Sulfatide binding properties of murine and human antiganglioside antibodies

Antiganglioside antibodies form an important component of the innate and adaptive B cell repertoire, where they provide antimicrobial activity through binding encapsulated bacterial glycans. In an aberrant role, they target peripheral nerve gangliosides to induce autoimmune nerve injury. An important characteristic of antiganglioside antibodies is their ability to selectively recognize highly defined glycan structures. Since sialylated and sulfated glycans often share lectin recognition patterns, we here explored the possibility that certain antiganglioside antibodies might also bind 3-O-sulfo-beta-D-galactosylceramide (sulfatide), an abundant constituent of plasma and peripheral nerve myelin, that could thereby influence any immunoregulatory or autoimmune properties. Out of 25 antiganglioside antibodies screened in solid phase assays, 20 also bound sulfatide (10(-5) to 10(-6) M range) in addition to their favored ganglioside glycan epitope ( approximately 10(-7) M range). Solution inhibition studies demonstrated competition between ganglioside and sulfatide, indicating close proximity or sharing of the antigen binding variable region domain. Sulfatide and 3-O-sulfo-beta-D-galactose were unique in having this property amongst a wide range of sulfated glycans screened, including 4- and 6-O-sulfo-beta-D-galactose analogues. Antiganglioside antibody binding to 3-O-sulfo-beta-D-galactose was highly dependent upon the spatial presentation of the ligand, being completely inhibited by conjugation to protein or polyacrylamide (PAA) matrices. Binding was also absent when sulfatide was incorporated into plasma membranes, including myelin, under conditions in which antibody binding to ganglioside was retained. These data demonstrate that sulfatide binding is a common property of antiganglioside antibodies that may provide functional insights into, and consequences for this component of the innate immune repertoire.     

Provision of first contact care out of hours in four urban areas in England.

In a typical two week period in 1984 in three urban areas with general practitioner deputising services roughly 40% of first contact patient encounters out of hours were with hospital accident and emergency departments, and only a quarter were with general practitioner deputising services, although 47%, 64%, and 97% of general practitioners in the areas had permission to use such services. Roughly a third only of the encounters were with the practices themselves, and even fewer occurred overnight (11 pm-7 am). In a fourth urban area where 68% of general practitioners formed an out of hours cooperative rota a third of the encounters were with the accident and emergency department and half (more overnight) were with the rota. The presence of a woman principal in a practice and large partnerships of four principals or more were associated with an increased proportion of encounters with the practice itself. Undue prominence may have been given to the role of deputising services in out of hours care. Paradoxically, the use of general practitioner cooperatives may result in even less personal care being given by the patient''s own practice.     

Assessment of protein stability in cerebrospinal fluid using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry protein profiling

Recent studies have evaluated proper acquisition and storage procedures for the use of serum or plasma for mass spectrometry (MS)-based proteomics. The present study examines the proteome stability of human cerebrospinal fluid (CSF) over time at 23°C (room temperature) and 4°C using surface-enhanced laser desorption/ionization time-of-flight MS. Data analysis revealed that statistically significant differences in protein profiles are apparent within 4 h at 23°C and between 6 and 8 h at 4°C. Inclusion of protease and phosphatase inhibitor cocktails into the CSF samples failed to significantly reduce proteome alterations over time. We conclude that MS-based proteomic analysis of CSF requires careful assessment of sample collection procedures for rapid and optimal sample acquisition and storage.     

The Microaerophilic Microbiota of De-Novo Paediatric Inflammatory Bowel Disease: The BISCUIT Study

Introduction

Children presenting for the first time with inflammatory bowel disease (IBD) offer a unique opportunity to study aetiological agents before the confounders of treatment. Microaerophilic bacteria can exploit the ecological niche of the intestinal epithelium; Helicobacter and Campylobacter are previously implicated in IBD pathogenesis. We set out to study these and other microaerophilic bacteria in de-novo paediatric IBD.

Patients and Methods

100 children undergoing colonoscopy were recruited including 44 treatment naïve de-novo IBD patients and 42 with normal colons. Colonic biopsies were subjected to microaerophilic culture with Gram-negative isolates then identified by sequencing. Biopsies were also PCR screened for the specific microaerophilic bacterial groups: Helicobacteraceae, Campylobacteraceae and Sutterella wadsworthensis.

Results

129 Gram-negative microaerophilic bacterial isolates were identified from 10 genera. The most frequently cultured was S. wadsworthensis (32 distinct isolates). Unusual Campylobacter were isolated from 8 subjects (including 3 C. concisus, 1 C. curvus, 1 C. lari, 1 C. rectus, 3 C. showae). No Helicobacter were cultured. When comparing IBD vs. normal colon control by PCR the prevalence figures were not significantly different (Helicobacter 11% vs. 12%, p = 1.00; Campylobacter 75% vs. 76%, p = 1.00; S. wadsworthensis 82% vs. 71%, p = 0.312).

Conclusions

This study offers a comprehensive overview of the microaerophilic microbiota of the paediatric colon including at IBD onset. Campylobacter appear to be surprisingly common, are not more strongly associated with IBD and can be isolated from around 8% of paediatric colonic biopsies. S. wadsworthensis appears to be a common commensal. Helicobacter species are relatively rare in the paediatric colon.

Trial Registration

This study is publically registered on the United Kingdom Clinical Research Network Portfolio (9633).     

Differences in durations of stay for surgery in the NHS and private sector in England and Wales.

Median durations of stay in England and Wales for eight "marker" operations were consistently shorter for patients in pay beds in the National Health Service than for patients in independent acute hospitals or public sector beds. This pattern was seen for both preoperative and postoperative stay and among specific age groups. Differential use of other hospitals for part of the period of care was not a factor, except for patients having hip replacement operations treated in NHS pay beds, one in seven of whom appeared to transfer to public sector care postoperatively. These findings suggest that there is scope for a possible further reduction in durations of stay in public sector beds and, within the private sector, for an assessment of whether spells in independent hospitals need be longer than in NHS pay beds.     

Patient characteristics and clinical caseload of short stay independent hospitals in England and Wales, 1992-3.

OBJECTIVE--To describe and quantify the patients and clinical activities of independent short stay hospitals. DESIGN--Retrospective survey of hospital records for sampled periods of one financial year and comparison with data from 1981 to 1986. SETTING--217 independent hospitals in England and Wales, 1992-3. MAIN OUTCOME MEASURES--Distributions of sex, age groups, and areas of residence of patients, clinical procedures, financial provision. RESULTS--Data were obtained from 201 (93%) hospitals. An estimated 429,172 inpatients (7% more than 1986) and 249,531 day cases (an increase of 154%) from 1986 were treated in the year. The number of overseas patients was half that in 1986. Clinical case mix remained similar to 1986. Abortion remained the commonest procedure (13% v 19% in 1986). Lens operations, heart operations, endoscopies, and non-surgical cases showed the largest increases from 1986. Proportionately more overseas patients had abortions (30% v 12% for England and Wales residents) and they received 41% of coronary artery bypass grafting. Three quarters of the patients were aged 15-64. The proportion of patients aged over 65 had changed little (19% v 17% in 1986). Estimated average bed occupancy was only 48%. Only one in 20 patients was treated under NHS contract; 90% of episodes were funded through private health insurance. CONCLUSIONS--The demand for treatment in private hospitals continues to increase despite additional investment in the NHS, but the overseas market is falling. Overall, the range of clinical activity has changed little.     

Induction of cellular stress overcomes the requirement of herpes simplex virus type 1 for immediate-early protein ICP0 and reactivates expression from quiescent viral genomes

Herpes simplex virus type 1 (HSV-1) mutants impaired in the activities of the structural protein VP16 and the immediate-early (IE) proteins ICP0 and ICP4 establish a quiescent infection in human fibroblasts, with most cells retaining an inactive, repressed viral genome for sustained periods in culture. To date, the quiescent state has been considered stable, since it has been reversed only by provision of herpesviral proteins, such as ICP0, not by alteration of the cell physiological state. We report that the interaction of HSV-1 with human fibroblasts can be altered significantly by transient treatment of cultures with sodium arsenite, an inducer of heat shock and oxidative stress, or gramicidin D, a toxin that selectively permeabilizes cell membranes, prior to infection. These regimens stimulated gene expression from IE-deficient HSV-1 mutants in a promoter sequence-independent manner and also overcame the replication defect of ICP0-null mutants. Reactivation of gene expression from quiescent HSV-1 genomes and the resumption of virus replication were observed following addition of arsenite or gramicidin D to cultures. Both agents induced reorganization of nuclear domain 10 structures, the sites of quiescent genomes, but appeared to do so through different mechanisms. The results demonstrate that the physiological state of the cell is important in determining the outcome of infection with IE-deficient HSV-1 and show novel methods for reactivating quiescent HSV-1 in fibroblasts with a high efficiency.     

Trans-generational specificity within a cnidarian–algal symbiosis

Ocean warming and other anthropogenic stresses threaten the symbiosis between tropical reef cnidarians and their dinoflagellate endosymbionts (Symbiodinium). Offspring of many cnidarians acquire their algal symbionts from the environment, and such flexibility could allow corals to respond to environmental changes between generations. To investigate the effect of both habitat and host genotype on symbiont acquisition, we transplanted aposymbiotic offspring of the common Caribbean octocoral Briareum asbestinum to (1) an environmentally different habitat that lacked B. asbestinum and (2) an environmentally similar habitat where local adults harbored Symbiodinium phylotypes that differed from parental colonies. Symbiont acquisition and establishment of symbioses over time was followed using a within-clade DNA marker (23S chloroplast rDNA) and a within-phylotype marker (unique alleles at a single microsatellite locus). Early in the symbiosis, B. asbestinum juveniles harbored multiple symbiont phylotypes, regardless of source (parent or site). However, with time (~4 yr), offspring established symbioses with the symbiont phylotype dominant in the parental colonies, regardless of transplant location. Within-phylotype analyses of the symbionts revealed a similar pattern, with offspring acquiring the allelic variant common in symbionts in the parental population regardless of the environment in which the offspring was reared. These data suggest that in this host species, host–symbiont specificity is a genetically determined trait. If this level of specificity is widespread among other symbiotic cnidarians, many cnidarian–algal symbioses may not be able to respond to rapid, climate change-associated environmental changes by means of between-generation switching of symbionts.