<A<em>b</em>stractText>Bariatric surgery improves glucose meta<em>b</em>olism. Recent data suggest that faecal micro<em>b</em>iota transplantation (FMT) using faeces from post<em>b</em>ariatric surgery diet-induced o<em>b</em>ese mice in germ-free mice improves glucose meta<em>b</em>olism and intestinal homeostasis. We here investigated whether allogenic FMT using faeces from post-Roux-en-Y gastric <em>b</em>ypass donors (RYGB-D) compared with using faeces from meta<em>b</em>olic syndrome donors (METS-D) has short-term effects on glucose meta<em>b</em>olism, intestinal transit time and adipose tissue inflammation in treatment-naïve, o<em>b</em>ese, insulin-resistant male su<em>b</em>jects.</A<em>b</em>stractText><p><div>(<em>b</em>)DESIGN</<em>b</em>)</div>Su<em>b</em>jects with meta<em>b</em>olic syndrome (n=22) received allogenic FMT either from RYGB-D or METS-D. Hepatic and peripheral insulin sensitivity as well as lipolysis were measured at <em>b</em>aseline and 2 weeks after FMT <em>b</em>y hyperinsulinaemic euglycaemic sta<em>b</em>le isotope (²H<su<em>b</em>)2</su<em>b</em>)-glucose and ²H<su<em>b</em>)5</su<em>b</em>)-glycerol) clamp. Secondary outcome parameters were changes in resting energy expenditure, intestinal transit time, faecal short-chain fatty acids (SCFA) and <em>b</em>ile acids, and inflammatory markers in su<em>b</em>cutaneous adipose tissue related to intestinal micro<em>b</em>iota composition. Faecal SCFA, <em>b</em>ile acids, glycaemic control and inflammatory parameters were also evaluated at 8 weeks.</p><A<em>b</em>stractText>We o<em>b</em>served a significant decrease in insulin sensitivity 2 weeks after allogenic METS-D FMT (median rate of glucose disappearance: from 40.6 to 34.0 µmol/kg/min; p&<em>lt</em>;0.01). Moreover, a trend (p=0.052) towards faster intestinal transit time following RYGB-D FMT was seen. Finally, we o<em>b</em>served changes in faecal <em>b</em>ile acids (increased lithocholic, deoxycholic and (iso)lithocholic acid after METS-D FMT), inflammatory markers (decreased adipose tissue chemokine ligand 2 (CCL2) gene expression and plasma CCL2 after RYGB-D FMT) and changes in several intestinal micro<em>b</em>iota taxa.</A<em>b</em>stractText><A<em>b</em>stractText>Allogenic FMT using METS-D decreases insulin sensitivity in meta<em>b</em>olic syndrome recipients when compared with using post-RYGB-D. Further research is needed to delineate the role of donor characteristics in FMT efficacy in human insulin-resistant su<em>b</em>jects.</A<em>b</em>stractText><A<em>b</em>stractText>NTR4327.</A<em>b</em>stractText>

<A<em>b</em>stractText>RTOG 0617 compared standard-dose (SD; 60 Gy) versus high-dose (HD; 74 Gy) radiation with concurrent chemotherapy and determined the efficacy of cetuxima<em>b</em> for stage III non-small-cell lung cancer (NSCLC).</A<em>b</em>stractText><A<em>b</em>stractText>The study used a 2 × 2 factorial design with radiation dose as 1 factor and cetuxima<em>b</em> as the other, with a primary end point of overall survival (OS).</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Median follow-up was 5.1 years. There were 3 grade 5 adverse events (AEs) in the SD arm and 9 in the HD arm. Treatment-related grade ≥3 dysphagia and esophagitis occurred in 3.2% and 5.0% of patients in the SD arm <i>v</i> 12.1% and 17.4% in the HD arm, respectively (<i>P</i> = .0005 and &<em>lt</em>; .0001). There was no difference in pulmonary toxicity, with grade ≥3 AEs in 20.6% and 19.3%. Median OS was 28.7 <i>v</i> 20.3 months (<i>P</i> = .0072) in the SD and HD arms, respectively, 5-year OS and progression-free survival (PFS) rates were 32.1% and 23% and 18.3% and 13% (<i>P</i> = .055), respectively. Factors associated with improved OS on mu<em>lt</em>ivaria<em>b</em>le analysis were standard radiation dose, tumor location, institution accrual volume, esophagitis/dysphagia, planning target volume and heart V5. The use of cetuxima<em>b</em> conferred no survival <em>b</em>enefit at the expense of increased toxicity. The prior signal of <em>b</em>enefit in patients with higher H scores was no longer apparent. The progression rate within 1 month of treatment completion in the SD arm was 4.6%. For comparison purposes, the resu<em>lt</em>ant 2-year OS and PFS rates allowing for that dropout rate were 59.6% and 30.7%, respectively, in the SD arms.</p><A<em>b</em>stractText>A 60-Gy radiation dose with concurrent chemotherapy should remain the standard of care, with the OS rate <em>b</em>eing among the highest reported in the literature for stage III NSCLC. Cetuxima<em>b</em> had no effect on OS. The 2-year OS rates in the control arm are similar to the PACIFIC trial.</A<em>b</em>stractText>

(<em>b</em>)Background:</<em>b</em>) Immunosenescence contri<em>b</em>utes to reduced vaccine response in elderly persons, and is worsened <em>b</em>y deficiencies in nutrients such as Vitamin (Vit-D). The immune system is a well-known target of Vit-D, which can <em>b</em>oth potentiate the innate immune response and inhi<em>b</em>it the adaptive system, and so modulate vaccination response. (<em>b</em>)O<em>b</em>jective:</<em>b</em>) This randomized place<em>b</em>o-controlled dou<em>b</em>le-<em>b</em>lind trial investigated whether Vit-D supplementation in deficient elderly persons could improve influenza seroprotection and immune response. (<em>b</em>)Design:</<em>b</em>) Deficient volunteers (Vit-D serum &<em>lt</em>;30 ng/mL) were assigned (V1) to receive either 100,000 IU/15 days of cholecalciferol (D, <i>n</i> = 19), or a place<em>b</em>o (P, <i>n</i> = 19), over a 3 month period. Influenza vaccination was performed at the end of this period (V2), and the vaccine response was evaluated 28 days later (V3). At each visit, serum cathelicidin, immune response to vaccination, plasma cytokines, lymphocyte phenotyping, and phagocyte ROS production were assessed. (<em>b</em>)Resu<em>lt</em>s:</<em>b</em>) Levels of serum 25-(OH)D increased after supplementation (D group, V1 vs. V2: 20.7 ± 5.7 vs. 44.3 ± 8.6 ng/mL, <i>p</i> &<em>lt</em>; 0.001). No difference was o<em>b</em>served for serum cathelicidin levels, anti<em>b</em>ody titers, and ROS production in D vs. P groups at V3. Lower plasma levels of TNFα (<i>p</i> = 0.040) and IL-6 (<i>p</i> = 0.046), and higher ones for TFGβ (<i>p</i> = 0.0028) were o<em>b</em>served at V3. The Th1/Th2 ratio was lower in the D group at V2 (D: 0.12 ± 0.05 vs. P: 0.18 ± 0.05, <i>p</i> = 0.039). (<em>b</em>)Conclusions:</<em>b</em>) Vit-D supplementation promotes a higher TGFβ plasma level in response to influenza vaccination without improving anti<em>b</em>ody production. This supplementation seems to direct the lymphocyte polarization toward a tolerogenic immune response. A deeper characterization of meta<em>b</em>olic and molecular pathways of these o<em>b</em>servations will aid in the understanding of Vit-D's effects on cell-mediated immunity in aging. This clinical trial was registered at clinica<em>lt</em>rials.gov as NCT01893385.

The effectiveness of COVID-19 vaccination in preventing new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in the general community is still unclear. Here, we used the Office for National Statistics COVID-19 Infection Survey-a large community-based survey of individuals living in randomly selected private households across the United Kingdom-to assess the effectiveness of the BNT162b2 (Pfizer-BioNTech) and ChAdOx1 nCoV-19 (Oxford-AstraZeneca; ChAdOx1) vaccines against any new SARS-CoV-2 PCR-positive tests, split according to self-reported symptoms, cycle threshold value (&lt;30 versus ≥30; as a surrogate for viral load) and gene positivity pattern (compatible with B.1.1.7 or not). Using 1,945,071 real-time PCR results from nose and throat swabs taken from 383,812 participants between 1 December 2020 and 8 May 2021, we found that vaccination with the ChAdOx1 or BNT162b2 vaccines already reduced SARS-CoV-2 infections ≥21 d after the first dose (61% (95% confidence interval (CI) = 54-68%) versus 66% (95% CI = 60-71%), respectively), with greater reductions observed after a second dose (79% (95% CI = 65-88%) versus 80% (95% CI = 73-85%), respectively). The largest reductions were observed for symptomatic infections and/or infections with a higher viral burden. Overall, COVID-19 vaccination reduced the number of new SARS-CoV-2 infections, with the largest benefit received after two vaccinations and against symptomatic and high viral burden infections, and with no evidence of a difference between the BNT162b2 and ChAdOx1 vaccines.

OBJECTIVE

We compared the effects of high-intensity interval (HIT) and moderate-intensity continuous (MIT) training (matched for total work) on changes in repeated-sprint ability (RSA) and muscle metabolism.

METHODS

Pre- and posttraining, VO(2peak), lactate threshold (LT), and RSA (5 x 6-s sprints, every 30 s) were assessed in 20 females. Before and immediately after the RSA test, muscle biopsies were taken from the vastus lateralis. Subjects were matched on RSA, randomly placed into the HIT (N = 10) or MIT (N = 10) group and performed 5 wk (3 d.wk(-1)) of cycle training; performing either HIT (6-10, 2-min intervals at 120-140% LT) or MIT (continuous, 20-30 min at 80-95% LT).

RESULTS

Both groups had significant improvements in VO(2peak) (10-12%; P < 0.05) and LT (8-10%; P < 0.05), with no significant differences between them. Both groups also had significant increases in RSA total work (kJ) (P < 0.05), with a significantly greater increase following HIT than MIT (13 vs 8.5%, respectively; P < 0.05). There was a significant decrease in resting [ATP] and an increase in postexercise [La(-)](b) for both groups, but no significant differences between them. There were no significant changes in resting or postexercise [PCr], [Cr], muscle [La(-)], or [H(+)] after the training period.

CONCLUSIONS

When total work is matched, HIT results in greater improvements in RSA than MIT. This results from an improved ability to maintain performance during consecutive sprints, which is not explained by differences in work done during the first sprint, aerobic fitness or metabolite accumulation at the end of the sprints.

Bacillus anthracis is a Gram-positive spore-forming bacterium that causes anthrax disease in humans and animals. Systemic infection is characterized by septicemia, toxemia, and meningitis, the main neurological complication associated with high mortality. We have shown previously that B. anthracis Sterne is capable of blood-brain barrier (BBB) penetration, establishing the classic signs of meningitis, and that infection is dependent on the expression of both major anthrax toxins, lethal toxin (LT) and edema toxin (ET). Here we further investigate the contribution of the individual toxins to BBB disruption using isogenic toxin mutants deficient in lethal factor, ΔLF, and edema factor, ΔEF. Acute infection with B. anthracis Sterne and the ΔLF mutant resulted in disruption of human brain microvascular endothelial cell (hBMEC) monolayer integrity and tight junction protein zona occludens-1, while the result for cells infected with the ΔEF mutant was similar to that for the noninfected control. A significant decrease in bacterial invasion of BBB endothelium in vitro was observed during infection with the ΔLF strain, suggesting a prominent role for LT in promoting BBB interaction. Further, treatment of hBMECs with purified LT or chemicals that mimic LT action on host signaling pathways rescued the hypoinvasive phenotype of the ΔLF mutant and resulted in increased bacterial uptake. We also observed that toxin expression reduced bacterial intracellular survival by inducing the bulk degradative autophagy pathway in host cells. Finally, in a murine model of anthrax meningitis, mice infected with the ΔLF mutant exhibited no mortality, brain bacterial load, or evidence of meningitis compared to mice infected with the parental or ΔEF strains.

OBJECTIVE

To assess the effect of long-term maintenance of steroids on HCV recurrence after liver transplantation (LT), that is still controversial, a prospective multicentre trial was conducted at the centres of Bologna and Padua, Italy.

METHODS

From September 2002, 47 eligible HCV positive LT recipients were randomized to receive 2 different steroid schedules in association with tacrolimus: group A: rapid tapering and withdrawal 91 days after LT group B: slow tapering and withdrawal 25 months after LT. Thirty-nine patients were assessable: 23 in group A and 16 in group B. Donor and recipient characteristics were similar in the two groups. Median follow-up was 841 days (130-1376). One hundred liver biopsies were performed, and every patient had a biopsy at month 12.

RESULTS

Twenty-two out of 23 (95, 65%) patients in group A and 15 out of 16 (93, 75%) in group B had histologically-confirmed HCV recurrence. Twelve-month histology showed advanced fibrosis (score 3 or 4) in 42.1% of the patients in group A versus 7.6% in group B (P=0.03). One-and 2-year advanced fibrosis-free survival were 65.2 and 60.8 in group A and 93.7% in group B (P=0.03 and =0.02, respectively).

CONCLUSIONS

Slow tapering of steroids reduced the progression of recurrent hepatitis C after LT.

In developing veterinary mucosal vaccines and vaccination strategies, mucosal adjuvants are one of the key players for inducing protective immune responses. Most of the mucosal adjuvants seem to exert their effect via binding to a receptor/or target cells and these properties were used to classify the mucosal adjuvants reviewed in the present paper: (1) ganglioside receptor-binding toxins (cholera toxin, LT enterotoxin, their B subunits and mutants); (2) surface immunoglobulin binding complex CTA1-DD; (3) TLR4 binding lipopolysaccharide; (4) TLR2-binding muramyl dipeptide; (5) Mannose receptor-binding mannan; (6) Dectin-1-binding ss 1,3/1,6 glucans; (7) TLR9-binding CpG-oligodeoxynucleotides; (8) Cytokines and chemokines; (9) Antigen-presenting cell targeting ISCOMATRIX and ISCOM. In addition, attention is given to two adjuvants able to prime the mucosal immune system following a systemic immunization, namely 1alpha, 25(OH)2D3 and cholera toxin.

Tumour necrosis factor (TNF) and related cytokines have been found to alter the phenotype of vascular endothelial cells so as to promote coagulation, inflammation and immunity. We have used recombinant human TNF, lymphotoxin (LT), interleukin 1 alpha (IL-1 alpha) and interleukin 1 beta (IL-1 beta) to study and compare the effects of these molecules on cultured human endothelial cells (HEC). All four mediators cause HEC monolayers to reorganize from an epithelioid to a fibroblastoid morphology. Reorganization is slow (days), reversible upon cytokine withdrawal and enhanced by co-addition of immune interferon. Coincident with morphological change, TNF and LT (but not IL-1 alpha or IL-1 beta) cause a marked increase in HLA-A, B mRNA and antigen expression. TNF and LT also induce a slow increase in the mRNA levels and cell-surface expression of IL-1 species. All four cytokines have been reported to enhance HEC adhesiveness for lymphocytes and inflammatory leucocytes; these changes temporally coincide with a rapid (hours) and sustained increase in expression of intercellular adhesion molecule 1 (ICAM-1), and with a rapid but transient de novo expression of an endothelial-leucocyte adhesion molecule (detected by antibody H4/18), respectively. TNF and LT induce reciprocal tachyphylaxis for the reinduction of H4/18 binding but do not inhibit induction by IL-1 alpha and IL-1 beta; similarly, IL-1 alpha and IL-1 beta induce reciprocal tachyphylaxis but do not inhibit TNF or LT. We have used the binding of H4/18 to explore the mechanism of action of TNF. Tumour-promoting phorbol esters, but not agents which increase cytoplasmic calcium concentrations, were found to induce binding, suggesting a possible involvement of the protein kinase C pathway in the response of HEC to TNF. Cells pretreated for 24 hours with phorbol esters cannot be reinduced to express H4/18 binding by phorbol esters yet retain full responsiveness to TNF. Thus TNF also appears to act on HEC through a pathway independent of protein kinase C activation. Collectively, these effects of TNF and related cytokines may be understood as examples of endothelial cell activation.

Alymphoplasia mice (aly/aly) have been shown to be deficient for a nuclear factor-kappaB-inducing kinase involved in signal transduction of lymphotoxin beta receptor (LT-betaR) and of CD40, resulting in structural defects of secondary lymphoid organs and highly increased susceptibility to viral infections. We analyzed the anti-viral immune response of bone marrow chimeras (BMC) between aly/aly mice and (C57BL/6 x DBA2)F1 mice (BB or T cell defects led to immunodeficiency in aly/aly mice. Transfer of aly/aly bone marrow into B>BB cell reconstitution of recipients. Antiviral cytotoxic T cell (CTL) responses of aly/aly->>BBMC were clearly improved compared to aly/aly mice whereas virus-neutralizing IgG reponses were virtually absent. Therefore, the inefficient CTL response was predominantly caused by the structural defect of secondary lymphoid organs and not by an intrinsic T cell defect. In contrast, B cells of aly/aly origin were unable to undergo isotype switch after viral infections, indicating an intrinsic B cell defect in vivo. Overall, aly/aly mice show the combined immunodeficient phenotype of mice deficient for LT-3R with B cells functionally deficient for CD40.

Bacillus anthracis must overcome host innate immune defences to establish a systemic anthrax infection. This is facilitated in part by lethal toxin (LT), a secreted virulence factor that consists of a cell-binding moiety, protective antigen (PA), and an enzymatic moiety, lethal factor (LF). PA binds cells through protein receptors and mediates the delivery of LF to the cytosol. LF is a protease that cleaves amino-terminal fragments from mitogen-activated protein kinase kinases (MAPKKs), preventing phosphorylation of their downstream targets. Here we report that LT reduces the amount of interleukin (IL)-8 produced and secreted by human endothelial cells. The reduction of IL-8 levels by LT was not attributable to reduced expression from the IL-8 promoter, but resulted from destabilization of IL-8 mRNA. Destabilization by LT was mediated through the 3' untranslated region of the IL-8 transcript and could be mimicked by pharmacological inhibitors of MAPK pathways. LT diminished the induction of IL-8 mRNA and protein by lipopolysaccharide, indicating that the toxin can impair the ability of these cells to initiate an immune response. Destabilization of a cytokine transcript represents a new interference strategy used by either a bacterial or viral pathogen to reduce cytokine expression and may help B. anthracis to evade host immune defences.

The use of mucosally administered killed bacteria or viruses as vaccines has a number of attractive features over the use of viable attenuated organisms, including safety, cost, storage and ease of delivery. Unfortunately, mucosally administered killed organisms are not usually effective as vaccines. The use of LT(R192G), a genetically detoxified derivative of LT, as a mucosal adjuvant enables the use of killed bacteria or viruses as vaccines by enhancing the overall humoral and cellular host immune response to these organisms, especially the Th1 arm of the immune response. With this adjuvant, protective responses equivalent to those elicited by live attenuated organisms can be achieved with killed organisms without the potential side effects. These findings have significant implications for vaccine development and further support the potential of LT(R192G) to function as a safe, effective adjuvant for mucosally administered vaccines. There are a number of unresolved issues regarding the use of LT and CT mutants as mucosal adjuvants. Both active-site and protease-site mutants of LT and CT have been constructed and adjuvanticity reported for these molecules in various animal models and with different antigens. There needs to be a side-by-side comparison of CT, LT, active-site mutants, protease-site mutants and recombinant B subunits regarding the ability to induce specific, targeted immunological outcomes as a function of route of immunization and nature of the co-administered antigen. Those side-by-side comparisons have not been carried out and there is a substantial body of evidence indicating that the outcomes may very well be different. With that information, vaccine strategies could be designed employing the optimum adjuvant/antigen formulation and route of administration for a variety of bacterial and viral pathogens. Also lacking is an understanding of the underlying cellular and intracellular signaling pathways activated by these different molecules and an understanding of the mechanisms of adjuvanticity at the cellular level. These are important issues because they take us beyond the phenomenological observations of "enhanced immunity" to a more clear understanding of the mechanisms of adjuvant activity.

We have studied the pathways of regulation of cytokine and cell cycle control proteins during infection of human B lymphocytes by Epstein-Barr virus (EBV). Among 30 cytokine RNAs analyzed by the RNase protection assay, tumor necrosis factor alpha (TNF-alpha), granulocyte colony-stimulating factor, lymphotoxin (LT), and LTbeta were found to be regulated within 20 h of EBV infection of primary B cells. Similar results were obtained using the estrogen-regulated EBNA-2 cell line EREBLT and TNF-alpha were induced within 6 h of activation of EBNA-2. Expression of Notch also caused an induction of TNF-alpha RNA. The induction of TNF-alpha RNA by EBNA-2 was indirect, and constitutive expression of either LMP-1 or c-myc proteins did not substitute for EBNA-2 in induction of TNF-alpha RNA. Cyclin D2 is also an indirect target of EBNA-2-mediated transactivation. EBNA-2 was found to activate the cyclin D2 promoter in a transient-transfection assay. A mutant of EBNA-2 that does not bind RBP-Jkappa retained some activity in this assay, and activation did not depend on the presence of B-cell-specific factors. Deletion analysis of the cyclin D2 promoter revealed that removal of sequences containing E-box c-myc consensus DNA binding sequences did not reduce EBNA-2-mediated activation of the cyclin D2 promoter in the transient-transfection assay. The results indicate that cytokines are an early target of EBNA-2 and that EBNA-2 can regulate cyclin D2 transcription in EBV-infected cells by mechanisms additional to the c-myc pathway.

OBJECTIVE

To examine the influence of cadence, cycling experience, and aerobic power on delta efficiency during cycling and to determine the significance of delta efficiency as a factor underlying the selection of preferred cadence.

METHODS

Delta efficiency (DE) was determined for 11 trained experienced cyclists (C), 10 trained runners (R), and 10 less-trained noncyclists (LT) at 50, 65, 80, 95, and 110 rpm. Preferred cadence (PC) was determined at 100, 150, and 200 W for C and R and at 75, 100, and 150 W for LT. Gas exchange at each power output (PO) was measured on a separate day, and the five cadences were randomly ordered on each occasion. It was hypothesized that: a) cyclists are most efficient at the higher cadences at which they are accustomed to training and racing, i.e., there will be a trend for DE to increase with increases in cadence; b) cyclists and runners will exhibit similar DE across the range of cadences tested; and c) DE of less-trained subjects will be lower than that of cyclists and runners.

RESULTS

PCs of C and R were similar and did not change appreciably with PO (100 W:C, 95.6 +/- 10.8; R, 92.0 +/- 8.5: 150 W:C, 94.4 +/- 10.3; R, 92.9 +/- 7.8: 200 W:C, 92.2 +/- 7.2; R, 91.8 +/- 7.9 rpm). The PC of LT was significantly lower and decreased with increases in power output (75 W: 80.0 +/- 15.3; 100 W; 77.5 +/- 15.1; 150 W; 69.1 +/- 11.9 rpm). The first hypothesis was rejected because analysis of the cyclists' data alone revealed no systematic increase in DE as cadence was increased [F(4,40) = 0.272, P = 0.894]. Repeated measures ANOVA on all three groups revealed no group x cadence interaction [F(8,112) = 0.589, P = 0.785]. Again there was no systematic effect of cadence on DE [F(4,112) = 1.058, P = 0.381]. The second and third hypotheses were also rejected since there was no group main effect, i.e., DE of cyclists, runners, and less-trained subjects were not significantly different [F(2,28) = 1.397, P = 0.264].

CONCLUSIONS

Pedaling cadence did not have a dramatic effect on DE in any group. Muscular efficiency, as measured indirectly by delta efficiency, appears to remain relatively constant at approximately 24%, regardless of cycling experience or fitness level.

OBJECTIVE

To summarize the annual nationwide survey on fulminant hepatitis (FH) and late-onset hepatic failure (LOHF) between 2004 and 2009 in Japan.

METHODS

The annual survey was performed in a two-step questionnaire process to detail the clinical profile and prognosis of patients in special hospitals.

RESULTS

Four hundred and sixty (n = 227 acute type; n = 233 subacute type) patients had FH and 28 patients had LOHF. The mean age of patients with FH and LOHF were 51.1 ± 17.0 and 58.0 ± 14.4 years, respectively. The causes of FH were hepatitis A virus in 3.0%, hepatitis B virus (HBV) in 40.2%, other viruses in 2.0%, autoimmune hepatitis in 8.3%, drug allergy-induced in 14.6% and indeterminate etiology in 29.6% of patients. HBV reactivation due to immunosuppressive therapy was observed in 6.8% of FH patients. The short-term survival rates of patients without liver transplantation (LT) were 48.7% and 24.2% for the acute and subacute type, respectively, and 13.0% for LOHF. The prognosis was poor in patients with HBV reactivation. The implementation rate for LT in FH patients was equivalent to that in the previous survey. The short-term survival rates of total patients, including LT patients, were 54.2% and 40.8% for the acute and subacute type, respectively, and 28.6% for LOHF.

CONCLUSIONS

The demographic features and etiology of FH patients has gradually changed. HBV reactivation due to immunosuppressive therapy is problematic. Despite advances in therapeutic approaches, the prognosis of patients without LT has not improved.

In the present study phosphodiesterase (PDE) isozymes in human airway smooth muscle were isolated, identified and characterized, and the functional roles of these isozymes in intact bronchi were evaluated by using isozyme-selective PDE inhibitors. PDE isozymes in human trachealis were isolated by using a combination of DEAE-Sepharose and calmodulin-Sepharose affinity chromatography, and were identified based upon their kinetic characteristics as well as their sensitivity to allosteric modulators and isozyme-selective PDE inhibitors. By using this approach, six distinct isozymes were identified: two calmodulin-stimulated PDEs (PDE I alpha and PDE I beta), cyclic GMP (cGMP)-stimulated PDE (PDE II), cGMP-inhibited PDE (PDE III), cyclic AMP (cAMP)-specific PDE (PDE IV) and cGMP-specific PDE (PDE V). PDEs III and IV were the major cAMP-hydrolyzing enzymes present, whereas PDEs I alpha, I beta and V accounted for most of the cGMP-hydrolytic activity. In carbachol-precontracted small (< 0.5-2 mm diameter) or large (4-15 mm diameter) human bronchus, zaprinast (10 nM-30 microM), the selective PDE V inhibitor, was without marked relaxant activity (< 13%), whereas rolipram (30 microM), the selective PDE IV inhibitor, produced approximately 25% relaxation in both preparations. Siguazodan was a significantly more effective relaxant than zaprinast or rolipram in large bronchus, producing a maximum relaxation of 77 +/- 15% at a concentration of 30 microM, whereas in small bronchus 30 microM siguazodan elicited 20 +/- 6% relaxation. Similar results were obtained in large bronchi contracted with leukotriene (LT) D4 (0.1 microM). The ability of isozyme-selective PDE inhibitors to potentiate agonist-induced relaxation was studied in LTD4-contracted large bronchi. Siguazodan (10 microM), but not rolipram (10 microM) or zaprinast (10 microM), potentiated the relaxant response in LTD4-contracted large bronchus to isoproterenol, a beta adrenoceptor agonist thought to induce relaxation via a cAMP-mediated mechanism. In contrast, zaprinast (10 microM), but not siguazodan (10 microM), potentiated relaxation induced by sodium nitroprusside, a nitrovasodilator that relaxes airway smooth muscle via a cGMP-mediated mechanism. The most striking observation from functional studies was that the combination of rolipram and siguazodan produced a much greater relaxation of small or large human bronchi than either agent alone, indicating an interaction between PDE III and PDE IV inhibitors that was at least additive and, in some cases, synergistic.(ABSTRACT TRUNCATED AT 400 WORDS)

Nuclear and mitochondrial genomes combine in ALR/Lt mice to produce systemically elevated defenses against free radical damage, rendering these mice resistant to immune-mediated pancreatic islet destruction. We analyzed the mechanism whereby isolated islets from ALR mice resisted proinflammatory stress mediated by combined cytokines (IL-1beta, TNF-alpha, and IFN-gamma) in vitro. Such damage entails both superoxide and NO radical generation, as well as peroxynitrite, resulting from their combination. In contrast to islets from other mouse strains, ALR islets expressed constitutively higher glutathione reductase, glutathione peroxidase, and higher ratios of reduced to oxidized glutathione. Following incubation with combined cytokines, islets from control strains produced significantly higher levels of hydrogen peroxide and NO than islets from ALR mice. Nitrotyrosine was generated in NOD and C3H/HeJ islets but not by ALR islets. Western blot analysis showed that combined cytokines up-regulated the NF-kappaB inducible NO synthase in NOD-Rag and C3H/HeJ islets but not in ALR islets. This inability of cytokine-treated ALR islets to up-regulate inducible NO synthase and produce NO correlated both with reduced kinetics of IkappaB degradation and with markedly suppressed NF-kappaB p65 nuclear translocation. Hence, ALR/Lt islets resist cytokine-induced diabetogenic stress through enhanced dissipation and/or suppressed formation of reactive oxygen and nitrogen species, impaired IkappaB degradation, and blunted NF-kappaB activation. Nitrotyrosylation of beta cell proteins may generate neoantigens; therefore, resistance of ALR islets to nitrotyrosine formation may, in part, explain why ALR mice are resistant to type 1 diabetes when reconstituted with a NOD immune system.

The common mucosal immune system (CMIS) consists of an integrated cross-communication pathway of lymphoid tissues made up of inductive and effector sites for host protection against pathogenic microorganisms. Major effector molecules of the CMIS include IgA antibodies and cytokines, chemokines and their corresponding receptors. Secretory IgA (S-IgA), the major immunoglobulin, is induced by gut-associated lymphoreticular tissue (GALT)-derived B cells with the help of Th1- and Th2-type CD4(+) T lymphocytes. Cytotoxic T lymphocytes (CTLs) in the mucosal epithelium, a subpopulation of intraepithelial lymphocytes (IELs), also help maintain the mucosal barrier. The CMIS is unique in that it can provide both positive and negative signals for the induction and regulation of immune responses in both the mucosal and systemic compartments after oral or nasal antigen exposure. Prevention of infection through mucosal surfaces can be achieved by the CMIS through connections between inductive (e.g. GALT) and effector tissues. When vaccine antigens are enterically administered together with mucosal adjuvants [e.g. cholera toxin (CT), heat-labile toxin produced by Escherichia coli (LT) and IL-12], antigen-specific Th1/Th2 and IgA B cell responses are induced simultaneously in the mucosal effector compartment. Since these antigen-specific immune responses are not generated by oral vaccine without mucosal adjuvant, safe and effective adjuvants for the induction of antigen-specific S-IgA and CTL responses are essential for the development of mucosal vaccines for protection against infectious diseases. Finally, recent findings suggest the presence of a CMIS-independent IgA induction pathway, which also must be considered in the development of mucosal vaccines.

Aeromonas hydrophila produces two haemolysins, now designated alpha- and beta-haemolysin (formerly aerolysin or cytotoxic protein), and one enterotoxin. These toxins were separated and purified by isoelectric focusing and gel chromatography. Alpha- and beta-haemolysin differ with regard to prerequisites for their elaboration, such as selection of A. hydrophila strains and temperature and time for cultivation. The nature of their lytic effects on erythrocytes and toxic effects on tissue culture cells differs significantly. Alpha- but not beta-haemolysin was inactivated by reducing agents and activated by oxygen, while beta-haemolysin was more resistant than alpha-haemolysin to proteolytic enzymes but was inactivated by crude gangliosides. When separated from the two haemolysins, the enterotoxin gave positive reactions in the rabbit intestinal loop test, the rabbit skin and the adrenal Y1 cell test, though the sensitivity of the Y1 cell was low as compared with cholera toxin and E. coli LT toxin. No cytotoxic effects were obtained in HeLa cells.

Homeostasis at the cellular level appears to be a complex state of equilibrium derived, in part, from multiple interactions among lymphokines, cytokines, growth factors, and hormones. Tumor necrosis factor (TNF-alpha) secreted by mitogen-stimulated macrophages and lymphotoxin (LT, TNF-beta) produced by mitogen-stimulated lymphocytes are two peptide hormones that share considerable homology in amino acid sequence and biological action. It has become increasingly evident that these factors, primarily involved in host defense, also mediate some responses to inflammatory, infectious, and neoplastic disease states. TNF-alpha is a low molecular weight (17,000-kdalton) peptide which seems to have a remarkably broad range of biological and immunologic effects, including antiviral action, growth regulation, and immunomodulation. These effects are shared by other lymphokines and cytokines such as interleukin 1 (IL-1), interferon alpha (IFN-alpha), interferon gamma (IFN-gamma), and TNF-beta. TNF-alpha also demonstrates both positive and negative cooperative growth-regulatory effects alone and in combination with antitumor drugs as well as with other bioactive peptides. Clinical trials are currently under way to determine the effectiveness of TNF-alpha as a single agent and in combination with biologic and chemotherapeutic agents against a variety of neoplastic diseases in man.

The heat-stable enterotoxin B (STB) of Escherichia coli is a 48-amino acid extracellular peptide that induces rapid fluid accumulation in animal intestinal models. Unlike other E. coli enterotoxins that elicit cAMP or cGMP responses in the gut [heat-labile toxin (LT) and heat-stable toxin A (STA), respectively], STB induces fluid loss by an undefined mechanism that is independent of cyclic nucleotide elevation. Here we studied the effects of STB on intracellular calcium concentration ([Ca2+]i), another known mediator of intestinal ion and fluid movement. Ca2+ and pH measurements were performed on different cell types including Madin-Darby canine kidney (MDCK), HT-29/C1 intestinal epithelial cells, and primary rat pituitary cells. Ca2+ and pH determinations were performed by simultaneous real-time fluorescence imaging at four emission wavelengths. This allowed dual imaging of the Ca(2+)- and pH-specific ratio dyes (indo-1 and SNARF-1, respectively). STB treatment induced a dose-dependent increase in [Ca2+]i with virtually no effect on internal pH in all of the cell types tested. STB-mediated [Ca2+]i elevation was not inhibited by drugs that block voltage-gated Ca2+ channels including nitrendipine, verapamil (L-type), omega-conotoxin (N-type), and Ni2+ (T-type). The increase in [Ca2+]i was dependent on a source of extracellular Ca2+ and was not affected by prior treatment of MDCK cells with thapsigargin or cyclopiazonic acid, agents that deplete and block internal Ca2+ stores. In contrast to these results, somatostatin and pertussis toxin pretreatment of MDCK cells completely blocked the STB-induced increase in [Ca2+]i. Taken together, these data suggest that STB opens a GTP-binding regulatory protein-linked receptor-operated Ca2+ channel in the plasma membrane. The nature of the STB-sensitive Ca2+ channel is presently under investigation.

We describe a general method for the analysis of ligand-macromolecule binding equilibria for cases in which the interaction is monitored by a change in a signal originating from the ligand. This method allows the absolute determination of the average degree of ligand binding per macromolecule without any assumptions concerning the number of modes or states for ligand binding or the relationship between the fractional signal change and the fraction of bound ligand. Although this method is generally applicable to any type of signal, we discuss the details of the method as it applies to the analysis of binding data monitored by a change in fluorescence of a ligand upon binding to a nucleic acid. We apply the analysis to the equilibrium binding of Escherichia coli single-strand binding (SSB) protein to single-stranded nucleic acids, which is monitored by the quenching of the intrinsic tryptophan fluorescence of the SSB protein. With this method, one can quantitatively determine the relationship between the fractional signal change of the ligand and the fraction of bound ligand, LB/LT, and rigorously test whether the signal change is directly proportional to LB/LT. For E. coli SSB protein binding to single-stranded nucleic acids in its (SSB)65 binding mode [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Chrysogelos, S., & Griffith, J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5803], we show that the fractional quenching of the SSB fluorescence is equal to the fraction of bound SSB.

The effect of beta-alanine (beta-Ala) alone or in combination with creatine monohydrate (Cr) on aerobic exercise performance is unknown. The purpose of this study was to examine the effects of 4 weeks of beta-Ala and Cr supplementation on indices of endurance performance. Fifty-five men (24.5 +/- 5.3 yrs) participated in a double-blind, placebo-controlled study and randomly assigned to one of 4 groups; placebo (PL, n = 13), creatine (Cr, n = 12), beta-alanine (beta-Ala, n = 14), or beta-alanine plus creatine (CrBA, n = 16). Prior to and following supplementation, participants performed a graded exercise test on a cycle ergometer to determine VO(2peak), time to exhaustion (TTE), and power output, VO(2), and percent VO(2peak) associated with VT and LT. No significant group effects were found. However, within groups, a significant time effect was observed for CrBa on 5 of the 8 parameters measured. These data suggest that CrBA may potentially enhance endurance performance.

Normal human epidermal melanocytes became swollen and more dendritic with an increase in the amount of tyrosinase and immunoreactive b-locus protein when they were cultured for 2 days with the following arachidonic acid metabolites: prostaglandin (PG) D2, leukotriene (LT) B4, LTC4, LTD4, LTE4, thromboxane (TX) B2 and 12-hydroxy eicosatetraenoic acid (12-HETE). The effect of LTC4 was particularly strong compared to that of PGE2, about which we have previously reported. On the other hand, PGE1, PGF2 alpha and 6-ketoPGF1 alpha did not show any significant stimulatory effect. These data suggest that arachidonate-derived chemical mediators, especially LTC4, may be responsible for the induction of post-inflammatory hyperpigmentation of the skin.