Rheumatoid arthritis (RA) is a chronic inflammatory disorder that primarily involves the joints. Accurate and frequent assessment of RA disease activity is critical to optimal treatment planning. A novel algorithm has been developed to determine a multi-biomarker disease activity (MBDA) score based upon measurement of the concentrations of 12 serum biomarkers in multiplex format. Biomarker assays from several different platforms were used in feasibility studies to identify biomarkers of potential significance. These assays were adapted to a multiplex platform for training and validation of the algorithm. In this study, the analytical performance of the underlying biomarker assays and the MBDA score was evaluated. Quantification of 12 biomarkers was performed with multiplexed sandwich immunoassays in three panels. Biomarker-specific capture antibodies were bound to specific locations in each well; detection antibodies were labeled with electrochemiluminescent tags. Data were acquired with a Sector Imager 6000, and analyte concentrations were determined. Parallelism, dynamic range, cross-reactivity, and precision were established for each biomarker as well as for the MBDA score. Interference by serum proteins, heterophilic antibodies, and common RA therapies was also assessed. The individual biomarker assays had 3-4 orders of magnitude dynamic ranges, with good reproducibility across time, operators, and reagent lots; the MBDA score had a median coefficient of variation of <2% across the score range. Cross-reactivity as well as interference by serum rheumatoid factor (RF), human anti-mouse antibodies (HAMA), or common RA therapies, including disease-modifying antirheumatic drugs and biologics, was minimal. The same MBDA score was observed in different subjects despite having different biomarker profiles, supporting prior literature reports that multiple pathways contribute to RA.

All 3 diagnostic tests are highly specific, and a positive test result is highly predictive of disease presence. IVCM is both highly sensitive and specific when performed by an experienced operator. PCR is a useful adjunct in the diagnosis of AK because of its wider availability compared with IVCM, and it may be used in combination with IVCM for microbiologic confirmation.

PTEN tumor suppressor gene failure in ras Ha -activated skin carcinogenesis was investigated by mating exon 5 floxed-PTEN (#5PTEN) mice to HK1.ras mice that expressed a RU486-inducible cre recombinase (K14.creP). PTEN inactivation in K14.cre/PTEN flx/flx keratinocytes resulted in epidermal hyperplasia/hyperkeratosis and novel 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted papillomas, whereas HK1.ras/K14.cre/PTEN flx/flx cohorts displayed a rapid onset of papillomatogenesis due to a synergism of increased AKT activity and extracellular signal-regulated kinase (ERK) elevation. High 5-bromo-4-deoxyuridine labeling in #5PTEN papillomas showed that a second promotion mechanism centered on failures in cell cycle control. Elevated cyclin D1 was associated with both HK1.ras/ERK-and #5PTEN-mediated AKT signaling, whereas cyclin E2 overexpression seemed dependent on PTEN loss. Spontaneous HK1.ras/ #5PTEN malignant conversion was rare, whereas TPA promotion resulted in conversion with high frequency. On comparison with all previous HK1.ras carcinomas, such TPAinduced carcinomas expressed atypical retention of keratin K1 and lack of K13, a unique marker profile exhibited by TPAinduced K14.cre/PTEN flx/flx papillomas that also lacked endogenous c-ras Ha activation. Moreover, in all PTEN-null tumors, levels of ras Ha -associated total ERK protein became reduced, whereas phosphorylated ERK and cyclin D1 were lowered in late-stage papillomas returning to elevated levels, alongside increased cyclin E2 expression, in TPA-derived carcinomas. Thus, during early papillomatogenesis, PTEN loss promotes rasHa initiation via elevation of AKT activity and synergistic failures in cyclin regulation. However, in progression, reduced ras Ha -associated ERK protein and activity, increased #5PTEN-associated cyclin E2 expression, and unique K1/K13 profiles following TPA treatment suggest that PTEN loss, rather than ras Ha activation, gives rise to a population of cells with greater malignant potential. (Cancer Res 2006; 66(3): 1302-12)

Olfactory ensheathing cells can develop into distinct subtypes in culture after incubation in serum-free medium conditioned by astrocytes, which have Schwann cell-like and astrocyte-like properties. It has not been possible so far to modulate and grow large numbers of these olfactory ensheathing cell subtypes. In this study, we have shown that astrocyte-conditioned medium, although promoting differentiation of the two olfactory ensheathing cell types, is growth-restrictive after 14 days, probably due to the upregulation of p16 and p27. Growth arrest can be overridden and cells maintained for a further 11 weeks, by a mitogen mix of fibroblast growth factor 2, forskolin, and heregulin (olfactory mitogen medium) combined with astrocyte-conditioned medium. In the absence of astrocyte-conditioned medium, combinations of the same factors can also override growth arrest but to a lesser extent. Olfactory mitogen medium combined with astrocyte-conditioned medium upregulates O4 and low-affinity nerve growth factor receptor expression on olfactory ensheathing cells, leading to a 100% Schwann cell--like phenotype. If cells are maintained in olfactory mitogen medium alone, or if they are treated with forskolin or fibroblast growth factor 2 diluted in serum-free medium, O4 and low-affinity nerve growth factor receptor expression remains at 100%, but there is also an increase in expression of E-NCAM, the astrocyte-like marker. Medium containing serum also overrides growth arrest, but for only 4 weeks, during which time most differentiation-specific markers disappear. These studies have allowed us to define conditions to modulate the olfactory ensheathing cell phenotype.

A Schistosoma mansoni cercarial antigen preparation (cercarial transformation fluid – SmCTF) was evaluated for detection of anti-schistosome antibodies in human sera in 4 collaborating laboratories. The performance of SmCTF was compared with that of S. mansoni egg antigens (SmSEA) in an indirect enzyme-immunoassay (ELISA) antigen assay, the latter being used routinely in 3 of the 4 participating laboratories to diagnose S. mansoni and S. haematobium infections. In the fourth laboratory the performance of SmCTF was compared with that of S. japonicum egg antigens (SjSEA) in ELISA for detection of anti-S. japonicum antibodies. In all 4 laboratories the results given by SmCTF in ELISA were very similar to those given by the antigen preparation routinely used in the respective laboratory to detect anti-schistosome antibodies in human infection sera. In so far as the ELISA results from SmCTF are thus so little different from those given by schistosome egg antigens and also cheaper to produce, the former is a potentially useful new diagnostic aid for schistosomiasis.

To investigate gene synergism in multistage skin carcinogenesis, the RU486-inducible cre/lox system was employed to ablate Pten function (K14.cre/Δ5Ptenflx) in mouse epidermis expressing activated Fos (HK1.Fos). RU486-treated HK1.Fos/Δ5Ptenflx mice exhibited hyperplasia, hyperkeratosis and tumours that progressed to highly differentiated keratoacanthomas, rather than to carcinomas, owing to re-expression of high p53 and p21WAF levels. Despite elevated MAP kinase activity, cyclin D1 and cyclin E2 overexpression, and increased AKT activity that produced areas of highly proliferative papillomatous keratinocytes, increasing levels of GSK3β inactivation induced a novel p53/p21WAF expression profile, which subsequently halted proliferation and accelerated differentiation to give the hallmark keratosis of keratoacanthomas. A pivotal facet to this GSK3β-triggered mechanism centred on increasing p53 expression in basal layer keratinocytes. This increase in expression reduced activated AKT expression and released inhibition of p21WAF, which accelerated keratinocyte differentiation, as indicated by unique basal layer expression of differentiation-specific keratin K1 alongside premature filaggrin and loricrin expression. Thus, Fos synergism with Pten loss elicited a benign tumour context where GSK3β-induced p53/p21WAF expression continually switched AKT-associated proliferation into differentiation, preventing further progression. This putative compensatory mechanism required the critical availability of normal p53 and/or p21WAF, otherwise deregulated Fos, Akt and Gsk3β associate with malignant progression.

BackgroundWe report a widespread foodborne outbreak of Cryptosporidium parvum in England and Scotland in May 2012. Cases were more common in female adults, and had no history of foreign travel. Over 300 excess cases were identified during the period of the outbreak. Speciation and microbiological typing revealed the outbreak strain to be C. parvum gp60 subtype IIaA15G2R1.MethodsHypothesis generation questionnaires were administered and an unmatched case control study was undertaken to test the hypotheses raised. Cases and controls were interviewed by telephone. Controls were selected using sequential digit dialling. Information was gathered on demographics, foods consumed and retailers where foods were purchased.ResultsSeventy-four laboratory confirmed cases and 74 controls were included in analyses. Infection was found to be strongly associated with the consumption of pre-cut mixed salad leaves sold by a single retailer. This is the largest documented outbreak of cryptosporidiosis attributed to a food vehicle.

Olfactory ensheathing cells can develop into distinct subtypes in culture after incubation in serum-free medium conditioned by astrocytes, which have Schwann cell-like and astrocyte-like properties. It has not been possible so far to modulate and grow large numbers of these olfactory ensheathing cell subtypes. In this study, we have shown that astrocyte-conditioned medium, although promoting differentiation of the two olfactory ensheathing cell types, is growth-restrictive after 14 days, probably due to the upregulation of p16 and p27. Growth arrest can be overridden and cells maintained for a further 11 weeks, by a mitogen mix of fibroblast growth factor 2, forskolin, and heregulin (olfactory mitogen medium) combined with astrocyte-conditioned medium. In the absence of astrocyte-conditioned medium, combinations of the same factors can also override growth arrest but to a lesser extent. Olfactory mitogen medium combined with astrocyte-conditioned medium upregulates O4 and low-affinity nerve growth factor receptor expression on olfactory ensheathing cells, leading to a 100% Schwann cell--like phenotype. If cells are maintained in olfactory mitogen medium alone, or if they are treated with forskolin or fibroblast growth factor 2 diluted in serum-free medium, O4 and low-affinity nerve growth factor receptor expression remains at 100%, but there is also an increase in expression of E-NCAM, the astrocyte-like marker. Medium containing serum also overrides growth arrest, but for only 4 weeks, during which time most differentiation-specific markers disappear. These studies have allowed us to define conditions to modulate the olfactory ensheathing cell phenotype.