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.

Inhibition of cobalt(II) carboxypeptidase A by ligands. Kinetic evidence for the formation of a ternary enzyme-metal-ligand complex.

Saturation kinetics are observed in the inhibition of cobalt carboxypeptidase A by the chelating agent 1,10-phenanthroline. The association constant K1 for the formation of the enzyme-metal-ligand ternary complex and k2, the rate of breakup of the ternary complex, have been obtained. A mechanism is proposed to account for the pH profile of the reaction which, in conjunction with K1, permits the calculation of the individual rate constants k1, k-1, k2, k3. The magnitude of the rate constant k1 suggests that cobalt(II) in CoCPA is five-coordinate. Similar but less extensive studies on inhibition by 2,2'-bipyridyl and 8-hydroquinoline-5-sulfonic acid have also been carried out.

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.

The distribution of Mycobacterium bovis in Queensland cattle herds with observations on the laboratory diagnosis of tuberculosis.

Of 1,791 tissues submitted from 1975 to 1978 for histological examination for tuberculosis, 78.5% were classified as tuberculosis, 8.1% as club-forming granulomas, 3.3% as hydatid cysts, 3.4% as other granulomas and 2.4% as squamous cell carcinomas. Cultural examination of 2,682 tissues for mycobacteria yielded Mycobacterium bovis from 42.88%, other mycobacteria from 7.64% and 0.89% Rhodococcus (Corynebacterium) equi. Of 1,478 tissues on which both examinations were performed, culture and histology results were in agreement on 94.1%. M. Bovis was isolated on one or more occasions from 564 herds with the highest herd prevalence being recorded in western areas of the state. Culture of 1,024 tissues from no visible lesion (NVL) reactors yielded 0.59% M. bovis, 15.04% other mycobacteria and 0.19% R. equi. Of 120 isolates of other mycobacteria from tissues of NVL reactors, 39 were Runyon Group III (20 M.avium complex) and 61 Runyon Group IV. Approximately half of the herds from which NVL tissues were collected were confirmed M. bovis infected properties.

Pathology and tuberculin sensitivity in cattle inoculated with Mycobacterium avium complex serotypes 6, 14 and 18.

Three strains of Mycobacterium avium complex organisms, serotypes 6, 14 and 18 isolated from typical tuberculous lesions in cattle were examined for pathogenicity and ability to sensitise cattle to avian and bovine tuberculin. Each strain caused tuberculoid granulomas at the site of subcutaneous inoculation but no lesions elsewhere. Sensitisation to bovine tuberculin was detected in the caudal fold test in 11 of 18 inoculated animals 8 weeks after injection. In a simultaneous comparative cervical test, reactions to avian tuberculin were much larger than reactions to bovine tuberculin in all inoculated animals.

Bovine melioidosis in South-Eastern Queensland.

Acute fatal infection with Pseudomonas pseudomallei caused pneumonia, placentitis and endometritis in a pregnant cow. Pneumonia was also present in the foetal lung. Mononuclear cell response with extensive karyorrhexis occurred in maternal and foetal lung and the same cellular pathology, but with diffuse distribution, was responsible for plaques on the endometrium. A more chronic disease with encapsulated caseous lesions in the lung, together with arthritis, occurred in a bull on the same property. Nutritional and physical stress caused by a flood were thought to be predisposing factors. An unusually wet summer with prolonged flooding apparently provided suitable environmental conditions for saprophytic growth of Ps. pseudomallei in an area far south of the region in which infection of animals with this organism commonly occur.

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.

Haematological investigation of a multiple case leucosis herd.

Adult cattle in a Queensland dairy herd with a history of deaths from lymphosarcoma were sampled regularly over a 4 year period for the identification of animals with persistent lymphocytosis (PL). Twenty-one of 94 animals that were sampled at least 6 times had PL. At the initial sampling 27% of the animals had lymphocytosis. Culling of haematologically positive animals in the first 18 months of the investigation reduced this to 5.3%, but cessation of the culling programme resulted in a gradual increase in the percentage of animals with lymphocytosis. Four deaths from lymphosarcoma occurred in adult animals, but only in the first 18 months of the investigation. Two of these animals had lymphocytosis and two lymphoblastic leukaemia. The calf of one of the latter cows developed lymphoblastic leukaemia and lymphosarcoma by the time it was 6 months of age. Although histological evidence of lymphosarcoma was lacking in a number of clinically normal animals with lymphocytosis, haematological investigation identified a group of animals within the herd that may develop lymphosarcoma.

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.

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.

Evaluation of an indirect fluorescent antibody test to diagnose Babesia equi infection in horses.

An indirect fluorescent antibody (IFA) test for the diagnosis of Babesia equi infections was evaluated. Antigen prepared by conventional methods was of high quality in one instance and of lesser quality in a second when possible autofluorescence of the horse blood caused inconvenience in reading tests. Tests on 14 horses shown by parasitological means to be either infected (9) or uninfected (5) produced reactions at dilutions of 1/270 to 1/7290 for infected and at 1/10 to 1/90 for uninfected animals. The accuracy of the test was further demonstrated during investigations of 701 horses in 3 states of Australia. The 30 horses reacting at 1/270 to 1/2430 were from 33 imported to 3 different farms in Australia from a common source. Investigations of crossreactivity between B. equi and B. bovis of cattle suggested that B. bovis would not interfere with the test for B. equi, but that the reverse was possible.

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.

Epidemiology and control of anaplasmosis in Australia.

Anaplasmosis occurs in those areas of northern and eastern Australia infested by the cattle tick Boophilus microplus but it has been studied intensively only in Queensland. Anaplasmosis is predominantly a disease of autumn and winter and of cattle greater than 1 year of age. The complement fixation test has been used in serological surveys of the tick-infested areas of the state. Both clinical and subclinical infections occur only in tick-infested areas and they are both more frequent in Bos taurus than in Bos indicus cattle probably due to the greater susceptibility of the former to ticks. Prevalence of infection is significantly greater in cattle exposed to heavy tick infestations than it is in cattle exposed to light tick infestations. B. microplus is considered to be the main vector with transmission being effected by transtadial and intrastadial but not transovarial means. Transtadial transmission by Rhipicephalus sanguineus has been demonstrated but attempts to demonstrate transmission by Haemaphysalis longicornis were unsuccessful. Vaccination with Anaplasma centrale is employed either as a routine preventative measure in young cattle or in the face of an outbreak. Attempts to attenuate a strain of A. marginale by adapting it to sheep were unsuccessful. Oxytetracycline and imidocarb have been used successfully to control the clinical disease.