, IL-6 and IL-8 were significantly elevated as compared to healthy subjects, whereas IL-4 showed an inverse relationship to periodontal status and higher amounts were found in the healthy group. The amounts of all four cytokines were positively correlated with probing depths. IL-4, IL-6 and IL-8 were significantly correlated to smoking while stress was associated with IL-1beta, IL-6 and IL-8 levels.

CONCLUSIONS

The present data suggest that crevicular IL-1beta, IL-6 and IL-8 reflect the activity of periodontal destruction, whereas IL-4 shows an inverse correlation to it. The enhanced production of inflammatory cytokines in the presence of smoking and stress may have clinical consequences.

The purpose of this article was to study the effect of hyaluronic acid (HA) on chondrocyte apoptosis in a rat osteoarthritis in vitro model (exposure to <em>IL</em>-1beta) and explore its mechanism. A rat in vitro model of osteoarthritis (OA) was established using 10 ng/mL <em>IL</em>-1beta as a modulating and chondrocyte apoptosis inducing agent. Different doses of HA (10, <em>20</em>, and 40 microg/mL) were added 1 h prior to the addition of <em>IL</em>-1beta to a monolayer culture of freshly isolated juvenile rat chondrocytes. The ratio of apoptotic cell death was surveyed by Annexin V-FITC and propidium iodide double-labeling FACS analysis. The mitochondrial membrane potential of chondrocytes was evaluated by rhodamine-123 fluorescence. The mitochondrial function was evaluated through detecting the ATP production by a luciferase assay. The reverse transcription polymerase chain reaction (RT-PCR) was performed to measure mRNA expression levels of inducible oxide synthase (iNOS). HA could inhibit <em>IL</em>-1beta-induced chondrocyte apoptosis in our cell culture model system. It was showed that addition of HA to the medium was able in a dose-dependent way to reduce the impairment of the mitochondrial membrane potential and to restore mitochondrial ATP production. This study shows that HA could suppress in a dose-dependent way chondrocyte apoptosis in our <em>IL</em>-1beta-induced osteoarthritis model. The suppression of inflammatory cytokine activity within the joint might be one important mechanism of the clinical action of intraarticular injection of HA in the treatment of OA.

The expression patterns of the <em>IL</em>-10 ligand and receptor genes were examined in normal and transformed cell lines of human hematopoietic and non-hematopoietic origin. <em>IL</em>-10 family ligands, <em>IL</em>-10, <em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22, <em>IL</em>-24 and <em>IL</em>-26 were predominantly expressed by hematopoietic cells. <em>IL</em>-10, <em>IL</em>-24 and <em>IL</em>-26 were produced by both monocytes and T cells, <em>IL</em>-19 and <em>IL</em>-<em>20</em> were produced by monocytes whereas <em>IL</em>-22 was produced mainly by activated T cells. The receptors of the <em>IL</em>-10 family, <em>IL</em>-10R1, <em>IL</em>-10R2, <em>IL</em>-<em>20</em>R1, <em>IL</em>-<em>20</em>R2, <em>IL</em>-22R1 and <em>IL</em>-22 BP were also expressed in a distinct pattern when probed on these cell lines. The expression of <em>IL</em>-10R2 was ubiquitous whereas <em>IL</em>-10R1 was predominantly expressed on hematopoietic cells, including, T cells, B cells, NK cells, monocytes and dendritic cells. <em>IL</em>-<em>20</em>R1, <em>IL</em>-<em>20</em>R2 and <em>IL</em>-22R1 were absent or expressed at extremely low levels on cells of the hematopoietic lineage. These receptors were mainly found on epithelial and stromal cells fibroblasts of various tissues. Interestingly, <em>IL</em>-22BP was quite specifically expressed by dendritic cells. These data point to a function of the novel <em>IL</em>-10 family members in communication and interaction between cells of the hematopoietic and non-hematopoietic lineages, a role quite distinct from the immunomodulating effects of <em>IL</em>-10 itself.

BACKGROUND

Alzheimer's disease is characterized by the accumulation of neuritic plaques, containing activated microglia and beta-amyloid peptides (Abeta). Fibrillar Abeta can activate microglia, resulting in production of toxic and inflammatory mediators like hydrogen peroxide, nitric oxide, and cytokines. We have recently found that microglial proliferation is regulated by hydrogen peroxide derived from NADPH oxidase. Thus, in this study, we investigated whether Abeta can stimulate microglial proliferation and cytokine production via activation of NADPH oxidase to produce hydrogen peroxide.

METHODS

Primary mixed glial cultures were prepared from the cerebral cortices of 7-day-old Wistar rats. At confluency, microglial cells were isolated by tapping, replated, and treated either with or without Abeta. Hydrogen peroxide production by cells was measured with Amplex Red and peroxidase. Microglial proliferation was assessed under a microscope 0, 24 and 48 hours after plating. TNF-alpha and IL-1beta levels in the culture medium were assessed by ELISA.

RESULTS

We found that 1 muM fibrillar (but not soluble) Abeta1-40 peptide induced microglial proliferation and caused release of hydrogen peroxide, TNF-alpha and IL-1beta from microglial cells. Proliferation was prevented by the NADPH oxidase inhibitor apocynin (10 microM), by the hydrogen peroxide-degrading enzyme catalase (60 U/ml), and by its mimetics EUK-8 and EUK-134 (20 microM); as well as by an antibody against TNF-alpha and by a soluble TNF receptor inhibitor. Production of TNF-alpha and IL-1beta, measured after 24 hours of Abeta treatment, was also prevented by apocynin, catalase and EUKs, but the early release (measured after 1 hour of Abeta treatment) of TNF-alpha was insensitive to apocynin or catalase.

CONCLUSIONS

These results indicate that Abeta1-40-induced microglial proliferation is mediated both by microglial release of TNF-alpha and production of hydrogen peroxide from NADPH oxidase. This suggests that TNF-alpha and NADPH oxidase, and its products, are potential targets to prevent Abeta-induced inflammatory neurodegeneration.

OBJECTIVE

To explore the mechanism of Etanercept in the treatment of rheumatoid arthritis (RA), we investigated whether the Th1/Th2 and Th17/regulatory T cells (Treg) imbalance could be reversed by Etanercept and whether the reversal was related to the improvement of clinical indications.

METHODS

We conducted a 12-week study in 40 active RA patients, of whom <em>20</em> were given a stable weekly dose of methotrexate (MTX) alone and the other ten received a combined therapy of Etanercept and MTX. Ten healthy donors were chosen as controls. Frequencies of Th1, Th2, Th17, and Treg were quantified using flow cytometry, and related serum cytokines were detected by enzyme-linked immunosorbent assay. The composite 28-joint count Disease Activity Score, erythrocyte sedimentation rate, and C-reactive protein were assessed at each visit.

RESULTS

Percentages of IFN-γ(+)Th1 and IL-17(+)Th17 among CD4(+) T cells were significantly higher, while CD4(+)CD25(high)Foxp3(+) Treg were significantly lower in RA patients compared with those in healthy control. After 12 weeks of therapy of MTX single or combination of MTX and Etanercept, the circulating Th17/Treg ratio significantly decreased, while no significant difference was observed in Th1/Th2 ratio. In combined therapy group, the Th17/Treg ratio was positively correlated with the remittance of disease activity. IL-1β, TNF-α, IL-6, IL-17, and IL-23 were significantly decreased, while TGF-β was significantly elevated. The Th17/Treg ratio was positively related to TGF-β, but negatively correlated with IL-6.

CONCLUSIONS

Etanercept in combination with MTX ameliorates RA activity by normalizing the distribution of Th17 and Treg, and their related cytokines, which may partly explain the mechanism of combined therapy of Etanercept plus MTX in RA treatment.