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The alternative (noncanonical) nuclear factor-κB (NF-κB) signaling pathway predominantly regulates the function of the p52/RelB heterodimer. GermlineiNfkb/i2 deficiency in mice leads to loss of p100/p52 protein and offers protection against a variety of gastrointestinal conditions, including azoxymethane/dextran sulfate sodium (DSS)-induced colitis-associated cancer and lipopolysaccharide (LPS)-induced small intestinal epithelial apoptosis. However, the common underlying protective mechanisms have not yet been fully elucidated. We applied high-throughput RNA-Seq and proteomic analyses to characterize the transcriptional and protein signatures of the small intestinal mucosa of naïve adultiNfkb2/isup-/-/supmice. Those data were validated by immunohistochemistry and quantitative ELISA using both small intestinal tissue lysates and serum. We identified a B-lymphocyte defect as a major transcriptional signature in the small intestinal mucosa and immunoglobulin A as the most downregulated protein by proteomic analysis iniNfkb2/isup-/-/supmice. Small intestinal immunoglobulins were dramatically dysregulated, with undetectable levels of immunoglobulin A and greatly increased amounts of immunoglobulin M being detected. The numbers of IgA-producing, cluster of differentiation (CD)138-positive plasma cells were also reduced in the lamina propria of the small intestinal villi ofiNfkb2/isup-/-/supmice. This phenotype was even more striking in the small intestinal mucosa ofiRelB/isup-/-/supmice, although these mice were equally sensitive to LPS-induced intestinal apoptosis as theiriRelB/isup+/+/supwild-type counterparts. NF-κB2/p52 deficiency confers resistance to LPS-induced small intestinal apoptosis and also appears to regulate the plasma cell population and immunoglobulin levels within the gut.bNEWamp; NOTEWORTHY/bNovel transcriptomic analysis of murine proximal intestinal mucosa revealed an unexpected B cell signature iniNfkb2/isup-/-/supmice. In-depth analysis revealed a defect in the CD38+ B cell population and a gut-specific dysregulation of immunoglobulin levels.

Aim To improve the quality and completion of consent forms for patients undergoing emergency laparotomy. Methods A standard for emergency laparotomy consent forms, guided by recommendations from the National Emergency Laparotomy Audit (NELA) was established. This standard was used to score consent forms. Our initial audit of 20 consent forms scored an average of 41.9%. A template sticker, specific to emergency laparotomy, was created. This contains all of the information deemed important by our standard. The sticker allows the surgeon completing the consent process to simply tick off the individual components once discussed with the patient and apply it to the regular consent form. The stickers were introduced to all of those involved in consenting patients, and their use was strongly encouraged. Results Consent forms were collected and scored over the following 2-months. Of the 18 consent forms, 33% made use of the consent form sticker, with an average score of 65.2% (improvement of 23.3%). In analysing scores for both the pre- and post-intervention consent forms, we found a P value of < 0.0001. Conclusions The use of consent templates specific to surgical procedures improves the quality of consent forms and ensures that all possible complications are discussed with patients. The use of stickers for emergency laparotomy has improved the quality of our consent forms. With ongoing use, we aim to ensure that all patients undergoing this procedure are adequately consented. There remains room for continued improvement, and we believe ongoing exposure to these stickers will lead to an up-take in use.

Aim To assess consent forms for appendicectomy and ensure that patients are consented for an intraoperative finding of a normal appendix. Method Retrospective audit assessing consent forms (for mention of Normal appendix finding) for patients who underwent appendicectomy over a 2-month period. Results A total of 68 patients underwent appendicectomy. The age range of these patients was 3 to 84 years old (median 20 years). 58 of these procedures were performed laparoscopically. 23 (33.8%) of the consent forms have mention of a normal appendix. The remaining 45 patients (66.2%) had no mention of normal appendix on the consent form. In analysis of the operative notes, 7 patients had no intraoperative finding of macroscopic inflammation. Of these 7 patients, only 1 was consented for a normal appendix finding. Conclusion During the consent process for an appendicectomy, it is important to warn the patient (or caregiver) about the possibility of finding a normal appendix. The appendix is routinely removed regardless of the intraoperative findings (normal or inflamed). The patient should be made aware, as the appendicectomy may not result in the resolution of their symptoms, or further procedures may be required. This audit found that there is currently a deficit in consenting patients, undergoing appendicectomy, about the possibility of finding a normal appendix intraoperatively. A quality improvement project is currently underway, wherein a combination of departmental education and patient information booklets aims to improve consenting practices.

Emerging high-content data modalities like functional genomics and spatial proteomics have enormous potential to reveal determinants of phenotypic plasticity that underlie variability in clinical outcomes, but to date these modalities are only collected in modestly sized research cohorts (< 200-400 patients), where we lack power to detect subtype-specific or prognostic signatures. To study intertumor heterogeneity on a much larger scale (>10,000 patients), we developed a machine learning framework based on self-supervised embeddings that allows scalable imputation of high-content data on large standard of care datasets. Our framework starts by learning a phenotypic embedding of tumor state based solely on H&E histology images, allowing the embedding to be trained on large patient cohorts regardless of availability of molecular covariates. It then learns to predict genomic or proteomic labels from the lower-dimensional phenotypic embeddings. This model can be used for imputation in much larger cohorts, where only clinical outcome and histology are available. To demonstrate our method, we use the TCGA ATAC-seq data, which is available for 400 patients across 23 cancer types. By learning self-supervised embeddings of histology, our framework was able to impute ATAC-seq for 5,000 peaks in 11,000 patients across 31 cancer types, with high accuracy in held out samples (R2 = 0.61). To our knowledge, this represents the broadest available pan-cancer chromatin landscape. The imputed ATAC-seq reveals a subset of peaks that are significantly associated with overall survival (OS) in multiple cancer types (e.g., Breast (BC) ATAC-only HR 1.75, p: 8.6E-3). Genes proximal to these peaks are strongly enriched for well characterized oncogenes, and also several novel genes with functions in cellular metabolism and chromatin remodeling whose expression is not known to be prognostic in our disease settings. Finally, we developed models to predict OS from H&E slide embeddings and from imputed ATAC-seq, both pan-cancer and in specific tumor types. Both models significantly outperform baseline stage and molecular subtype clinical risk predictors (e.g., BC baseline HR: 2.44, p: 3E-6 vs. embedding/imputed ATAC HR 3.78, 2E-9, p for improvement 2E-9) and, interestingly, we find that adding an ATAC-seq based risk score to an embedding-based risk score significantly improves disease-specific survival prediction (HCC embedding-only HR: 2.13, p: 8E-5 vs. HCC embedding/imputed ATAC HR: 2.65, p: 1E-6). This suggests that histopathology images are a rich source of prognostic information beyond that which is captured by traditional pathologist grading. Overall, our work highlights the ability to use self-supervised embeddings of histopathology to impute biological covariates on large, standard-of-care cohorts, empowering novel insights into disease mechanisms and patient outcome. Citation Format: Christopher S. Probert, Zachary R. McCaw, Navami Jain, Daphne Koller. Learned phenotypic embeddings enable scalable imputation of high-content molecular data elucidating prognostic chromatin signatures. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5375.

Background The aforementioned service provides specialist intestinal failure care to patients in Leicestershire and surrounding counties. Aim To study the epidemiology of IF cases referred to the service & review outcomes particularly the indications and delivery of parenteral nutrition (PN). To review catheter-related sepsis (CRS) rates and identify areas for improvement. Method Service data from January 2016 to November 2017 was analysed retrospectively. Descriptive and inferential statistical analysis was carried out. Chi-square/ Fisher Exact tests were used to identify significance on categorical data and non-parametric settling was used for qualitative data analysis. Results 365 patients were referred to the service from January 2016 to November 2017. 58% patients had grade I intestinal failure. 34% and 24% referrals were made by the colorectal and hepato-pancreato-biliary (HPB) units respectively. Gut rest (27%) and bowel obstruction (25%) were the most common indications for nutritional support. Duration of PN requirements varied greatly, but most patients (39%) patients required it for up to 7 days. Single lumen PICC line (64%) and cephalic vein (54%) were the most commonly used catheter and site of vascular access respectively. Chronicity of IF and number of vascular catheters required to provide PN were significantly associated with rates of CRS. Conclusions CRS was found to be statistically significantly associated with chronic IF and number of vascular access catheters required to provide PN. Cost implication and morbidity of CRS could be analysed further to identify other ways to provide safer and cost-efficient parental nutrition for patients with intestinal failure.

Background Amyand’s hernia, first described by Claudius Amyand in 1735, continues to remain a diagnostic & therapeutic challenge. By definition includes inguinal herniae that contain the vermiform appendix. Aim This up-to-date review aims to define and classify Amyand’s hernia and suggest appropriate management options in the current era. Aim to shed light on the value of pre-operative cross-sectional imaging and the use of laparoscopic surgery to provide a one stop solution. Method Published English literature was reviewed (1735–2021), using PubMed and Embase, combining the MeSH terms “hernia”, “inguinal”, “appendix”, “Amyand”, “low dose CT” and “laparoscopic herniotomy”. Most cited open access reviews, case series and case reports were chosen to provide a framework. A recent case is described with selected images (radiological, histology and intra operative). Results Since the original publication, by Claudius Amyand in 1735, there exists a gulf on this condition in the available British medical literature. Nearly 100,000 hernia repairs are performed in the UK annually. If Amyand’s hernia account for 1% of these (according to the most stated estimate), there should be a thousand Amyand’s herniae repaired annually in the UK alone. Unfortunately, the management options remain nonstandard. Conclusions Computerized tomography (CT) allows for pre-operative diagnosis and should be considered more often, even in children. Laparoscopic appendicectomy combined with a laparoscopic herniotomy is a safe one stop solution, in experienced hands, for paediatric patients.

Predicting when a patient with advanced cancer is dying is a challenge and currently no prognostic test is available. We hypothesised that a dying process from cancer is associated with metabolic changes and specifically with changes in volatile organic compounds (VOCs). We analysed urine from patients with lung cancer in the last weeks of life by headspace gas chromatography mass spectrometry. Urine was acidified or alkalinised before analysis. VOC changes in the last weeks of life were identified using univariate, multivariate and linear regression analysis; 12 VOCs increased (11 from the acid dataset, 2 from the alkali dataset) and 25 VOCs decreased (23 from the acid dataset and 3 from the alkali dataset). A Cox Lasso prediction model using 8 VOCs predicted dying with an AUC of 0.77, 0.78 and 0.85 at 30, 20 and 10 days and stratified patients into a low (median 10 days), medium (median 50 days) or high risk of survival. Our data supports the hypothesis there are specific metabolic changes associated with the dying. The VOCs identified are potential biomarkers of dying in lung cancer and could be used as a tool to provide additional prognostic information to inform expert clinician judgement and subsequent decision making.

In 2019, the United States (US) experienced the highest number of measles importations and cases in the postelimination era. More than a quarter of imported cases entered the US through California. Measles surveillance efforts in California resulted in the identification of 26 importations, 6 outbreaks, and 72 cases in 2019. Only genotype B3 and D8 measles strains were detected. Genotype-specific differences were noted in the incidence of vaccine failures, hospitalizations, and severe complications among cases. A targeted whole genome sequencing approach provided higher-resolution discrimination between epidemiologically linked and sporadically introduced strains than conventional N450 sequencing. Our report underscores the importance of ensuring appropriate measles vaccination status, especially prior to international travel to measles-endemic regions, and highlights the value of a strong measles surveillance system in minimizing outbreaks and preserving measles elimination status in the US.

The heterogeneous nature of inflammatory bowel disease (IBD) presents challenges, particularly when choosing therapy. Activation of the NF-κB transcription factor is a highly-regulated, dynamic event in IBD pathogenesis. We expressed the human NF-κB/p65 subunit in blood-derived macrophages, using lentivirus. Confocal imaging of p65 activation revealed that a higher proportion of macrophages from Crohn’s patients responded to lipid-A compared to controls. In contrast, cells from ulcerative colitis (UC) patients exhibited a shorter duration of p65 nuclear localisation compared to healthy controls and Crohn’s donors. Using a similar lentivirus approach, NF-κB-regulated luciferase was expressed in patient macrophages, isolated from frozen peripheral blood mononuclear cell samples. Following activation, samples could be segregated into three clusters based on the NF-κB-regulated luciferase response. The majority of UC samples appeared in hypo-responsive cluster 1, with Crohn’s patients representing the majority of hyper-responsive cluster 3. A positive correlation was seen between NF-κB-induced luciferase activity and cytokine levels released to medium from stimulated macrophages, but not in serum or biopsy. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between Crohn’s patients who smoked and hyper-activation of p65. Thesein vitrodynamic assays of NF-κB activation in blood-derived macrophages segregate IBD patients into groups with different phenotypes and therefore may help determine response to therapy.Significance statementThis manuscript describes two dynamic assays of NF-κB activation in blood-derived macrophages that can segregate IBD patients into groups with different phenotypes. For the first time we introduce the use of dynamic measurements of a transcription factor activation as a method to stratify patients and we are confident that our approach will lead in future to early patient stratification and prediction of treatment outcome.

Laboratory surveillance plays an important role in the detection and control of hepatitis A outbreaks and requires the application of rapid and accurate molecular diagnostic tools for hepatitis A virus (HAV) RNA detection, subgenotype identification, and sequence-based genotyping. We describe the development and validation of a triplex real-time, reverse transcription-PCR (triplex rRT-PCR) assay for the identification and discrimination of HAV subgenotypes IA, IB, and IIIA and a singleplex rRT-PCR assay designed to detect all HAV genotypes infecting humans. Overall, the accuracy, sensitivity, and specificity of the new assays were >97% for serum and plasma specimens collected during unrelated outbreaks of HAV in California and Michigan compared to a nested RT-PCR genotyping assay and the ISO 15216-1 rRT-PCR method for HAV detection. The new assays will permit the rapid detection of HAV RNA and discrimination among subgenotypes IA, IB, and IIIA in serum and plasma specimens, which will strengthen public health surveillance efforts for HAV outbreak detection and response.

Microsporidia are ubiquitous parasites that infect a wide range of animal hosts, and these fungal-related microbes undergo their entire replicative lifecycle inside of host cells. Despite being widespread in the environment and causing medical and agricultural harm, virtually nothing is known about the host factors important to facilitate their growth and development inside of host cells. Here, we perform a genetic screen to identify host transcription factors important for development of the microsporidian pathogen Nematocida parisii inside intestinal cells of its natural host, the nematode Caenorhabditis elegans. Through this screen, we identified the C. elegans Myc family of transcription factors as key host regulators of microsporidia growth and development. The Mad-like transcription factor MDL-1, and the Max-like transcription factors MXL-1 and MXL-2 promote pathogen levels, while the Myc-Mondo-like transcription factor MML-1 inhibits pathogen levels. We used epistasis analysis to show that MDL-1 and MXL-1, which are thought to function as a heterodimer, appear to be acting canonically. In contrast, MXL-2 and MML-1, which are also thought to function as a heterodimer, appear to be acting in separate pathways (noncanonically) in the context of pathogen infection. We also found that both MDL-1::GFP and MML-1::GFP are expressed in intestinal cells during infection. These findings provide novel insight into the host transcription factors that regulate microsporidia development.

Despite high coverage with measles, mumps, and rubella vaccine in the United States, outbreaks of mumps occur in close contact settings such as schools, colleges, and camps. Starting in late 2015, outbreaks were reported from several universities, and by the end of 2017, greater than 13,800 cases had been reported nation-wide. In 2013, the CDC and the Association of Public Health Laboratories contracted four Vaccine Preventable Diseases Reference Centers (VPD-RCs) to perform real-time reverse transcription PCR (RT-qPCR) to detect mumps RNA in clinical samples and to determine the genotype. Twelve genotypes of mumps virus are currently recognized by the World Health Organization, and the standard protocol for genotyping requires sequencing the entire gene coding for the small hydrophobic (SH) protein. Phylogenetic analysis of the 1862 mumps samples genotyped from 2015 through 2017 showed that the overall diversity of genotypes detected was low. Only 0.8 % of the sequences were identified as genotypes C, H, J, or K, and 0.5 % were identified as vaccine strains in genotypes A or N, while most sequences (98.7 %) were genotype G. The majority of the genotype G sequences could be included into one of two large groups with identical SH sequences. Within genotype G, a small number of phylogenetically significant outlier sequences were associated with epidemiologically distinct chains of transmission. These results demonstrate that molecular and epidemiologic data can be used to track transmission pathways of mumps virus; however, the limited diversity of the SH sequences may be insufficient for resolving transmission in all outbreaks.

Large randomized controlled trials (RCTs) in preterm infants offer unique opportunities for mechanistic evaluation of the risk factors leading to serious diseases, as well as the actions of interventions designed to prevent them. Necrotizing enterocolitis (NEC) a serious inflammatory gut condition and late-onset sepsis (LOS) are common feeding and nutrition-related problems that may cause death or serious long-term morbidity and are key outcomes in two current UK National Institutes for Health Research (NIHR) trials. Speed of increasing milk feeds trial (SIFT) randomized preterm infants to different rates of increases in milk feeds with a primary outcome of survival without disability at 2 years corrected age. Enteral lactoferrin in neonates (ELFIN) randomizes infants to supplemental enteral lactoferrin or placebo with a primary outcome of LOS. This is a protocol for the mechanisms affecting the gut of preterm infants in enteral feeding trials (MAGPIE) study and is funded by the UK NIHR Efficacy and Mechanistic Evaluation programme. MAGPIE will recruit ~480 preterm infants who were enrolled in SIFT or ELFIN. Participation in MAGPIE does not change the main trial protocols and uses non-invasive sampling of stool and urine, along with any residual resected gut tissue if infants required surgery. Trial interventions may involve effects on gut microbes, metabolites (e.g., short-chain fatty acids), and aspects of host immune function. Current hypotheses suggest that NEC and/or LOS are due to a dysregulated immune system in the context of gut dysbiosis, but mechanisms have not been systematically studied within large RCTs. Microbiomic analysis will use next-generation sequencing, and metabolites will be assessed by mass spectrometry to detect volatile organic and other compounds produced by microbes or the host. We will explore differences between disease cases and controls, as well as exploring the actions of trial interventions. Impacts of this research are multiple: translation of knowledge of mechanisms promoting gut health may explain outcomes or suggest alternate strategies to improve health. Results may identify new non-invasive diagnostic or monitoring techniques, preventative or treatment strategies for NEC or LOS, or provide data useful for risk stratification in future studies. Mechanistic evaluation might be especially informative where there are not clear effects on the primary outcome (ISRCTN 12554594).

Background Methotrexate is an immunosuppressant used in the treatment of patients with Crohn’s disease who are intolerant or refractory to azathioprine. Aim To present clinical experience in terms of response, side effects and reason for cessation of methotrexate treatment as well as adherence to monitoring regimens. Design Retrospective review of case records of 37 patients taking methotrexate for Crohn’s disease at the Bristol Royal Infirmary, a large UK teaching hospital. Thirty-five patients used oral methotrexate. Results Response rate to methotrexate was 78% at 3 months. Adverse events were uncommon and rarely dangerous. Monitoring of treatment did not adhere to British Society of Gastroenterology guidelines. Conclusion Oral methotrexate is effective and safe in the treatment of Crohn’s disease. This is in contrast to findings of previous series, which have mainly reported on intramuscular methotrexate use. Monitoring of treatment is currently inadequate and consensus should be sought for a pragmatic monitoring schedule to resolve this important governance issue.

We report the development of a single-tube, multi-locus variable number tandem repeat analysis (MLVA) assay for simultaneous speciation and strain typing of Brucella, the etiologic agent of brucellosis. Our MLVA assay consists of eight loci, two of which are species-specific markers that allow for definitive identification of Brucella melitensis, B. abortus, and Brucella species while ruling out related pathogenic bacterial genera. The remaining six loci are moderately variable loci capable of discriminating between Brucella strains originating within our study area. We applied the assay to a collection of 110 B. melitensis isolates of primarily Mexican origin and to smaller sample sizes of four other Brucella species for a total of 161 isolates. Simpson's index of diversity was 0.985 for B. melitensis and 0.938 for B. abortus. The assay accurately distinguished seven epidemiologically-linked clusters of B. melitensis infections and ascertained the source of infection in several laboratory-acquired cases. This assay is accessible to limited-resource settings due to its technological and economical feasibility. The timely and accurate information provided by this assay will potentially aid brucellosis control efforts, improve patient outcomes, and reduce the occurrence of laboratory-acquired infections.

A comparative analysis of the Bordetella pertussis , B. bronchiseptica , and B. parapertussis genome assemblies permitted the identification of regions with significant sequence divergence and the design of two new real-time PCR assays, BP283 and BP485, for the specific detection of B. pertussis . The performance characteristics of these two assays were evaluated and compared to those of culture and an existing real-time PCR assay targeting the repetitive element IS 481 . The testing of 324 nasopharyngeal specimens indicated that, compared to culture, the BP283 assay had a sensitivity and specificity of 100 and 96.8% and the BP485 assay had a sensitivity and specificity of 92.3 and 97.1%. Notably, B. holmesii was isolated from two specimens that were positive by the IS 481 assay but negative by the BP283 and BP485 assays. These two assays represent an improvement in specificity over those of PCR assays targeting only IS 481 and may be duplexed or used in conjunction with existing PCR assays to improve the molecular detection of B. pertussis .

Members of the genus Brucella are known worldwide as pathogens of wildlife and livestock and are the most common organisms of zoonotic infection in humans. In general, brucellae exhibit a range of host specificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella species are highly conserved, which makes the differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus enabled us to develop real-time PCR assays based around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate with its previously determined Brucella clade. Six of the seven clade-specific assays detected DNA concentrations of less than 10 fg, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons to be made among the lineages of clonal bacteria and providing a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.

Culture-negative bacterial meningitis with secondary complications remains a significant challenge. Optimal treatment requires identification of the infecting organism. While the gold standard for diagnosis remains cerebrospinal fluid culturing, a significant number of cultures remain negative despite clinical evidence of meningitis. This patient illustrates the usefulness of polymerase chain reaction technology in identifying a specific organism, in an otherwise culture-negative bacterial meningitis with spinal cord abscess.

Molecular subtyping is of significant importance to the recognition of outbreaks of meningococcal disease caused by serogroup C Neisseria meningitidis. We describe the application of multilocus variable number tandem repeat analysis (MLVA) for the molecular subtyping of N. meningitidis and compare its performance to that of pulsed-field gel electrophoresis (PFGE). For MLVA, a multiplex PCR assay targeting five variable number tandem repeat regions was developed and evaluated using a panel of sporadic and outbreak-associated serogroup C N. meningitidis isolates. MLVA was highly reproducible and provided results within 6 h. Overall, the discriminatory power of MLVA was equivalent to that of PFGE. The utilization of MLVA for subtyping N. meningitidis isolates provides a rapid and safer alternative to PFGE for identifying outbreaks of meningococcal disease. As such, it may provide public health officials with timely information that may minimize the spread of outbreak-related cases through prophylaxis.

A collection of 52 strains belonging to the Hafnia alvei complex were subjected to molecular (16S rRNA gene sequencing) and biochemical analysis. Based upon 16S rRNA gene sequencing results, two genetic groups were identified which correspond with previously recognized DNA hybridization group 1 (ATCC 13337 T and ATCC 29926; n = 23) and DNA hybridization group 2 (ATCC 29927; n = 29). Of 46 biochemical tests used to characterize hafniae, 19 reactions (41%) yielded variable results. Of these 19 tests, 6 were determined to have discriminatory value in the separation of DNA groups 1 and 2, with malonate utilization found to be the most differential test. Test results of malonate utilization alone correctly assigned 90% of Hafnia isolates to their correct DNA group.

The identification of Brucella can be a time-consuming and labor-intensive process that places personnel at risk for laboratory-acquired infection. Here, we describe a real-time PCR assay for confirmation of presumptive Brucella isolates. The assay was designed in a multiplex format that will allow the rapid identification of Brucella spp., B. abortus , and B. melitensis in a single test.

The description of Massilia timonae , a nonfermentative aerobic gram-negative rod, was based on a single strain. A subsequent report of a second isolate has been recently published. Phenotypic descriptions of these two strains were based primarily on commercial test kit results. We have identified three additional strains as M. timonae by 16S rRNA sequence analysis and have characterized them phenotypically in parallel with the type strain of M. timonae , CIP 105350, by conventional test methods. A fourth strain, designated M. timonae -like, was also characterized. All four strains were isolated from human patients: two were blood isolates, one was isolated from cerebrospinal fluid, and one was isolated from bone. The four strains and the type strain were quite similar phenotypically. However, in contrast to the original description, the strains were found to be oxidase positive and arginine dihydrolase negative and to have lateral flagella as well as a single polar flagellum. Additionally the strains produced acid oxidatively from some carbohydrates. Other phenotypic characteristics, including cellular fatty acids, agreed with the original description. Based on our emended description, M. timonae and M. timonae -like strains can be differentiated from other aerobic nonfermentative gram-negative rods by conventional biochemical tests combined with cellular fatty acid analysis.

The incidence of serogroup Y meningococcal disease has increased recently in the United States. Here, we describe the development of a 5′ exonuclease assay for the detection of serogroup Y Neisseria meningitidis and demonstrate the usefulness of this assay for resolving serogroup identification of strains that are resistant to conventional serogrouping and for the nonculture identification of serogroup Y meningococcal disease.

We placed 43 isolates belonging to the Proteus vulgaris complex into proposed DNA groups (genomovars) using five previously recommended tests (tests for esculin hydrolysis, production of acid from salicin, l -rhamnose fermentation, and elaboration of DNase and lipase). On the basis of the results of these five tests, 49% of the isolates fell into DNA groups 5 and 6, 37% fell into DNA group 2, and the remaining 14% fell into DNA groups 3 and 4. Sequencing of the 16S rRNA genes of 11 members of DNA groups 5 and 6 indicated that 10 of these isolates (91%) could be unambiguously assigned to one of these two genomospecies. Overall expression of selected enzymatic and virulence-associated characteristics did not differ significantly among DNA groups.

The attachment of pathogenic microorganisms to host cells and tissues is often mediated through the expression of surface receptors recognizing components of the extracellular matrix (ECM). Here, we investigate the ability of Borrelia spirochaetes to bind the ECM constituent, fibronectin. Borrelia lysates were separated by SDS-PAGE, transferred to nitrocellulose and probed with alkaline phosphatase-labelled fibronectin (fibronectin-AP). Five of six Borrelia species and four of eight B. burgdorferi sensu lato isolates expressed one or more fibronectin-binding proteins. Borrelia burgdorferi isolate B31 expressed a 47 kDa (P47) fibronectin-binding protein that was localized to the outer envelope based on susceptibility to proteinase K. The interaction of P47 with fibronectin was specific, and the region of fibronectin bound by P47 mapped to the gelatin/collagen binding domain. P47 was purified by affinity chromatography, digested with endoproteinase Lys-C, and the peptide fragments analysed by liquid chromatography/tandem mass spectroscopy. A search of protein databases disclosed that the P47 peptide mass profile matched that predicted for the bbk32 gene product of B. burgdorferi isolate B31. The bbk32 gene was cloned into Escherichia coli, and the ability of recombinant BBK32 to bind fibronectin and inhibit the attachment of B. burgdorferi was demonstrated. The identification of BBK32 as a receptor for fibronectin binding may enhance our understanding of the pathogenesis and chronic nature of Lyme disease.

We reviewed clinical and epidemiologic features of 56 human Capnocytophaga canimorsus isolates submitted during a 32-year period to California's Microbial Diseases Laboratory for identification. An increasing number of isolates identified as C. canimorsus have been submitted since 1990. Many laboratories still have difficulty correctly identifying this species.

The phenotype of T cells associated with the common bile duct (CBD) is unknown. We investigated the hypothesis that they behave like other intraepithelial lymphocytes (IEL). Thus, we sought to determine the phenotype, TCR repertoire, and epithelial recognition of T cells obtained during endoscopic retrograde cholangiopancreatography. Three subjects were studied: two with primary sclerosing cholangitis and one normal control. After establishing a short-term T cell line, cells were 1) stained with mAbs for flow cytometric analysis, 2) analyzed for TCRB chain transcript expression, and 3) used as effector cells for cytotoxicity and proliferation. Flow cytometry revealed that for all the subjects 98% of the T cells were TCR-alpha beta-positive. Immunohistology of the CBD showed that the epithelium and lamina propria contained significant numbers of CD3+ CD43+ CD45RO+ lymphocytes. Complementarity-determining region 3 length displays suggested that the CBD-derived lines were oligoclonal. This was confirmed by cloning and random sequencing of PCR amplification products using TCRBV region family-specific primers; TCRB chain sequences were reiterated in all transcripts analyzed. In one case, two expanded TCRB clones could be identified that were persistent in the bile duct over a 1-yr period. The CBD-derived lines were cytolytic in a redirected lysis assay and caused cytolysis of an intestinal epithelial cell line (Caco-2). This recognition was likely preferential for intestinal epithelial cells, since a CBD-derived line exhibited proliferation to two intestinal epithelial cell lines (HT-29 and Caco-2) but not three other lines (HepG2, human foreskin fibroblast, and KD). We conclude that the CBD contains IELs that share several characteristics with intestinal IELs.

It is increasingly recognized that chronic Ag exposure may lead to clonal expansions of T cells, including those within the peripheral blood. Inflammatory bowel disease is a chronic, multisystemic disease of unknown origin that predominantly affects the intestine. We sought to determine whether clonal expansions of T cells are present in the peripheral blood of patients with inflammatory bowel disease by an examination of TCR usage. Positively selected CD4+ and CD8+ peripheral blood T cells were isolated from subjects with active ulcerative colitis, Crohn's disease, and diverticulitis and from normal controls. Analysis of complementarity determining region 3 lengths of 24 TCR-beta chain V region families from CD4+ and CD8+ peripheral blood T cells showed a skewed distribution in the three inflammatory groups, consistent with expansion of T cell clones, in comparison to the normally distributed pattern observed among the control donors. Random sequencing of the PCR amplification products of CD4+ peripheral blood T cells from the subjects with ulcerative colitis, Crohn's disease, and diverticulitis revealed reiterative TCR-beta chain sequences that were not found in the normal donors. In subjects with Crohn's disease, the reiterative TCR-beta chain sequences from the CD4+ peripheral blood T cells were persistent over at least a 1-yr period. The persistently expanded TCR-beta chain sequences of CD4+ peripheral blood T cells were identifiable in genomic DNA isolated from archival tissue of intestine from subjects with Crohn's disease and ulcerative colitis by Southern blotting and direct DNA sequencing. An identical twin pair, concordant for Crohn's disease, shared the same reiterative TCR-beta chain sequences in their CD4+ peripheral blood T cells. These studies show that chronic intestinal inflammation is associated with expansions of CD4+ peripheral blood T cells. Furthermore, in inflammatory bowel disease these T cell clonal expansions are persistent and shared among HLA-identical individuals, implicating a response to specific, persistent, and stimulating Ags in these diseases.

The human intestine contains two populations of anatomically distinct T cells, intraepithelial lymphocytes and lamina propria lymphocytes (LPLs), both of which preferentially use the TCR-alpha beta. Recent studies of TCR alpha- and beta-chain usage by intestinal intraepithelial lymphocytes, which are predominantly CD8+ T cells, have demonstrated that these cells are oligoclonal in normal intestine. This report examined the TCR beta-chains expressed by purified CD4+ and CD8+ T cells from normal colonic lamina propria and from the intestinal mucosa of patients with active ulcerative colitis (UC). The selective expansion of CD8+ T cell clones, and to a lesser extent CD4+ T cell clones, was observed among both normal LPLs and mucosal T cells in UC. These expanded LPL clones from normal donors were all distinct, but the mucosal T cells isolated from five of nine patients with UC contained CD8+ T cells expressing related V beta 3-J beta 1.6 TCRs. These observations provide evidence for an Ag-specific mucosal T cell response in UC. Further studies will be required to identify this Ag and address whether the T cell response to it plays a primary role in initiating the disease or is secondary to the inflammatory response.

Five Mycobacterium tuberculosis complex isolates in California were identified as M. africanum by spoligotyping, single nucleotide polymorphisms, a deletion mutation, and phenotypic traits, confirming it as a cause of tuberculosis in the United States. Three of the five patients from whom M. africanum was isolated had lived in Africa.

In a “utilization deficiency,” a child spontaneously produces an appropriate strategy but receives little or no benefit from it for recall. Three studies provided evidence for two causes—children's failure to relate the task situation to their event knowledge or to link the strategy to a second strategy, in this case linking a selective attention strategy to a labeling strategy. In Study 1, 83 kindergartners and first graders were asked to remember the locations of 6 objects (e.g., animals) from a set of 12 objects (animals, household objects). The most efficient study strategy is a selective strategy—looking only at objects to be remembered. Selectivity predicted recall when the task was embedded in a story about a familiar context but not in the absence of this context. In Studies 2 and 3, instructing spontaneously selective preschool, kindergarten, and first-grade children to label the relevant objects improved recall. We discuss the possible mechanisms underlying the influence of knowledge and strategy linking and the role of production, mediational, and utilization deficiencies in the development of strategies.

The sequence and characterization of a chromosomal gene from the Lyme disease agent Borrelia burgdorferi and the antigen it encodes are described. The gene was cloned and expressed in transformed Escherichia coli cells. The gene is composed of 597 bases and expresses a predicted protein of 199 amino acids. Antibodies specific for the recombinant antigen reacted with a single B. burgdorferi protein with a molecular mass of approximately 22 kDa. The protein was not susceptible to proteinase digestion but was extracted by n-butanol phase partitioning, suggesting a periplasmic location of the antigen. Sera from humans and canines seropositive for B. burgdorferi reacted with the recombinant antigen. The antigen characterized in this report appears to be immunologically significant in naturally infected hosts.

In order to provide an overview and evaluation of the ways in which constructs might be elicited, a number of procedures are reviewed including: the original triad procedure, dyadic and monadic procedures, ordinal elicitation procedures, and self-characterization procedures. In addition, two recently developed procedures are presented: 1) a focusing elicitation procedure using a modification of Gendlin's focusing technique which allows it to be applied to the triadic elicitation procedure, 2) a storytelling elicitation procedure that uses an original board game to aid a small group of participants in examining their personal and interpersonal constructions. In conclusion, some of the ways that these elicitation procedures have been compared and evaluated are reviewed.

We report the second human case of infection caused by an organism identified as the proposed Bartonella species, “ B . washoensis .” The organism was isolated from a blood sample from a patient presenting with meningitis and early sepsis. Oropsylla montana fleas were implicated as the vector for disease transmission in this case.

This study examined whether spontaneously strategic children from two age groups differed in the capacity required to execute a strategy. The strategic behavior of younger (grades K and 1) and older (grades 4 and 5) children (N = 104) was assessed on a selective memory task. Children selected objects to view from a larger pool of objects. The most mature strategy (i.e., selectivity) involves opening doors that reveal objects to be remembered and not opening those that do not. In a dual-task procedure in a second session, children performed a finger tapping task simultaneously with the selective strategy. The reduction of rate of finger tapping from the finger tapping baseline to the dual-task trials provided an assessment of the capacity required to execute the strategy. A control group followed the same procedure, but with a simpler door opening selective strategy. A significant age difference in the capacity required for the strategy, even among spontaneously strategic children, suggested that there is further development of strategies even after children spontaneously produce them. Results were discussed in terms of increased strategy effectiveness during development.

To the Editor: Brucellosis is a zoonotic disease that remains endemic to many parts of the world. There are 6 classic Brucella species described with different preferred hosts. Human disease is most commonly associated with consumption of unpasteurized dairy products or with occupational exposure for veterinarians, agricultural workers, laboratory workers, meat industry workers, and hunters. In recent years, it has become clear that novel members of the genus, yet to be formally named, are associated with a variety of marine mammal species, particularly dolphins, porpoises, and seals (1). To date there are 3 reports in the literature of naturally acquired infection of humans with Brucella species originating from marine mammals (2,3) One other case, representing infection of a laboratory worker, has also been reported (4). Two of the naturally acquired cases were reported in Peru (2). One person had consumed raw shellfish and swam in the Pacific Ocean but did not report any direct contact with marine mammals; the second person reported infrequent visits to the coast and no contact with marine mammals but had consumed raw shellfish. An additional naturally acquired case was recently reported from New Zealand, where extensive molecular testing characterized the strain involved as a marine mammal type (3). This patient again reported no exposure to marine mammals but did report that he fished regularly, had contact with uncooked fish bait, and consumed raw snapper. The cases in Peru were notable for severe, atypical symptoms; both patients had symptoms of neurobrucellosis. The New Zealand case was associated with spinal osteomyelitis (3). In contrast, the laboratory-acquired infection was mild and uncomplicated (4). We have characterized these isolates by a variety of molecular approaches in conjunction with ongoing studies, which examine genetic diversity within Brucella species isolated from marine mammals. Multilocus sequence analysis (5) showed that all 3 isolates from naturally acquired human infection with Brucella species from marine mammals shared an identical genotype (ST27). In previous characterization of 56 Brucella isolates from marine mammals, ST27 was found only once. Strain F5/99, originally isolated from an aborted bottlenose dolphin fetus off the western coast of the United States (6), shares this genotype. Use of an alternative typing approach, based on restriction fragment length polymorphism analysis of outer membrane protein–encoding genes (7), gave identical findings. Again, all 3 isolates derived from naturally acquired human infection represent an identical genotype. This genotype is shared only by strain F5/99 among a collection of 120 Brucella isolates from marine mammals characterized by this method. Finally, isolates were characterized by a variable number of tandem repeats–based typing approach (8). When comparing profiles at 6 loci with a relatively slow evolutionary speed, previously shown to be useful for dividing Brucella isolates into species groups (8), we determined that F5/99 and the 3 naturally acquired human isolates share a unique profile not seen in any of >1,400 isolates of marine or terrestrial Brucella species examined to date. In contrast, the strain associated with laboratory-acquired infection (4) was not a member of ST27 but belonged to ST23, a genotype that is predominantly associated with porpoises (9). It is clear from these findings that the 3 cases of naturally acquired infection with Brucella species originating from marine mammals reported to date were caused by closely related organisms. The particular genotype concerned, ST27, is rare in our collection, having been noted only once in marine mammals. However, this may reflect the fact that most isolates examined to date originated from northern Europe; only 5 isolates in our collection originated from Pacific waters. It is possible that isolates of this genotype are predominantly or exclusively associated with regions other than those extensively sampled to date. Indeed, examination of the literature provides further evidence for the presence of this genotype in marine mammals in the Pacific. BLAST (www.ncbi.nlm.nih.gov/blast) comparison of outer membrane protein sequences from a minke whale isolate originating in the North Pacific (10) showed a close match with the equivalent sequence from F5/99. Although numbers are currently small, the isolation of an identical genotype from all 3 cases of naturally acquired human infection derived from marine mammal Brucella species raises the possibility of increased zoonotic potential associated with this genotype. Furthermore, where diagnosis is based on serologic testing alone, it is possible that human infection with marine mammal Brucella species may go unnoticed. Members of ST27 may be more pathogenic to man, per se. Alternatively, they may be associated with natural hosts or circulate through intermediaries that make contact with humans more likely. Notably, none of the 3 patients reported direct contact with marine mammals, though all had consumed raw seafood. These findings clearly suggest that more extensive studies of the presence and distribution of marine mammal Brucella genotypes, particularly ST27, in waters other than those of northern Europe would be valuable for clarifying the natural habitat of ST27. Furthermore, relevant authorities should be aware of the potential for zoonotic disease caused by this Brucella genotype particularly, but not exclusively, where occupation or lifestyle may make exposure more likely. Brucellosis research at the Veterinary Laboratories Agency is supported by the UK Department of Environment, Food and Rural Affairs (Defra).

Truncated versions of the human cytomegalovirus (CMV) strain Towne glycoprotein B (gB) gene were stably expressed in CHO cell lines. The calcium-specific ionophore A23187 inhibited proteolytic cleavage of C-terminal-truncated gB expressed by cell line 67.77. These inhibition studies also showed that the 93-kilodalton cleavage product most likely represents the N-terminal cleavage fragment of gB. The ionophore carboxyl cyanide m-chlorophenyl-hydrazone was used to show that proteolytic cleavage of gB did not occur in the endoplasmic reticulum. Two-dimensional polyacrylamide gel electrophoresis demonstrated that the N- and C-terminal cleavage products of gB remained associated by disulfide linkages after cleavage. Expression studies using constructs in which 80% or all of the N terminus was deleted demonstrated that the N terminus was required for secretion of the gB molecule. The amino acid sequence at the site of cleavage was shown to be critical for cleavage by a cellular protease. Our results indicate that an arginine-to-threonine change at either amino acid 457 or 460, a lysine-to-glutamine change at amino acid 459, or all three substitutions together block gB cleavage. The effect on proteolysis of the arginine-to-threonine amino acid change at residue 457 (position -4 relative to the cleavage site) demonstrated that a basic pair of amino acids at the endoproteolytic processing site is not the only requirement in cis for gB cleavage.

An 11-year-old girl presented to a central California children's hospital with a 3-day history of erythematous lesions on her forehead, neck, and trunk, abdominal pain, persistent emesis, and decreased urinary output. One day prior to admission she had a mild bout of diarrhea with a small amount of blood in her stool. Upon admission her condition rapidly worsened with acute renal failure, anemia, and thrombocytopenia. One of the possible causes of this condition included hemolytic uremic syndrome. Stool cultures of this patient tested at the children's hospital and at a state reference laboratory were repeatedly negative for Escherichia coli O157:H7. However, the state reference laboratory detected a toxigenic strain of Hafnia alvei active on Vero cells from two consecutive stool cultures during the acute phase of her illness.

The slow-growing nature of Mycobacterium tuberculosis complex hinders the improvement of turnaround time for phenotypic drug susceptibility testing. We designed a set of molecular beacons for the detection of isoniazid and rifampin resistance mutations in M. tuberculosis complex organisms from cultures or from N -acetyl- l -cysteine-NaOH-treated, smear-positive specimens. The performance of the molecular beacons was characterized by studying a total of 196 clinical isolates (127 drug-resistant isolates and 69 drug-susceptible isolates). For detection of isoniazid resistance, the sensitivity and specificity of the assay were 82.7 and 100%, and the positive predictive value (PPV) and negative predictive value (NPV) at a resistance prevalence of 10% were 100 and 98.11%, respectively. For detection of rifampin resistance, the sensitivity and specificity of the assay were 97.5 and 100%, and the PPV and NPV at a resistance prevalence of 2.0% were 100 and 99.95%, respectively.

The present study characterised 73 Hafnia alvei isolates and five Escherichia isolates (originally identified as H. alvei) isolated from cases of diarrhoeal disease by the International Centre for Diarrhoeal Disease Research Branch (ICDDRB) in Bangladesh. Based upon the hydrolysis of arbutin and aesculin and the fermentation of salicin and D-arabinose, four distinct biotypes could be recognised among the 73 H. alvei isolates tested; biotype 1 (D-(-)-arabinose-positive only) accounted for 75% of all isolates analysed. Hydrolysis of aglycone compounds such as arbutin, salicin and aesculin appeared to be associated with expression of beta-glucosidase activity. ICDDRB isolates, when compared with type or reference strains of H. alvei, were shown not to belong to the genus Hafnia based upon resistance to Hafnia-specific bacteriophage 1672, possession of the phoE gene, expression of glutamate decarboxylase activity and significant 16S rDNA sequence divergence (approximately 8%) from the type strain, ATCC 13337T. True H. alvei strains, implicated in outbreaks of diarrhoeal disease in Canada, lacked the eaeA gene in contrast to ICDDRB isolates. Twenty-two H. alvei isolates were selected for further study. Based upon partial 16S rDNA sequencing, these 22 isolates fell into two genomic groups (genomospecies), identical to DNA groups previously established by DNA hybridisation studies. Markers such as motility, biotype, or enzymic or carbohydrate fermentation patterns did not correlate totally with DNA grouping, although malonate utilisation appeared to be the single best discriminatory phenotype. The results indicate that the genus Hafnia is heterogeneous and there do not appear to be any laboratory data available specifically linking these organisms to gastro-enteritis.

Background Sugar Sweetened Beverages contribute to obesity in older children but whether highly calorific Fruit Drinks (FD) contributes to preschool child obesity is unknown. We therefore quantified the beverage intake of preschool children and their parent’s knowledge of the sugar content. Methods Parents of children aged 1–5 years attending three North East England Acute and Outpatient Paediatric centres over six months completed questionnaires. Volume and types of beverages consumed, recommended daily intake (RDI) and parental knowledge of calorie content of three popular FDs were collected. FD calories, as a percentage of RDI, were calculated and compared with the child’s BMI. Results 304 questionnaires were analysed. 61% reported daily FDs with 33% exceeding their RDI. 28% were overweight or obese with the proportion rising from 24% in the under twos to 31% in the older children. Mean FD calorie intake as% of RDI was 5.5% with no association to increased BMI (p = 0.32. Mann Whitney U). Children in the lower and higher BMI centiles constituted the largest groups drinking >10% Fruit Drink RDI. Parents (99%) had no knowledge of their child’s calorie intake or RDI with 76% unable to identify the highest calorie FD. Conclusion No association between Fruit Drink intake and obesity was found. 61% of children drank Fruit Drink daily with 33% in excess of RDI. Overweight and underweight children constituted the largest groups drinking >10% RDI of Fruit Drinks Parents were unaware of their children’s calorie intake, RDI or FD calorie content.