Immunochemical characterization of a polysaccharide antigen of Bacteroides fragilis with an IgM monoclonal antibody

An IgM mouse monoclonal antibody (McAb) Bf4 was produced to a surface polysaccharide of Bacteroides fragilis NCTC 9343. Immunoblotting showed that McAb Bf4 reacted strongly with a high molecular mass structure which was sensitive to oxidation with periodate but resisted protease treatment. An inhibition enzyme-linked immunosorbent assay (ELISA) indicated that McAb Bf4 did not cross react with the sixteen Bacteroides species and strains tested. Cells of B. fragilis NCTC 9343 recovered from the various interfaces of a Percoll discontinuous density gradient were tested in the inhibition ELISA. Bacteria from the 0-20%, 20-40% and 40-60% interfaces inhibited the ELISA; however, cells from the 60-80% interface did not. Electron microscopy with immunogold labelling showed that McAb Bf4 did not react with the extracellular fibrous network on bacteria recovered from the 0-20% interface, or the extracellular electron dense layer on cells from the 60-80% interface; however, it was associated with a surface structure on cells from the 20-40% interface. Growth in vivo did not enrich for bacteria with this structure.

Skin Dendritic Cell Targeting via Microneedle Arrays Laden with Antigen-Encapsulated Poly-D, L-lactide-co-Glycolide Nanoparticles Induces Efficient Antitumor and Antiviral Immune Responses

The efficacious delivery of antigens to antigen-presenting cells (APCs), in particular, to dendritic cells (DCs), and their subsequent activation remains a significant challenge in the development of effective vaccines. This study highlights the potential of dissolving microneedle (MN) arrays laden with nanoencapsulated antigen to increase vaccine immunogenicity by targeting antigen specifically to contiguous DC networks within the skin. Following in situ uptake, skin-resident DCs were able to deliver antigen-encapsulated poly-d,l-lactide-co-glycolide (PGLA) nanoparticles to cutaneous draining lymph nodes where they subsequently induced significant expansion of antigen-specific T cells. Moreover, we show that antigen-encapsulated nanoparticle vaccination via microneedles generated robust antigen-specific cellular immune responses in mice. This approach provided complete protection in vivo against both the development of antigen-expressing B16 melanoma tumors and a murine model of para-influenza, through the activation of antigen-specific cytotoxic CD8(+) T cells that resulted in efficient clearance of tumors and virus, respectively. In addition, we show promising findings that nanoencapsulation facilitates antigen retention into skin layers and provides antigen stability in microneedles. Therefore, the use of biodegradable polymeric nanoparticles for selective targeting of antigen to skin DC subsets through dissolvable MNs provides a promising technology for improved vaccination efficacy, compliance, and coverage.

Pathobiology of rabies virus and the European bat lyssaviruses in experimentally infected mice

A comparison of the clinicopathology of European bat lyssavirus (EBLV) types-1 and -2 and of rabies virus was undertaken. Following inoculation of mice at a peripheral site with these viruses, clinical signs of rabies and distribution of virus antigen in the mouse brain were examined. The appearance of clinical signs of disease varied both within and across the different virus species, with variation in incubation periods and weight loss throughout disease progression. The distribution of viral antigen throughout the regions of the brain examined was similar for each of the isolates during the different stages of disease progression, suggesting that antigen distribution was not associated with clinical presentation. However, specific regions of the brain including the cerebellum, caudal medulla, hypothalamus and thalamus, showed notable differences in the proportion of virus antigen positive cells present in comparison to other brain regions suggesting that these areas are important in disease development irrespective of virus species.

Analysis of Betaine and Choline Contents of Aleurone, Bran, and Flour Fractions of Wheat (Triticum aestivum L.) Using 1H Nuclear Magnetic Resonance (NMR) Spectroscopy

In conventional milling, the aleurone layer is combined with the bran fraction. Studies indicate that the bran fraction of wheat contains the majority of the phytonutrients betaine and choline, with relatively minor concentrations in the refined flour. This present study suggests that the wheat aleurone layer (Triticum aestivum L. cv. Tiger) contains the greatest concentration of both betaine and choline (1553.44 and 209.80 mg/100 g of sample, respectively). The bran fraction contained 866.94 and 101.95 mg/100 g of sample of betaine and choline, respectively, while the flour fraction contained 23.30 mg/100 g of sample (betaine) and 28.0 mg/100 g of sample (choline). The betaine content for
the bran was lower, and the choline content was higher compared to previous studies, although it is known that there is large variation in betaine and choline contents between wheat cultivars. The ratio of betaine/choline in the aleurone fraction was approximately 7:1; in the bran, the ratio was approximately 8:1; and in the flour fraction, the ratio was approximately 1:1. The study further
emphasizes the superior phytonutrient composition of the aleurone layer.
INTRODUCTION
Wheat is a valuable source of betaine, choline (1, 2), B
vitamins, vitamin E, and a number of minerals, including iron,
zinc, magnesium, and phosphorus (3). Epidemiological studies
indicate that whole-grain consumption is protective against
several chronic diseases (4-12). It has not been fully elucidated
how whole-grain cereals or specific fractions (13) exert their
protective effect, but it is thought to be due to their content of
several nutrients associated with the reduced risk of disease.
Conventionally, whole grain is separated during milling into
bran, germ, and flour (14). The nutrient composition of these
fractions differ markedly; refined wheat flour contains approximately
50% less vitamins and minerals than whole-grain
flour (

Rotavirus enterotoxin NSP4 has mucosal adjuvant properties

Rotavirus nonstructural protein 4 (NSP4) is a protein with pleiotropic properties. It functions in rotavirus morphogenesis, pathogenesis, and is the first described viral enterotoxin. Since many bacterial toxins function as potent mucosal adjuvants, we evaluated whether baculovirus-expressed recombinant simian rotavirus SA11 NSP4 possesses adjuvant activity by co-administering NSP4 with keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or ovalbumin (OVA) as model antigens in mice. Following intranasal immunization, NSP4 significantly enhanced both systemic and mucosal immune responses to model immunogens, as compared to the control group, in an antigen-specific manner. Both full-length and a cleavage product of SA11 NSP4 had adjuvant activity, localizing this activity to the C-terminus of the protein. NSP4 forms from virulent and avirulent porcine rotavirus OSU strain, and SA11 NSP4 localized within a 2/6-virus-like particle (VLP) also exhibited adjuvant effects. These studies suggest that the rotavirus enterotoxin NSP4 can function as an adjuvant to enhance immune responses for a co-administered antigen.

Antigen-specific IgA and IgG responses in calves inoculated intranasally with ovalbumin encapsulated in poly(DL-lactide-co-glycolide) microspheres

The immunogenicity of proteins encapsulated in poly(DL-lactide-co-glycolide) (PLG) microspheres has not been investigated to any extent in large animal models. In this study, IgG and IgA responses to ovalbumin (OVA), encapsulated in microspheres was investigated following intranasal inoculation into calves. Scanning electron microscopy and flow cytometric analysis demonstrated a uniform microsphere population with a diameter of <2.5 micrometers. Ovalbumin was released steadily from particles stored in PBS almost in a linear fashion, and after 4 weeks many particles showed cracks and fissures in their surface structure. Following intranasal inoculation of calves with different doses of encapsulated antigen, mean levels of ovalbumin-specific IgA were observed to increase steadily but significant differences in IgA levels (from the pre-inoculation level) were only observed following a second intranasal inoculation. With 0.5 and 1.0mg doses of antigen, ovalbumin-specific IgG was also detected in serum. Ovalbumin-specific IgA persisted in nasal secretions for a considerable period of time and were still detectable in four out of seven animals, 6 months after inoculation.

Temporal and geographical distribution of measles virus genotypes

The nucleotide sequence encoding the C terminus of the nucleocapsid protein of measles virus (MV) is the most variable in the genome. The sequence of this region is reported for 21 new MV strains and for virus RNA obtained from cases of subacute panencephalitis (SSPE) tissue. The nucleotide sequence of a total of 65 MV strains has been analysed using the CLUSTAL program to determine the relationships between the strains. An unrooted tree shows that eight different genotypes can be discerned amongst the sequences analysed so far. The data show that the C-terminal coding sequence of the nucleocapsid gene, although highly variable between strains, is stable in a given strain and does not appear to diverge in tissue culture. It therefore provides a good 'signature' sequence for specific genotypes. The sequence of this region can be used to discriminate new imported viruses from old 'endemic' strains of MV in a geographical area. The different genotypes are not geographically restricted although some appear to be the mainly 'endemic' types in large areas of the world. In global terms there appears to be at least four co-circulating genotypes of MV. The low level of divergence in the Edmonston lineage group isolated before 1970 indicates that some isolates are probably laboratory contaminants. This applies to some SSPE isolates such as the Halle, Mantooth and Horta-Barbosa strains as well as some wild-type isolates from that period.

Prognostic value of TP53, KRAS and EGFR mutations in nonsmall cell lung cancer:The EUELC cohort

Nonsmall cell lung cancer samples from the European Early Lung Cancer biobank were analysed to assess the prognostic significance of mutations in the TP53, KRAS and EGFR genes. The series included 11 never-smokers, 86 former smokers, 152 current smokers and one patient without informed smoking status. There were 110 squamous cell carcinomas (SCCs), 133 adenocarcinomas (ADCs) and seven large cell carcinomas or mixed histologies. Expression of p53 was analysed by immunohistochemistry. DNA was extracted from frozen tumour tissues. TP53 mutations were detected in 48.8% of cases and were more frequent among SCCs than ADCs (p

Fasciola hepatica:development of monoclonal antibodies and their use to characterize a glycocalyx antigen in migrating flukes

Using mice harbouring early Fasciola hepatica infections, six monoclonal antibodies were prepared against a tegumental antigen present in T1 granules and glycocalyx of flukes. Blocking tests indicated that all monoclonals bound the same T1 epitope (or epitopes in close proximity on the antigen molecule), but this was not the determinant recognized by sheep and cattle. Localization of antibody binding at light and electron microscope levels showed that T1-type antigen also occurred in metacercarial tegument and in glycocalyx of gut cells and excretory ducts in juvenile and adult flukes. This indicates that the natural host-antibody response to F. hepatica may be to one antigen early in the infection. Protein A-gold labelling of monoclonal treated fluke sections revealed that the epitope was probably a polypeptide, unmodified by glycosylation in Golgi bodies. When isolated by immunoadsorption and separated electrophoretically under reducing conditions T1-type antigen was found to consist of a polypeptide mol. wt. 50 000, possibly linked to smaller entities mol. wt. 25-40 000. Tissue-specific variations in the antigen molecule might be conferred by linkage of different polypeptides or carbohydrate side-chains to an antigenic core polypeptide. A component of T1-type antigen was found to have mol. wt. of 25 000, possibly resembling a polypeptide of mol. wt. 24 000 from Schistosoma mansoni tegument.

Monospecific antibody to the haemagglutinin of measles virus

A hybrid between a murine myeloma cell line and spleen cells from a mouse immunized with measles has been produced. Two stable clones produce antibody with identical immunochemical and biological properties. This antibody reacts with the 76,000 mol. wt. protein present in the lysates and on the surface of cells persistently infected with measles. It exhibits HAI and neutralizing activity.

Non-steroidal anti-inflammatory drug use and brain tumour risk:a case-control study within the Clinical Practice Research Datalink

Purpose: The aetiology of primary brain tumours is largely unknown; the role of non-steroidal anti-inflammatory drugs (NSAIDs) or aspirin use and glioma risk has been inconclusive, but few population-based studies with reliable prescribing data have been conducted, and the association with meningioma risk has yet to be assessed. Methods: The UK Clinical Practice Research Datalink was used to assess the association between aspirin and non-aspirin NSAID use and primary brain tumour risk using a nested case-control study design. Conditional logistic regression analysis was performed on 5,052 brain tumour patients aged 16 years and over, diagnosed between 1987 and 2009 and 42,678 controls matched on year of birth, gender and general practice, adjusting for history of allergy and hormone replacement therapy use in the glioma and meningioma models, respectively.

Results: In conditional logistic regression analysis, excluding drug use in the year preceding the index date, there was no association with non-aspirin NSAID use (OR 0.96, 95 % CI 0.81-1.13) or glioma risk comparing the highest category of daily defined dose to non-users; however, non-aspirin NSAID use was positively associated with meningioma risk (OR 1.35, 95 % CI 1.06-1.71). No association was seen with high- or low-dose aspirin use irrespective of histology.

Conclusions: This large nested case-control study finds no association between aspirin or non-aspirin NSAID use and risk of glioma but a slight increased risk with non-aspirin NSAIDs and meningioma. © 2013 Springer Science+Business Media Dordrecht.

Determination of Spontaneous Mutation Frequencies in Measles Virus under Nonselective Conditions

There is a paradox between the remarkable genetic stability of measles virus (MV) in the field and the high mutation rates implied by the frequency of the appearance of monoclonal antibody escape mutants generated when the virus is pressured to revert in vitro (S. J. Schrag, P. A. Rota, and W. J. Bellini, J. Virol. 73: 51-54, 1999). We established a highly sensitive assay to determine frequencies of various categories of mutations in large populations of wild-type and laboratory-adapted MVs using recombinant viruses containing an additional transcription unit (ATU) encoding enhanced green fluorescent protein (EGFP). Single and double mutations were made in the fluorophore of EGFP to ablate fluorescence. The frequencies of reversion mutants in the population were determined by measuring the appearance of fluorescence indicating a revertant virus. This allows mutation rates to be measured under nonselective conditions, as phenotypic reversion to fluorescence requires only either a single-or a double-nucleotide change and amino acid substitution, which does not affect the length of the nonessential reporter protein expressed from the ATU. Mutation rates in MV are the same for wild-type and laboratory-adapted viruses, and they are an order of magnitude lower than the previous measurement assessed under selective conditions. The actual mutation rate for MV is approximately 1.8 x 10(-6) per base per replication event. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

Squirrelpox Virus: Assessing Prevalence, Transmission and Environmental Degradation

Red squirrels (Sciurus vulgaris) declined in Great Britain and Ireland during the last century, due to habitat loss and the introduction of grey squirrels (Sciurus carolinensis), which competitively exclude the red squirrel and act as a reservoir for squirrelpox virus (SQPV). The disease is generally fatal to red squirrels and their ecological replacement by grey squirrels is up to 25 times faster where the virus is present. We aimed to determine: (1) the seropositivity and prevalence of SQPV DNA in the invasive and native species at a regional scale; (2) possible SQPV transmission routes; and, (3) virus degradation rates under differing environmental conditions. Grey (n = 208) and red (n = 40) squirrel blood and tissues were sampled. Enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR) techniques established seropositivity and viral DNA presence, respectively. Overall 8% of squirrels sampled (both species combined) had evidence of SQPV DNA in their tissues and 22% were in possession of antibodies. SQPV prevalence in sampled red squirrels was 2.5%. Viral loads were typically low in grey squirrels by comparison to red squirrels. There was a trend for a greater number of positive samples in spring and summer than in winter. Possible transmission routes were identified through the presence of viral DNA in faeces (red squirrels only), urine and ectoparasites (both species). Virus degradation analyses suggested that, after 30 days of exposure to six combinations of environments, there were more intact virus particles in scabs kept in warm (25°C) and dry conditions than in cooler (5 and 15°C) or wet conditions. We conclude that SQPV is present at low prevalence in invasive grey squirrel populations with a lower prevalence in native red squirrels. Virus transmission could occur through urine especially during warm dry summer conditions but, more notably, via ectoparasites, which are shared by both species.

Local recurrence in patients with large and locally advanced breast cancer treated with primary chemotherapy

Background: Primary chemotherapy is being given in the treatment of large and locally advanced breast cancers, but a major concern is local relapse after therapy. This paper has examined patients treated with primary chemotherapy and surgery (either breast-conserving surgery or mastectomy) and has examined the role of factors which may indicate those patients who are subsequently more likely to experience local recurrence of,disease.

Methods: A consecutive series of 173 women, with data available for 166 of these, presenting with large and locally advanced breast cancer (T2 >4 cm, T3, T4, or N2) were treated with primary chemotherapy comprising cyclophosphamide, vincristine, doxorubicin, and prednisolone and then surgery (either conservation or mastectomy with axillary surgery) followed by radiotherapy were examined.

Results: The clinical response rate of these patients was 75% (21% complete and 54% partial), with a complete pathological response rate of 15%. A total of 10 patients (6%) experienced local disease relapse, and the median time to relapse was 14 months (ranging from 3 to 40). The median survival in this group was 27 months (ranging from 13 to 78). In patients having breast conservation surgery, local recurrence occurred in 2%, and in those undergoing mastectomy 7% experience local relapse of disease. Factors predicting patients most likely to experience local recurrence were poor clinical response and residual axillary nodal disease after chemotherapy.

Conclusions: Excellent local control of disease can be achieved in patients with large and locally advanced breast cancers using a combination of primary chemotherapy, surgery and radiotherapy. However, the presence of residual tumor in the axillary lymph nodes after chemotherapy is a predictor of local recurrence and patients with a better clinical response were also less likely to experience local disease recurrence. The size and degree of pathological response did not predict patients most likely to experience recurrence of disease. (C) 2003 Excerpta Medica, Inc. All rights reserved.

Freehand core biopsy in breast cancer: an accurate predictor of tumour grade following neoadjuvant chemotherapy?

Core biopsy is an increasingly used technique in the pre-operative diagnosis of breast carcinoma, as it provides useful prognostic information with respect to tumour type and grade. Neoadjuvant chemotherapy is being used in the treatment of large and locally advanced breast cancers but little is known regarding the correlation between tumour histology on pre-treatment core biopsy and that in residual tumour following primary chemotherapy and surgery. This study aimed to evaluate the accuracy of core biopsy in predicting these features in patients treated with primary chemotherapy. One hundred and thirty-three patients with carcinoma of the breast diagnosed on clinical, radiological and cytological examination underwent core biopsy, followed by primary chemotherapy (with cyclophosphamide, vincristine, doxorubicin and prednisolone) and surgery. The false-negative rate for pre-treatment core biopsy was 14%, with 91% agreement between the grade demonstrated on core biopsy and that in the residual tumour following completion of chemotherapy. Tumour type in the residual post-chemotherapy tumour was predicted by core biopsy in 84%. This study suggests that pre-treatment core biopsy histology accurately predicts residual tumour histology following primary chemotherapy and surgery in patients with breast cancer. (C) 2002 Elsevier Science Ltd. All rights reserved.