Analysis of the B cell receptor repertoire in six immune-mediated diseases.

B cells are important in the pathogenesis of many, and perhaps all, immune-mediated diseases. Each B cell expresses a single B cell receptor (BCR)1, and the diverse range of BCRs expressed by the total B cell population of an individual is termed the 'BCR repertoire'. Our understanding of the BCR repertoire in the context of immune-mediated diseases is incomplete, and defining this could provide new insights into pathogenesis and therapy. Here, we compared the BCR repertoire in systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Crohn's disease, Behçet's disease, eosinophilic granulomatosis with polyangiitis, and immunoglobulin A (IgA) vasculitis by analysing BCR clonality, use of immunoglobulin heavy-chain variable region (IGHV) genes and-in particular-isotype use. An increase in clonality in systemic lupus erythematosus and Crohn's disease that was dominated by the IgA isotype, together with skewed use of the IGHV genes in these and other diseases, suggested a microbial contribution to pathogenesis. Different immunosuppressive treatments had specific and distinct effects on the repertoire; B cells that persisted after treatment with rituximab were predominately isotype-switched and clonally expanded, whereas the inverse was true for B cells that persisted after treatment with mycophenolate mofetil. Our comparative analysis of the BCR repertoire in immune-mediated disease reveals a complex B cell architecture, providing a platform for understanding pathological mechanisms and designing treatment strategies.

Effects of supervised and unsupervised physical activity programmes for weight loss.

OBJECTIVES: Physical activity is important for weight management. However, it remains unclear what type of physical activity prescription/programme is optimal for increasing physical activity during a standard behavioural weight loss intervention. This study examined changes in physical activity after a 12-week supervised programme prescribed in minutes per week (SUP-PA), an unsupervised programme prescribed in minutes per week (UNSUP-PA) and an unsupervised programme prescribed in steps per day (STEP). METHODS: Fifty-two adults who were overweight or obese (age: 43.5 ± 10.1 years, BMI: 31.5 ± 3.5 kg·m-2) were randomized to STEP (n = 18), UNSUP-PA (n = 17) and SUP-PA (n = 17). Subjects attended weekly in-person group intervention sessions and were prescribed a calorie-restricted diet (1,200-1,800 kcals·day-1) combined with increased physical activity (150 min·week-1 or 10,000 steps·day-1 with 2,500 brisk steps·day-1). RESULTS: All three groups significantly increased moderate-to-vigorous physical activity (STEP: 80.6 ± 218.5 min·week-1, UNSUP-PA: 112.9 ± 180.4 min·week-1 and SUP-PA: 151.1 ± 174.0 min·week-1, p < 0.001) with no differences between groups (p = 0.94) or group by time interaction (p = 0.81). In addition, there were no significant differences in weight loss between the groups (p = 0.81). CONCLUSIONS: In this short-term study, all three physical activity programmes increased physical activity and elicited modest weight loss when combined with a standard behavioural weight loss intervention.

Applying a technology-based system for weight loss in adults with obesity.

OBJECTIVE: The aim of this study was to compare an in-person, group-based behavioral weight loss intervention to technology-based interventions in adults with obesity. METHODS: Adults (N = 39; body mass index: 39.5 ± 2.8 kg m-2; age: 39.9 ± 11.5 years) participated in a 6-month program with randomization to one of three intervention groups: standard behavioral weight loss, a technology-based system combined with a monthly intervention telephone call (TECH) or an enhanced technology-based system combined with a monthly intervention telephone call (EN-TECH). All groups were prescribed an energy-restricted diet and physical activity. Assessments occurred at 0, 3 and 6 months. Separate mixed-effects models using unstructured dependence structure were fit to the outcomes. RESULTS: Weight loss (least square means ± standard error) at 6 months was -6.57 ± 1.65 kg in standard behavioral weight loss, -5.18 ± 1.72 kg in TECH and -6.25 ± 1.95 kg in EN-TECH (p-value for time effect ≤ 0.0001). A similar pattern was observed for change in body mass index, waist circumference and percent body fat. There was a decrease in total energy intake (p = 0.0005) and percent dietary fat intake (p = 0.0172), and physical activity increased (p = 0.0003). CONCLUSIONS: Findings provide initial information on the use of technology-based interventions that include wearable devices combined with brief monthly telephone calls for weight loss in adults with obesity.

Eye on the B-ALL: B-cell receptor repertoires reveal persistence of numerous B-lymphoblastic leukemia subclones from diagnosis to relapse.

The strongest predictor of relapse in B-cell acute lymphoblastic leukemia (B-ALL) is the level of persistence of tumor cells after initial therapy. The high mutation rate of the B-cell receptor (BCR) locus allows high-resolution tracking of the architecture, evolution and clonal dynamics of B-ALL. Using longitudinal BCR repertoire sequencing, we find that the BCR undergoes an unexpectedly high level of clonal diversification in B-ALL cells through both somatic hypermutation and secondary rearrangements, which can be used for tracking the subclonal composition of the disease and detect minimal residual disease with unprecedented sensitivity. We go on to investigate clonal dynamics of B-ALL using BCR phylogenetic analyses of paired diagnosis-relapse samples and find that large numbers of small leukemic subclones present at diagnosis re-emerge at relapse alongside a dominant clone. Our findings suggest that in all informative relapsed patients, the survival of large numbers of clonogenic cells beyond initial chemotherapy is a surrogate for inherent partial chemoresistance or inadequate therapy, providing an increased opportunity for subsequent emergence of fully resistant clones. These results frame early cytoreduction as an important determinant of long-term outcome.

Tumor necrosis factor-alpha selectively induces MnSOD expression via mitochondria-to-nucleus signaling, whereas interleukin-1beta utilizes an alternative pathway.

Mitochondrial levels of the anti-oxidant enzyme, manganese superoxide dismutase (MnSOD), are dramatically elevated in response to stimulation with tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and lipopolysaccharide (LPS). However, the precise intracellular signaling pathways responsible for this inducible expression are poorly understood. MnSOD expression in pulmonary epithelial and endothelial cells, treated with inflammatory mediators and various inhibitors, was studied by Northern analysis. The mitochondrial electron transport chain inhibitors, antimycin A and myxothiazol, selectively blocked TNF-alpha-inducible expression of MnSOD but not that of IL-1beta or LPS, indicating different signaling pathways. N-Acetylcysteine could reliably decrease inducible MnSOD expression by TNF-alpha, but not IL-1, linking reactive oxygen species (ROS) to the TNF-alpha signaling pathway. Elevated levels of arachidonic acid have been demonstrated previously to generate mitochondrial ROS. A specific cytoplasmic phospholipase A(2) inhibitor reduced stimulated MnSOD expression by TNF-alpha, but not by IL-1beta, further supporting the role of ROS. Other investigators have shown that MnSOD expression may be regulated by NF-kappaB. Our results with a specific inhibitory kappa-kinase inhibitor indicate that NF-kappaB modulates IL-1beta signaling but not the TNF-alpha pathway. Thus, we have demonstrated that although inducible MnSOD transcription may appear similar at the messenger RNA level, the intracellular signaling pathways are differentially regulated.

Cytokine-inducible enhancer with promoter activity in both the rat and human manganese-superoxide dismutase genes.

Diverse pro-inflammatory mediators regulate transcription of the gene (MnSOD) encoding the mitochondrial anti-oxidant protein manganese-superoxide dismutase. Understanding the regulation of this gene is crucial to comprehending its role in cytoprotection. In transfected lung epithelial cells, a human-growth-hormone reporter gene system was utilized to identify a potential enhancer in the MnSOD genomic fragment previously shown to contain multiple DNase-I-hypersensitive sites. Northern analysis demonstrated a 10-20-fold increase in response to pro-inflammatory mediators. Inclusion of the MnSOD genomic fragment in reporter constructs was necessary to mimic these stimulus-dependent endogenous levels. The inducible enhancer element was localized to a 260 bp fragment in intron 2, coinciding with a previously defined DNase-I-hypersensitive site. This element functions in an orientation- and position-independent manner as well as with the heterologous thymidine kinase promoter. In addition, we have demonstrated that a homologous sequence within the human MnSOD gene exhibits identical enhancer activity. A novel characteristic of the rat and human enhancer elements involves the ability to promote cytokine-inducible transcription in the absence of a classical promoter.

In vivo architecture of the manganese superoxide dismutase promoter.

Mitochondrial manganese superoxide dismutase (Mn-SOD) is the primary cellular defense against damaging superoxide radicals generated by aerobic metabolism and as a consequence of inflammatory disease. Elevated expression of Mn-SOD therefore provides a potent cytoprotective advantage during acute inflammation. Mn-SOD contains a GC-rich and TATA/CAAT-less promoter characteristic of a housekeeping gene. In contrast, however, Mn-SOD expression is dramatically regulated in a variety of cells by numerous proinflammatory mediators, including lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1. To understand the underlying regulatory mechanisms controlling Mn-SOD expression, we utilized DNase I-hypersensitive (HS) site analysis, which revealed seven hypersensitive sites throughout the gene. Following high resolution DNase I HS site analysis, the promoter was found to contain five HS subsites, including a subsite that only appears following stimulus treatment. Dimethyl sulfate in vivo footprinting identified 10 putative constitutive protein-DNA binding sites in the proximal Mn-SOD promoter as well as two stimulus-specific enhanced guanine residues possibly due to alterations in chromatin structure. In vitro footprinting data implied that five of the binding sites may be occupied by a combination of Sp1 and gut-enriched Kr uppel-like factor. These studies have revealed the complex promoter architecture of a highly regulated cytoprotective gene.

Infection of humans and horses by a newly described morbillivirus.

OBJECTIVE: To describe the clinical and epidemiological features of an outbreak of a viral infection affecting humans and horses. SETTING: Stables in Hendra, a suburb of Brisbane. SUBJECTS: Affected horses and humans, and at-risk human contacts. RESULTS: A pregnant mare died two days after arrival from a paddock elsewhere in Brisbane. Eight to 11 days later, illness (depression, anorexia, fever, dyspnoea, ataxia, tachycardia, tachypnoea and nasal discharge) was reported among 17 other horses from the same or an adjoining stable. Fourteen horses died or were put down. Five and six days after the index mare's death, a stable-hand and then a horse-trainer, both of whom had had close contact with the sick mare's mucous secretions, developed influenza-like illnesses. The stable-hand recovered but the trainer developed pneumonitis, respiratory failure, renal failure and arterial thrombosis, and died from a cardiac arrest seven days after admission to hospital. A morbillivirus cultured from his kidney was identical to one isolated from the lungs of five affected horses. The two affected humans and eight other horses were seropositive for the infection, which was reproduced in healthy horses following challenge by spleen/lung homogenates from infected horses. There was no serological evidence of infection in 157 humans who had had contact with the stables or the sick horses or humans. CONCLUSIONS: A previously undescribed morbillivirus infected a probable 21 horses and two humans; one human and 14 horses died. That no further cases were detected among humans suggests that the virus was of low infectivity. The source of infection remains undetermined.

The comparative pathogenicity of four serovars of Actinobacillus (Haemophilus) pleuropneumoniae.

The pathogenicity of 2 isolates of each of serovars 7, 3, 1 and 2 of Actinobacillus pleuropneumoniae was tested by intranasal inoculation into 60, 6-week-old large white pigs. Four dose rates varying from 0.27 to 560 x 10(6) organisms per pig with 10-fold serial dilutions were used. Surviving pigs were necropsied 7 days after inoculation. The proportion of pigs dying and developing gross lesions following infection was significantly greater for pigs given serotype 1 than for each of the other 3 serotypes, which did not differ significantly from each other. Twelve of 16 pigs given either of the 2 isolates of serovar 1 died after acute illness and 1 of 44 pigs given either of the 2 isolates each of serovars 7, 3 and 2 died. Pigs given serovar 1 showed high temperatures, severe respiratory distress, frothy haemorrhagic nasal discharge and weight loss. Lung lesions were produced in all 16 pigs given serovar 1, in 7 of 14 pigs given serovar 7, 7 of 14 pigs receiving serovar 3 and in 5 of 16 pigs given serovar 2. The lethal infections were characterised by a severe acute fibrinohaemorrhagic necrotising pleuropneumonia, whereas non-lethal cases had lung lesions ranging from necrotising purulent pleuropneumonia to abscessation. Significant differences between isolates in proportions of tissues culture positive for A. pleuropneumoniae for serovars 7 and 2, but not for serovars 3 and 1 suggested that isolates may vary in virulence within serovars, but more detailed studies are needed to clarify this point.

An evaluation of three serological tests for antibody to Brucella suis in pigs.

The Rose Bengal Plate Agglutination test (RBT), the complement fixation text (CFT) and the tube agglutination test (TAT) were applied to serums from 345 feral and 80 domestic pigs sampled at slaughter. At least 2 of the 3 serological tests were applied to each serum. Tissues from all pigs were cultured for Brucella suis and the degree of culture effort was categorised from 1 to 4 in decreasing order. Fifty-eight feral and 35 domestic pigs were culture-positive. A greater proportion of culture-positive pigs was obtained for category 1 and 2 culture effort. Tissues yielding B. suis most often were mandibular, gastrohepatic and external iliac lymph nodes, spleen and various abdominal organs. Infection in domestic pigs was associated with exposure to feral pigs. The sensitivity (Se) in culture-positive pigs of the RBT (79.1%) was significantly greater than that of either the CFT (49.1%) or TAT (51.1%). The specificities (Sp) in culture-negative pigs were 81.2% for the RBT, 90.8% for the CFT and 81.0% for the TAT. A more realistic estimate of Sp for the RBT was considered to be 97.6%, based on serological results from 31,326 domestic pigs routinely tested for regulatory purposes. The RBT was clearly superior to the other 2 tests in this study. However, a more sensitive screening test would be preferable for use in a test and slaughter eradication program. The RBT would be a suitable confirmatory test.

National alpine ski team career and education survey.

Past and current National Alpine Ski Team (NAST) members were surveyed via a mailed questionnaire regarding their educational progress and attainment, career path, parental education and income, as well as their perceptions as to the effect of skiing on dimensions of personal development. The questionnaire was completed by 86% of the total possible (64 of 74) respondents. Although it was found that educational progress was retarded by involvement in elite level skiing, this conclusion could only be drawn if it was assumed that in skiers between 13 and 21 years of age, each one year change in chronological age is 'normally' accompanied by similar academic progress. It was found that upon retirement from NAST 78% of the skiers continued their formal education, with a high level of success. These results were compared to other studies concerning educational attainment.

Bovine leucosis virus contamination of a vaccine produced in vivo against bovine babesiosis and anaplasmosis.

Contamination of a batch of tick fever (babesiosis and anaplasmosis) vaccine with bovine leucosis virus (BLV) was detected when a herd, in the final stages of an enzootic bovine leucosis (EBL) accreditation program, developed a large number of seropositive cattle following use of tick fever vaccine. Investigations incriminated a single calf used to produce Anaplasma centrale vaccine from which 13,959 doses were distributed. The failure of this calf to give a positive agar gel immunodiffusion (AGID) test before use was not fully explained. A total of 22,627 cattle from 111 herds receiving contaminated vaccine was tested to validate claims for compensation. Results showed infection rates of 62% and 51.8% in vaccinated dairy and beef cattle, respectively, compared with 6.1% and 1.5% in non-vaccinated cattle in the same herds. The results also indicated that infection did not spread from vaccinated to non-vaccinated in-contact cattle. Heavy reliance is now placed on purchase of calves for vaccine production from EBL accredited-free herds and on transmission tests from the calves to sheep to prevent a recurrence of contamination. The need for a BLV antigen detection test, with the sensitivity of the sheep transmission test but simpler and faster to perform, is evident.

A Clostridium botulinum type B vaccine for prevention of shaker foal syndrome.

A toxoid was prepared from type B toxin of Clostridium botulinum by treatment with 0.6% formalin for 6 weeks. The toxoid was adsorbed to aluminium hydroxide and this vaccine was evaluated for safety in guinea pigs, mice and horses, and for immunogenicity in guinea pigs and horses. Neutralising antitoxin was demonstrated in adult horses receiving two 2 ml subcutaneous doses 6 weeks apart, and in a foal which suckled its vaccinated dam. Another vaccinated mare and the passively immunised foal were protected against subcutaneous injection of 1600 and 2000 mouse lethal doses of toxin per kg respectively.

Theophylline absorption from two sustained-release products. Implications for therapeutic drug monitoring.

Theophylline absorption from 2 sustained-release theophylline (S-RT) formulations was examined over 2 consecutive days during continuous therapy in 8 clinically stable hospitalized patients with moderate to severe asthma. Theo-24, a formulation intended for once-daily dosing, produced larger fluctuations in serum theophylline concentration (STC) than did twice-daily Theo-Dur (214 +/- 106% versus 89 +/- 33%, p less than 0.02). Bioavailability for both formulations was essentially complete over 2 consecutive study days (95 +/- 22% and 87 +/- 8% for Theo-Dur versus 88 +/- 21% and 87 +/- 24% for Theo-24 on Days 1 and 2, respectively). The time of maximal STC (Tmax) and minimal STC (Tmin) over a 24-h period were relatively predictable for Theo-24 with Tmax 6 to 14 h postdose, and Tmin occurring at the time of dose. However, only Tmax (4 to 10 h post-A.M. dose) was predictable for Theo-Dur. The mean maximal STC over a 24-h period (Cmax) for Theo-Dur was 13.9 (range, 8.9 to 20.7 micrograms/ml), whereas the mean 6-h post-AM dose STC was 13.0 (range, 8.0 to 20.7 micrograms/ml), indicating that the STC at this 6-h time point represents a very close estimate of the true Cmax. Similarly, the mean Cmax for Theo-24 was 14.5 (range, 6.2-20.4 micrograms/ml), and the mean 10-h post-dose STC was 13.7 (range, 3.6 to 20.4 micrograms/ml), suggesting that this time point approximates the true Cmax.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in the lymphoproliferative response of cattle and sheep to bovine leucosis virus infection.

Lymphoblastic leukaemia, preceded by a significantly increasing percentage of prolymphocytes in peripheral blood smears for from 12 to 68 weeks before death was a feature of sheep which developed lymphosarcoma following inoculation with the Australian strain of bovine leucosis virus (BLV). Lymphocytosis and/or the appearance of immature cells were a reliable predictor of tumour formation in sheep, but not in cattle. There was a terminal lymphoblastic leukaemia in only 43 of 84 cattle with lymphosarcoma. Differences in the morphological appearance and glycogen content of the leukaemic lymphoblasts of sheep and cattle were observed. In spite of these differences the high frequency of lymphocytosis and lymphosarcoma in experimentally infected sheep suggests that they could be a useful model for studying the pathological and immunological responses to BLV infection.

Inconsistent absorption from a sustained-release theophylline preparation during continuous therapy in asthmatic children.

During routine monitoring of hospitalized children with asthma receiving a sustained-release theophylline formulation, we frequently observe unpredictable fluctuations in serum theophylline concentration (STC). We evaluated eight asthmatic patients (ages 4 to 17 years) with inconsistent STCs to determine the cause of this phenomenon. Only minimal variation in STC and therefore theophylline clearance was noted during a 24-hour period of continuous intravenous aminophylline infusion. However, marked variability in STC was observed when measured every 2 hours for 48 hours after 6 days of continuous therapy orally. In addition, the time required to reach peak and trough STCs varied from dose to dose. Inasmuch as clearance was shown to be constant, the variability was attributed to inconsistent theophylline absorption. Unpredictable fluctuations of STC secondary to variable absorption from this sustained-release theophylline preparation may occur in certain patients. Appreciation of this potential variability is necessary for the proper interpretation of STC measurements and subsequent dosage adjustment.