BACKGROUND

Most of the studies investigating the role of leukotrienes (LTs) and prostaglandins (PGs) in asthma have used invasive (eg, bronchoalveolar lavage fluid) or semi-invasive (eg, sputum induction) techniques. Others have measured eicosanoids in plasma or urine, probably reflecting systemic rather than lung inflammation. Collection of exhaled breath condensate (EBC) is a noninvasive method to collect airway secretions.

OBJECTIVE

We sought to investigate whether eicosanoids are measurable in EBC, to show possible differences in their concentrations in asthmatic patients and healthy subjects, and to investigate whether exhaled eicosanoids correlate with exhaled nitric oxide (NO), a marker of airway inflammation.

METHODS

Twelve healthy nonsmokers and 15 steroid-naive patients with mild asthma were studied. Subjects attended on one occasion for pulmonary function tests, collection of EBC, and exhaled NO measurements. Exhaled LTB(4)-like immunoreactivity, LTE(4)-like immunoreactivity, PGE(2)-like immunoreactivity, PGD(2)-methoxime, PGF(2)(alpha)-like immunoreactivity, and thromboxane B(2)-like immunoreactivity were measured by means of enzyme immunoassays.

RESULTS

LTE(4)-like immunoreactivity and LTB(4)-like immunoreactivity were detectable in EBC in healthy subjects, and their levels in asthmatic patients were increased about 3-fold (P <.0001) and 2-fold (P <.0005), respectively. Exhaled NO was increased in asthmatic patients compared with healthy subjects (P <.0001). There was a correlation between exhaled LTB(4) and exhaled NO (r = 0.56, P <.04) in patients with asthma. When measurable, prostanoid levels were similar in asthmatic patients and control subjects.

CONCLUSIONS

Exhaled LTE(4) and LTB(4) are increased in steroid-naive patients with mild asthma. EBC may be proved to be a novel method to monitor airway inflammation in asthma.

<A<em>b</em>stractText>Recent evidence shows that long non-coding RNAs (lncRNAs) are closely related to hepatogenesis and a few aggressive features of hepatocellular carcinoma (HCC). Increasing studies demonstrate that lncRNAs are potential prognostic factors for HCC. Moreover, several studies reported the com<em>b</em>ination of lncRNAs for predicting the overall survival (OS) of HCC, <em>b</em>ut the resu<em>lt</em>s varied. Thus, more effort including more accurate statistical approaches is needed for exploring the prognostic value of lncRNAs in HCC.</A<em>b</em>stractText><A<em>b</em>stractText>To develop a ro<em>b</em>ust lncRNA signature associated with HCC recurrence to improve prognosis prediction of HCC.</A<em>b</em>stractText><A<em>b</em>stractText>Univariate COX regression analysis was performed to screen the lncRNAs significantly associated with recurrence-free survival (RFS) of HCC in GSE76427 for the least a<em>b</em>solute shrinkage and selection operator (LASSO) modelling. The esta<em>b</em>lished lncRNA signature was validated and developed in The Cancer Genome Atlas (TCGA) series using Kaplan-Meier curves. The expression values of the identified lncRNAs were compared <em>b</em>etween the tumor and non-tumor tissues. Pathway enrichment of these lncRNAs was conducted <em>b</em>ased on the significantly co-expressed genes. A prognostic nomogram com<em>b</em>ining the lncRNA signature and clinical characteristics was constructed.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>The lncRNA signature consisted of six lncRNAs: <i>MSC-AS1</i>, <i>POLR2J4</i>, <i>EIF3J-AS1</i>, <i>SERHL</i>, <i>RMST</i>, and <i>PVT1</i>. This risk model was significantly associated with the RFS of HCC in the TCGA cohort with a hazard ratio (HR) <em>b</em>eing 1.807 (95%CI [confidence interval]: 1.329-2.457) and log-rank <i>P</i>-value <em>b</em>eing less than 0.001. The <em>b</em>est candidates of the six-lncRNA signature were younger male patients with HBV infection in relatively early tumor-stage and <em>b</em>etter physical condition <em>b</em>ut with higher preoperative alpha-fetoprotein. All the lncRNAs were significantly upregulated in tumor samples compared to non-tumor samples (<i>P</i> &<em>lt</em>; 0.05). The most significantly enriched pathways of the lncRNAs were TGF-<em>β</em> signaling pathway, cellular apoptosis-associated pathways, <i>etc</i>. The nomogram showed great utility of the lncRNA signature in HCC recurrence risk stratification.</p><A<em>b</em>stractText>We have constructed a six-lncRNA signature for prognosis prediction of HCC. This risk model provides new clinical evidence for the accurate diagnosis and targeted treatment of HCC.</A<em>b</em>stractText>

It has been suggested that exercise training and probiotic supplementation could decelerate the progress of functional and biochemical deterioration in APP/PS1 transgenic mice (APP/PS1^TG). APP/PS1^TG mice were subjected to exercise training and probiotic treatments and functional, biochemical and microbiome markers were analyzed. Under these conditions the mice significantly outperformed controls on The Morris Maze Test, and the number of beta-amyloid plaques decreased in the hippocampus. B. thetaiotaomicron levels correlated highly with the results of the Morris Maze Test (p &lt; 0.05), and this group of bacteria was significantly elevated in the microbiome of the APP/PS1^TG mice compared to the wild type. L. johnsonii levels positively correlated with the beta amyloid content and area. Data revealed that exercise and probiotic treatment can decrease the progress of Alzheimer's Disease and the beneficial effects could be partly mediated by alteration of the microbiome.

<A<em>b</em>stractText>Patients with advanced or metastatic oesophageal squamous cell carcinoma have poor prognosis and few treatment options after first-line therapy. We aimed to assess efficacy and safety of the anti-PD-1 anti<em>b</em>ody camrelizuma<em>b</em> versus investigator's choice of chemotherapy in previously treated patients.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>ESCORT is a randomised, open-la<em>b</em>el, phase 3 study of patients aged 18 to 75 years with a histological or cytological diagnosis of advanced or metastatic oesophageal squamous cell carcinoma done at 43 hospitals in China. Eligi<em>b</em>le patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and had progressed on, or were intolerant to, first-line standard therapy. Patients were randomly assigned (1:1) to camrelizuma<em>b</em> (200 mg every 2 weeks) or chemotherapy with docetaxel (75 mg/m² every 3 weeks) or irinotecan (180 mg/m² every 2 weeks), all given intravenously. Central randomisation was done using the Randomization and Trial Supply Management system with <em>b</em>lock size randomly generated as four or six and stratified <em>b</em>y disease and ECOG performance status. The primary endpoint was overall survival, assessed in randomised patients who had received at least one dose of treatment. Safety was assessed in all treated patients. The trial is registered with ClinicalTrials.gov, NCT03099382, and is closed to new participants.</p><A<em>b</em>stractText>From May 10, 2017, to July 24, 2018, 457 (75%) of 607 screened patients were randomly assigned to treatment, of whom 228 received camrelizuma<em>b</em> treatment and 220 received chemotherapy. As of data cutoff on May 6, 2019, with a median follow-up time of 8·3 months (IQR 4·1-12·8) in the camrelizuma<em>b</em> group and 6·2 months (3·6-10·1) in the chemotherapy group, median overall survival was 8·3 months (95% CI 6·8-9·7) in the camrelizuma<em>b</em> group and 6·2 months (5·7-6·9) in the chemotherapy group (hazard ratio 0·71 [95% CI 0·57-0·87]; two-sided p=0·0010). The most common treatment-related adverse events of grade 3 or worse were anaemia (camrelizuma<em>b</em> vs chemotherapy: six [3%] vs 11 [5%]), a<em>b</em>normal hepatic function (four [2%] vs one [&<em>lt</em>;1%]), and diarrhoea (three [1%] vs nine [4%]). Serious treatment-related adverse events occurred in 37 (16%) of 228 patients in the camrelizuma<em>b</em> group, and in 32 (15%) of 220 patients in the chemotherapy group. Ten treatment-related deaths occurred, seven (3%) in the camrelizuma<em>b</em> group (three deaths from unknown causes, one enterocolitis, one hepatic function a<em>b</em>normal, one pneumonitis, and one myocarditis) and three (1%) in the chemotherapy group (two deaths from unknown causes, and one gastrointestinal haemorrhage).</A<em>b</em>stractText><A<em>b</em>stractText>Second-line camrelizuma<em>b</em> significantly improved overall survival in patients with advanced or metastatic oesophageal squamous cell carcinoma compared with chemotherapy, with a managea<em>b</em>le safety profile. It might represent a potential option of standard second-line treatment for patients with oesophageal squamous cell carcinoma in China.</A<em>b</em>stractText><A<em>b</em>stractText>Jiangsu Hengrui Medicine.</A<em>b</em>stractText>

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that primarily affects motoneurons in the brain and spinal cord. Dominant mutations in superoxide dismutase-1 (SOD1) cause a familial form of ALS. Mutant SOD1-damaged glial cells contribute to ALS pathogenesis by releasing neurotoxic factors, but the mechanistic basis of the motoneuron-specific elimination is poorly understood. Here, we describe a motoneuron-selective death pathway triggered by activation of lymphotoxin-β receptor (LT-βR) by LIGHT, and operating by a novel signaling scheme. We show that astrocytes expressing mutant SOD1 mediate the selective death of motoneurons through the proinflammatory cytokine interferon-γ (IFNγ), which activates the LIGHT-LT-βR death pathway. The expression of LIGHT and LT-βR by motoneurons in vivo correlates with the preferential expression of IFNγ by motoneurons and astrocytes at disease onset and symptomatic stage in ALS mice. Importantly, the genetic ablation of Light in an ALS mouse model retards progression, but not onset, of the disease and increases lifespan. We propose that IFNγ contributes to a cross-talk between motoneurons and astrocytes causing the selective loss of some motoneurons following activation of the LIGHT-induced death pathway.

The development and function of secondary lymphoid tissue require signaling by tumor necrosis factor and lymphotoxins. Mice deficient in LTbetaR show defective organogenesis of lymph nodes and Peyer's patches and a severely disturbed splenic architecture. In contrast, TNF or p55TNF-R deficiency does not affect the organogenesis of peripheral lymphoid organs but interferes with the formation of B cell follicles and the appearance of FDC networks and germinal centers in all secondary lymphoid organs. Based on these differences, we have previously hypothesized that the role of TNF in lymphoid structure is distinct from that of LT and restricted in regulating cellular interactions that allow the differentiation and/or correct positioning of FDCs. In the present study we show that, in addition to the defects in follicular structure, TNF or p55TNF-R knockout mice exhibit defects in the formation of the macrophage populations and of the sinus lining cells of the splenic marginal zone. Interestingly, a large number of dendritic-shaped cells stained with FDC-specific markers and able to trap immune complexes are retained within the defective marginal zone of TNF and p55TNF-R knockout spleens. We conclude that the primary defect in the lymphoid phenotype of TNF or p55TNF-R knockout mice is the failure of FDC precursors to migrate through the disorganized marginal sinus and to home properly into the splenic follicular areas where they would promote the formation of B cell follicles and germinal centers.

Influenza virus affects the respiratory tract by direct viral infection or by damage from the immune system response. In humans, the respiratory epithelium is the only site where the hemagglutinin (HA) molecule is effectively cleaved, generating infectious virus particles. Virus transmission occurs through a susceptible individual's contact with aerosols or respiratory fomites from an infected individual. The inability of the lung to perform its primary function of gas exchange can result from multiple mechanisms, including obstruction of the airways, loss of alveolar structure, loss of lung epithelial integrity from direct epithelial cell killing, and degradation of the critical extracellular matrix.Approximately 30-40% of hospitalized patients with laboratory-confirmed influenza are diagnosed with acute pneumonia. These patients who develop pneumonia are more likely to be &lt; 5 years old, > 65 years old, Caucasian, and nursing home residents; have chronic lung or heart disease and history of smoking, and are immunocompromised.Influenza can primarily cause severe pneumonia, but it can also present in conjunction with or be followed by a secondary bacterial infection, most commonly by Staphylococcus aureus and Streptococcus pneumoniae. Influenza is associated with a high predisposition to bacterial sepsis and ARDS. Viral infections presenting concurrently with bacterial pneumonia are now known to occur with a frequency of 30-50% in both adult and pediatric populations. The H3N2 subtype has been associated with unprecedented high levels of intensive care unit (ICU) admission.Influenza A is the predominant viral etiology of acute respiratory distress syndrome (ARDS) in adults. Risk factors independently associated with ARDS are age between 36 and 55 years old, pregnancy, and obesity, while protective factors are female sex, influenza vaccination, and infections with Influenza A (H3N2) or Influenza B viruses.In the ICU, particularly during the winter season, influenza should be suspected not only in patients with typical symptoms and epidemiology, but also in patients with severe pneumonia, ARDS, sepsis with or without bacterial co-infection, as well as in patients with encephalitis, myocarditis, and rhabdomyolysis.

The cytokines lymphotoxin (LT) alpha, beta and their receptor (LTbetaR) belong to the tumor necrosis factor (TNF) superfamily, whose founder-TNFalpha-was initially discovered due to its tumor necrotizing activity. LTbetaR signaling serves pleiotropic functions including the control of lymphoid organ development, support of efficient immune responses against pathogens due to maintenance of intact lymphoid structures, induction of tertiary lymphoid organs, liver regeneration or control of lipid homeostasis. Signaling through LTbetaR comprises the noncanonical/canonical nuclear factor-kappaB (NF-kappaB) pathways thus inducing chemokine, cytokine or adhesion molecule expression, cell proliferation and cell survival. Blocking LTbetaR signaling or Fcgamma-receptor mediated immunoablation of LT-expressing cells was demonstrated to be beneficial in various infectious or noninfectious inflammatory or autoimmune disorders. Only recently, LTbetaR signaling was shown to initiate inflammation-induced carcinogenesis, to influence primary tumorigenesis and to control reemergence of carcinoma in various cancer models through distinct mechanisms. Indeed, LTbetaR signaling inhibition has already been used as efficient anti-inflammatory, anti-cancer therapy in some experimental models. Here, we review the pleiotropic functions attributed to LT, the effects of its deregulation and extensively discuss the recent literature on LT's link to carcinogenesis.

<A<em>b</em>stractText>Vitamin D deficiency is a common, potentially reversi<em>b</em>le contri<em>b</em>utor to mor<em>b</em>idity and mortality among critically ill patients. The potential <em>b</em>enefits of vitamin D supplementation in acute critical illness require further study.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>We conducted a randomized, dou<em>b</em>le-<em>b</em>lind, place<em>b</em>o-controlled, phase 3 trial of early vitamin D<su<em>b</em>)3</su<em>b</em>) supplementation in critically ill, vitamin D-deficient patients who were at high risk for death. Randomization occurred within 12 hours after the decision to admit the patient to an intensive care unit. Eligi<em>b</em>le patients received a single enteral dose of 540,000 IU of vitamin D<su<em>b</em>)3</su<em>b</em>) or matched place<em>b</em>o. The primary end point was 90-day all-cause, all-location mortality.</p><A<em>b</em>stractText>A total of 1360 patients were found to <em>b</em>e vitamin D-deficient during point-of-care screening and underwent randomization. Of these patients, 1078 had <em>b</em>aseline vitamin D deficiency (25-hydroxyvitamin D level, &<em>lt</em>;20 ng per milliliter [50 nmol per liter]) confirmed <em>b</em>y su<em>b</em>sequent testing and were included in the primary analysis population. The mean day 3 level of 25-hydroxyvitamin D was 46.9±23.2 ng per milliliter (117±58 nmol per liter) in the vitamin D group and 11.4±5.6 ng per milliliter (28±14 nmol per liter) in the place<em>b</em>o group (difference, 35.5 ng per milliliter; 95% confidence interval [CI], 31.5 to 39.6). The 90-day mortality was 23.5% in the vitamin D group (125 of 531 patients) and 20.6% in the place<em>b</em>o group (109 of 528 patients) (difference, 2.9 percentage points; 95% CI, -2.1 to 7.9; P = 0.26). There were no clinically important differences <em>b</em>etween the groups with respect to secondary clinical, physiological, or safety end points. The severity of vitamin D deficiency at <em>b</em>aseline did not affect the association <em>b</em>etween the treatment assignment and mortality.</A<em>b</em>stractText><p><div>(<em>b</em>)CONCLUSIONS</<em>b</em>)</div>Early administration of high-dose enteral vitamin D<su<em>b</em>)3</su<em>b</em>) did not provide an advantage over place<em>b</em>o with respect to 90-day mortality or other, nonfatal outcomes among critically ill, vitamin D-deficient patients. (Funded <em>b</em>y the National Heart, Lung, and Blood Institute; VIOLET ClinicalTrials.gov num<em>b</em>er, NCT03096314.).</p>

Background: The Coronavirus disease 2019 (COVID-19) is an infectious disease presenting a major threat to public health. This study aims to assess Internet use characteristics and objectively examine the potential psychological factors associated with Internet addiction (IA) during the COVID-19 epidemic.

Methods: A cross-sectional, anonymized, self-reported survey was conducted among Chinese children and adolescents aged 6 to 18 years old. Participants completed questionnaires containing Young's Internet Addiction Test (IAT) and the Depression, Anxiety, and Stress Scale (DASS-21), and questions regarding demographic information and Internet use characteristics.

Results: A total of 2050 participants (mean age:12.34 ± 4.67 years old, female: 48.44%) were enrolled. Fifty-five (2.68%) participants met the criterion for addictive Internet use (IAT≥70), while 684 (33.37%) participants were classified as problematic Internet users (69≥IAT≥40). Internet usage had grown during the COVID-19 epidemic, including the frequency and duration of recreational Internet use, and the frequency of stay-up Internet use. A linear regression analysis showed female gender (β=-0.091, p&lt;0.001), age (β=0.066, p=0.001), depression (β=0.257, p&lt;0.001), and stress (β=0.323, p&lt;0.001) were significantly correlated with the IAT total scores (R=0.539, R^{2 =} 0.291, p&lt;0.001).

Conclusions: We observed excessive Internet use among Chinese children and adolescents during the outbreak of COVID-19. Age, gender, depression, and stress were the potential key factors affecting IA. Extended family and professional support should be considered for vulnerable individuals during these unprecedented times.

Keywords: Internet addiction (IA); anxiety; children and adolescents; depression; stress.

Background: Metastatic castration-resistant prostate cancer remains fatal despite recent advances. Prostate-specific membrane antigen (PSMA) is highly expressed in metastatic castration-resistant prostate cancer. Lutetium-177 (¹⁷⁷Lu)-PSMA-617 is a radioligand therapy that delivers beta-particle radiation to PSMA-expressing cells and the surrounding microenvironment.

Methods: We conducted an international, open-label, phase 3 trial evaluating ¹⁷⁷Lu-PSMA-617 in patients who had metastatic castration-resistant prostate cancer previously treated with at least one androgen-receptor-pathway inhibitor and one or two taxane regimens and who had PSMA-positive gallium-68 (⁶⁸Ga)-labeled PSMA-11 positron-emission tomographic-computed tomographic scans. Patients were randomly assigned in a 2:1 ratio to receive either ¹⁷⁷Lu-PSMA-617 (7.4 GBq every 6 weeks for four to six cycles) plus protocol-permitted standard care or standard care alone. Protocol-permitted standard care excluded chemotherapy, immunotherapy, radium-223 (²²³Ra), and investigational drugs. The alternate primary end points were imaging-based progression-free survival and overall survival, which were powered for hazard ratios of 0.67 and 0.73, respectively. Key secondary end points were objective response, disease control, and time to symptomatic skeletal events. Adverse events during treatment were those occurring no more than 30 days after the last dose and before subsequent anticancer treatment.

Results: From June 2018 to mid-October 2019, a total of 831 of 1179 screened patients underwent randomization. The baseline characteristics of the patients were balanced between the groups. The median follow-up was 20.9 months. ¹⁷⁷Lu-PSMA-617 plus standard care significantly prolonged, as compared with standard care, both imaging-based progression-free survival (median, 8.7 vs. 3.4 months; hazard ratio for progression or death, 0.40; 99.2% confidence interval [CI], 0.29 to 0.57; P<0.001) and overall survival (median, 15.3 vs. 11.3 months; hazard ratio for death, 0.62; 95% CI, 0.52 to 0.74; P<0.001). All the key secondary end points significantly favored ¹⁷⁷Lu-PSMA-617. The incidence of adverse events of grade 3 or above was higher with ¹⁷⁷Lu-PSMA-617 than without (52.7% vs. 38.0%), but quality of life was not adversely affected.

Conclusions: Radioligand therapy with ¹⁷⁷Lu-PSMA-617 prolonged imaging-based progression-free survival and overall survival when added to standard care in patients with advanced PSMA-positive metastatic castration-resistant prostate cancer. (Funded by Endocyte, a Novartis company; VISION ClinicalTrials.gov number, NCT03511664.).

Objective: To analyze the most frequent radiographic features of COVID-19 pneumonia and assess the effectiveness of chest X-ray (CXR) in detecting pulmonary alterations.

Materials and methods: CXR of 240 symptomatic patients (70% male, mean age 65 ± 16 years), with SARS-CoV-2 infection confirmed by RT-PCR, was retrospectively evaluated. Patients were clustered in four groups based on the number of days between symptom onset and CXR: group A (0-2 days), 49 patients; group B (3-5), 75 patients; group C (6-9), 85 patients; and group D (> 9), 31 patients. Alteration's type (reticular/ground-glass opacity (GGO)/consolidation) and distribution (bilateral/unilateral, upper/middle/lower fields, peripheral/central) were noted. Statistical significance was tested using chi-square test.

Results: Among 240 patients who underwent CXR, 180 (75%) showed alterations (group A, 63.3%; group B, 72%; group C, 81.2%; group D, 83.9%). GGO was observed in 124/180 patients (68.8%), reticular alteration in 113/180 (62.7%), and consolidation in 71/180 (39.4%). Consolidation was significantly less frequent (p < 0.01). Distribution among groups was as follows: reticular alteration (group A, 70.9%; group B, 72.2%; group C, 57.9%; group D, 46.1%), GGO (group A, 67.7%; group B, 62.9%; group C, 71%; group D, 76.9%), and consolidation (group A, 35.5%; group B, 31.4%; group C, 47.8%; group D, 38.5%). Alterations were bilateral in 73.3%. Upper, middle, and lower fields were involved in 36.7%, 79.4%, and 87.8%, respectively. Lesions were peripheral in 49.4%, central in 11.1%, or both in 39.4%. Upper fields and central zones were significantly less involved (p < 0.01).

Conclusions: The most frequent lesions in COVID-19 patients were GGO (intermediate/late phase) and reticular alteration (early phase) while consolidation gradually increased over time. The most frequent distribution was bilateral, peripheral, and with middle/lower predominance. Overall rate of negative CXR was 25%, which progressively decreased over time.

Key points: • The predominant lung changes were GGO and reticular alteration, while consolidation was less frequent. • The typical distribution pattern was bilateral, peripheral, or both peripheral and central and involved predominantly the lower and middle fields. • Chest radiography showed lung abnormalities in 75% of patients with confirmed SARS-CoV-2 infection, range varied from 63.3 to 83.9%, respectively, at 0-2 days and > 9 days from the onset of symptoms.

Keywords: COVID-19; Pneumonia; Radiography; Severe acute respiratory syndrome coronavirus 2; Thorax.

The nucleotide sequence of the LT-BH cistron (eltBH) from an enterotoxigenic Escherichia coli strain infectious for humans was determined and compared with the LT-B cistron sequence from a porcine E. coli isolate. Both cistrons were shown to comprise 375 nucleotide base pairs, and discrepancies were detected at eight positions. Of the nonhomologous base pairs, six resulted in codon changes that would lead to amino acid variations. The nucleotide sequence distal to both LT-B cistrons was also determined, and only three differences were detected in 197 base pairs. An HhaI site unique to eltBH was shown to be present in all the heat-labile (LT) genes from 31 human isolates surveyed, whereas the restriction enzyme recognition site was absent in the gene from 46 porcine E. coli isolates. The results suggest that two genetically discernable LT groups are identifiable and that the groups are also distinguishable by the isolation source (human or porcine) of the infecting E. coli strains.

We have recently shown that de novo formation of lymphoid structures resembling B-cell follicles occurs in the inflamed central nervous system (CNS) meninges in a subset of patients with secondary progressive multiple sclerosis and in SJL mice with relapsing-remitting experimental autoimmune encephalomyelitis (EAE). Because lymphotoxin (LT) alpha(1)beta(2) is essential for lymphoid tissue organization, we used real-time PCR to examine LTbeta and LTbeta receptor (LTbetaR) gene expression in the CNS of SJL mice immunized with PLP 139-151 peptide. Moreover, we used the decoy receptor LTbetaR-immunoglobulin fusion protein to block the interaction of lymphotoxin (LT) alpha(1)beta(2) with the LTbeta receptor (LTbetaR) in mice with established EAE and evaluate the effect of systemic and local treatments with the fusion protein on disease progression, CNS lymphocytic infiltration and formation of meningeal B-cell follicles. The present findings indicate that both LTbeta and LTbetaR are upregulated at EAE onset and during subsequent relapses and that systemic and local blockade of the LT pathway with LTbetaR-Ig results in protracted and transient inhibition of EAE clinical signs, respectively. LTbetaR-Ig treatment also reduces T- and B-cell infiltration and prevents the induction of the chemokines CXCL10 and CXCL13 and the formation of organized ectopic follicles in the EAE-affected CNS. Targeting of molecules involved in lymphoid organogenesis could represent a valid strategy to inhibit CNS inflammation and formation of ectopic follicles, which may play a role in maintaining an abnormal, intrathecal humoral immune response in CNS autoimmune disease.

The bacterial enterotoxins, cholera toxin and the heat labile toxin of E. coli, are well known adjuvants for mucosal immune response. Their common A chain mediates the toxigenic mechanism by causing ADP ribosylation of G proteins and subsequent elevation of cAMP in target cells. A large IgA and IgG antibody response to admixed protein antigen (Ag) is the hallmark of these adjuvants and is clearly associated with the A chain activity. Expansion of Ag-specific B and T cells, alteration of T cell cytokine production, and changes in regulatory T cells have been reported as adjuvant mechanisms. The B chain derivatives of these toxins can also weakly enhance immune response, especially if covalently associated with Ag and used for nasophyrangeal immunization. Importantly, these toxins or their B chain derivatives can alter the normal immune regulation that produces oral tolerance. This indicates that they modulate mechanisms operative between the mucosal and systemic immune systems. There are some discrepancies between in vitro models of CT or LT activity and in vivo manifestations of their adjuvant activities. Interpretation of current data regarding in vivo mechanism is hampered by an incomplete understanding of how mucosal B and T cells can interact with systemic lymphoid tissue and vice versa. More important, there is no clear understanding of the early effects of the toxins on the local (and draining) mucosal lymphoid tissues. This is especially true in the critical areas of antigen presentation, T and B cell activation, and cytokine production.

The amount of crude Campylobacter jejuni enterotoxin present in culture products was quantitated by comparing the response of these preparations with that of pure Escherichia coli heat-labile toxin (LT) in the Chinese hamster ovary assay and in enzyme-linked immunosorbent assays that used GM ganglioside or antisera to LT or both. Maximum C. jejuni enterotoxin production was achieved by growth at 42 degrees C for 24 h under agitation in supplemented GC medium. Adding polymyxin separately to either the broth supernatant or the cells enhanced the recovery of toxin; the yield from cell lysates was much lower. The quantity of C. jejuni enterotoxin produced by clinical isolates obtained locally or provided from Mexico varied widely, over a spectrum from none to large amounts; quantitative values for the amount of C. jejuni enterotoxin determined by the Chinese hamster ovary and enzyme-linked immunosorbent assays correlated with the degree of secretory potency of this material in ligated rat ileal loops. The cytotonic activity of C. jejuni enterotoxin in Chinese hamster ovary cells was abolished by heating at 96 degrees C for 10 min and by preincubation either with GM ganglioside or with LT or cholera toxin antisera. The secretory activity of C. jejuni enterotoxin in ligated rat ileal loops was passively neutralized by antiserum to LT, and immunizing rats with either LT or its B subunit significantly (P less than 0.001) reduced fluid response to active challenge with C. jejuni enterotoxin in ligated ileal loops. These observations indicate that strains of C. jejuni vary in their capacity to elaborate a heat-labile enterotoxin that has close immunological homology with LT and cholera toxin.

Cytokines are thought to contribute to the induction of pancreatic beta-cell destruction in insulin-dependent diabetes mellitus. The molecular mechanisms that underlie beta-cell death were investigated by studying cytokine-induced cell death in beta-cell lines. A combination of three cytokines (interleukin-1 beta, tumour necrosis factor-alpha, and interferon-gamma) induced apoptotic cell death in the mouse pancreatic beta-cell line beta TC1, as judged from the appearance of cells with hypodiploid nuclei and oligonucleosomal DNA fragmentation. The same treatment also induced apoptosis in the mouse pancreatic alpha-cell line alpha TC1 and the NOD/Lt mouse beta-cell line NIT-1, although to a lesser extent than in beta TC1 cells. The abundance of endogenous Bcl-2 in beta TC1 cells was lower than that in the other two cell lines. Overexpression of human Bcl-2 in beta TC1 cells partially protected them from cytokine-induced cell death. These results suggest that apoptosis may be responsible, at least in part, for cytokine-induced beta-cell destruction and that Bcl-2 prevents apoptosis in pancreatic islet cells.

Two synergistic plastic mechanisms have recently been identified in rat medial vestibular nucleus (MVN) neurons during 'vestibular compensation', the behavioral recovery that follows damage to the vestibular receptors or nerve of one inner ear. Ipsi-lesional MVN neurons develop a significant increase in their intrinsic excitability, and a marked decrease in the functional efficacy of GABA(A) and GABA(B) receptors, within 4 h of unilateral vestibular deafferentation. These mechanisms presumably counteract the disfacilitation and excessive commissural inhibition of the ipsi-lesional cells after deafferentation, and thus promote the recovery of resting activity. In this study, we investigated the intrinsic membrane properties and spike firing characteristics of rostral ipsi-lesional MVN neurons in slices from animals that underwent vestibular compensation for either 24-72 h or 7-10 days. Significant changes were observed in the spontaneous in vitro discharge rate, resting membrane potentials and voltage-activated membrane conductances of type B cells, but not type A cells. There was a significant increase in the number of type B(LTS) cells compared to normal. These findings indicate that during vestibular compensation marked changes occur in ion channel expression and function selectively in type B MVN neurons. These changes are appropriate to increase the responsiveness of type B cells both to their own intrinsic pacemaker-like membrane conductances and excitatory synaptic inputs. Together with the downregulation of inhibitory receptor efficacy, this increased intrinsic excitability may be sufficient to restore the resting discharge of the deafferented neurons in vivo. These results therefore provide further evidence for synaptic and neuronal plasticity in ipsi-lesional MVN neurons during vestibular compensation.

Type I and type II interferons (IFNs) play a critical role in control of a number of viral infections. To study whether altered and reduced functional capacities of type I and type II IFNs would affect rotavirus-induced diarrhea and viral replication, we obtained signal transducers and activators of transcription 1 (Stat1) knock-out mice (Stat1(-/-)) that lack many IFN-induced responses. We found that suckling Stat1(-/-) and immunocompetent mice orally infected with rotavirus experienced diarrhea and shed rotavirus with similar intensity. However, adult Stat1(-/-) mice shed up to 100-fold more homologous murine rotavirus and heterologous rhesus rotavirus antigen in their stools than did immunocompetent mice 2-6 days after infection. Clearance of rotavirus in stools from adult Stat1(-/-) mice occurred at the same time as in wild-type (WT) control mice. Clearance in Stat1(-/-) mice correlated with a potent antibody response and a mixed Th1 and Th2 response, whereas in WT control mice, clearance correlated with a weaker antibody response and a polarized Th1 response. Stat1(-/-) mice were fully protected against subsequent challenge. Moreover, vaccination of adult Stat1(-/-) mice with a rotavirus VP6 protein and the mucosal adjuvant Escherichia coli heat-labile toxin LT (R192G) elicited 94% protection, as measured by the total reduction in viral shedding for the group in comparison to unimmunized controls. Thus, modulating IFN function through the loss of Stat1 caused a defective innate immune response in adult mice but had no effect on rotavirus-induced diarrhea and replication in suckling mice. Furthermore, adult Stat1(-/-), IFN-gamma, and IFN-alpha/beta receptor(-/-) (IFNAR-2(-/-)) mice infected with rotavirus or vaccinated with VP6 vaccine and adjuvant were fully protected against rotavirus shedding following a subsequent challenge with rotavirus.

The type IIb heat-labile enterotoxin of Escherichia coli (LT-IIb) and its nontoxic pentameric B subunit (LT-IIb-B(5)) display different immunomodulatory activities, the mechanisms of which are poorly understood. We investigated mechanisms whereby the absence of the catalytically active A subunit from LT-IIb-B(5) renders this molecule immunostimulatory through TLR2. LT-IIb-B(5), but not LT-IIb, induced TLR2-mediated NF-kappaB activation and TNF-alpha production. These LT-IIb-B(5) activities were antagonized by LT-IIb; however, inhibitors of adenylate cyclase or protein kinase A reversed this antagonism. The LT-IIb antagonistic effect is thus likely dependent upon the catalytic activity of its A subunit, which causes elevation of intracellular cAMP and activates cAMP-dependent protein kinase A. Consistent with this, a membrane-permeable cAMP analog and a cAMP-elevating agonist, but not catalytically defective point mutants of LT-IIb, mimicked the antagonistic action of wild-type LT-IIb. The mutants moreover displayed increased proinflammatory activity compared with wild-type LT-IIb. Additional mechanisms for the divergent effects on TLR2 activation by LT-IIb and LT-IIb-B(5) were suggested by findings that the latter was significantly stronger in inducing lipid raft recruitment of TLR2 and interacting with this receptor. The selective use of TLR2 by LT-IIb-B(5) was confirmed in an assay for IL-10, which is inducible by both LT-IIb and LT-IIb-B(5) at comparable levels; TLR2-deficient macrophages failed to induce IL-10 in response to LT-IIb-B(5) but not in response to LT-IIb. These differential immunomodulatory effects by LT-IIb and LT-IIb-B(5) have important implications for adjuvant development and, furthermore, suggest that enterotoxic E. coli may suppress TLR-mediated innate immunity through the action of the enterotoxin A subunit.

BACKGROUND

Lipid mediators play an important pathophysiologic role in atopic asthmatic children, but their role in the airways of atopic nonasthmatic children is unknown.

OBJECTIVE

We sought (1) to measure leukotriene (LT) E 4 , LTB 4 , 8-isoprostane, prostaglandin E 2 , and thromboxane B 2 concentrations in exhaled breath condensate in atopic asthmatic and atopic nonasthmatic children; (2) to measure exhaled nitric oxide (NO) as an independent marker of airway inflammation; and (3) to study the effect of inhaled corticosteroids on exhaled eicosanoids.

METHODS

Twenty healthy children, 20 atopic nonasthmatic children, 30 steroid-naive atopic asthmatic children, and 25 atopic asthmatic children receiving inhaled corticosteroids were included in a cross-sectional study. An open-label study with inhaled fluticasone (100 microg twice a day for 4 weeks) was undertaken in 14 steroid-naive atopic asthmatic children.

RESULTS

Compared with control subjects, exhaled LTE 4 ( P <.001), LTB 4 ( P <.001), and 8-isoprostane ( P <.001) levels were increased in both steroid-naive and steroid-treated atopic asthmatic children but not in atopic nonasthmatic children (LTE 4 , P=.14; LTB 4 , P=.23; and 8-isoprostane, P=.52). Exhaled NO levels were increased in steroid-naive atopic asthmatic children ( P <.001) and, to a lesser extent, in atopic nonasthmatic children ( P <.01). Inhaled fluticasone reduced exhaled NO (53%, P <.0001) and, to a lesser extent, LTE 4 (18%, P <.01) levels but not LTB 4 , prostaglandin E 2 , or 8-isoprostane levels in steroid-naive asthmatic children. Conclusions Exhaled LTE 4 , LTB 4 , and 8-isoprostane levels are increased in atopic asthmatic children but not in atopic nonasthmatic children. In contrast to exhaled NO, these markers seem to be relatively resistant to inhaled corticosteroids.

Alzheimer's disease (AD) is the most common form of dementia worldwide, yet there is currently no effective treatment or cure. Extracellular deposition of amyloid-beta protein (Abeta) in brain is a key neuropathological characteristic of AD. In 1999, Schenk et al. first reported that an injected Abeta vaccine given to PDAPP mice, an AD mouse model displaying Abeta deposition in brain, led to the lowering of Abeta levels in brain. In 2000, we demonstrated that intranasal (i.n.) immunization with human synthetic Abetabeta burden in PDAPP mice; serum Abeta antibody titers were low (approximately 26 microg/ml). More recently, we have optimized our i.n. Abeta immunization protocol in wild-type (WT) mice. When low doses Escherichia coli heat-labile enterotoxin (LT) were given as a mucosal adjuvant with Abeta i.n., there was a dramatic 12-fold increase in Abeta antibody titers in WT B6D2F1 mice treated two times per week for 8 weeks compared to those of mice receiving i.n. Abeta without adjuvant. A non-toxic form of LT, designated LT(R192G), showed even better adjuvanticity; anti-Abeta antibody titers were 16-fold higher than those seen in mice given i.n. Abeta without adjuvant. In both cases, the serum Abeta antibodies recognized epitopes within Abetabeta vaccine protocol is now being tested in AD mouse models with the expectation that higher Abeta antibody titers may be more effective in reducing cerebral Abeta levels.

Leukotrienes (LTs) are lipid mediators implicated in asthma and other inflammatory diseases. LTB(4) and LTD(4) also participate in antimicrobial defense by stimulating phagocyte functions via ligation of B leukotriene type 1 (BLTLT type 1 (cysLTBLTLTLT receptors, or to other G protein-coupled receptors, in primary cells. In this study we sought to define the role of specific G proteins in pulmonary alveolar macrophage (AM) innate immune responses to LTB(4) and LTD(4). LTB(4) but not LTD(4) reduced cAMP levels in rat AM by a pertussis toxin (PTX)-sensitive mechanism. Enhancement of FcgammaR-mediated phagocytosis and bacterial killing by LTB(4) was also PTX-sensitive, whereas that induced by LTD(4) was not. LTD(4) and LTB(4) induced Ca(2+) and intracellular inositol monophosphate accumulation, respectively, highlighting the role of Galpha(q) protein in mediating PTX-insensitive LTD(4) enhancement of phagocytosis and microbicidal activity. Studies with liposome-delivered G protein blocking Abs indicated a dependency on specific Galpha(q/11) and Galpha(i3) subunits, but not Galpha(i2) or G(beta)gamma, in LTB(4)-enhanced phagocytosis. The selective importance of Galpha(q/11) protein was also demonstrated in LTD(4)-enhanced phagocytosis. The present investigation identifies differences in specific G protein subunit coupling to LT receptors in antimicrobial responses and highlights the importance of defining the specific G proteins coupled to heptahelical receptors in primary cells, rather than simply using heterologous expression systems.

Lymphocytes from lymphotoxin (LT) alpha-deficient mice, which lack segregation of their B- and T-cell areas, acquire normal organization following adoptive transfer into RAG-deficient recipients, identifying a non-B non-T cell in the segregation process. Here we show that a CD4+CD3- accessory cell is tightly associated with discrete VCAM-1-expressing stromal cells in B- and T-cell areas of the mouse spleen. CD4+CD3- cells express high levels of LTalpha, LTbeta, and tumor necrosis factor (TNF) alpha, which are the ligands for the LTbeta receptor and TNFR1 expressed by stromal cells. The expression of these ligands is functional, as transferring CD4+CD3- cells derived from either embryonic or adult tissues into LTalpha-deficient mice organizes B/T segregation and up-regulates CCL21 protein expression in areas where T cells are segregated from B cells. We propose that the function of CD4+CD3- cells is to form a link between primed CD4 T cells and the underlying stromal elements, creating distinct microenvironments in which they enable effector responses.