Biomedical subjects

R Harrop

Publications and source records attributed to R Harrop.

At least 19 recordsLinked to original sources

PubMed2001

The schistosome egg: development and secretions.

We have investigated the development of the schistosome egg and its secretions in order to understand how it migrates through gut tissues and also initiates pathology in the liver. We show by electron microscopy that the subshell envelope is absent in the newly deposited egg, but appears very early and differentiates as development progresses. In the mature egg, this nucleated envelope contains extensive endoplasmic reticulum, suggestive of a protein synthetic capacity. Furthermore, Reynolds' layer only appears between the envelope and the egg-shell in the mature egg and may represent its accumulated secretions. We have biosynthetically labelled and collected the secretions (ESP) released by mature but not immature eggs during culture. Their fractionation by SDS-PAGE reveals a simple pattern of 6 bands, differing markedly in composition from soluble egg antigen preparations. Electrophoresis in casein substrate gels demonstrates the presence of 2 distinct proteases in the egg secretions. By immunocytochemistry, ESP localized predominantly to the envelope of the mature egg, suggesting that this layer rather than the miracidium is the source of egg secretions.

Animals↗

PubMed2000

Characterization, cloning and immunogenicity of antigens released by transforming cercariae of Schistosoma mansoni.

A schistosome infection is initiated when the parasite penetrates the skin of a susceptible host. Relatively large quantities of protein are released by transforming cercariae compared to later larval stages. This represents the first parasite material to which the host's immune system is exposed, yet little is known about the proteins which are released during the first few hours post-transformation. We have shown that antiserum raised against such molecules was capable of imparting protection against a schistosome challenge infection upon passive transfer to naïve mice. By screening a cercarial cDNA library with this serum, 38 positive clones were identified. Sequence analysis showed these to represent 8 different molecules which included Schistosoma mansoni 21-7 kDa antigen, calcium-binding-protein and the vaccine candidate glutathione S-transferase (Sm28GST). In addition, 5 clones were isolated, 1 of which had significant homology to many cytochrome C proteins, another with leukocyte elastase inhibitors and 3 which represented novel molecules. Four clones were expressed in a prokaryotic high-level expression vector, sera produced against each purified recombinant protein and used subsequently to probe Western blots and parasite sections. The leukocyte elastase inhibitor homologue and 2 unknowns induced significant proliferation by lymph node cells recovered from mice vaccinated with irradiated cercariae. More strikingly, the 2 novel proteins stimulated very high levels of interferon gamma (IFNgamma) secretion both by lymph node cells and those recovered by broncho-alveolar lavage from the lungs of vaccinated mice. Such results will be discussed in the context of vaccine development.

Amino Acid Sequence↗

PubMed2000

Performance of the dynamic single photon emission computed tomography (dSPECT) method for decreasing or increasing activity changes.

Radionuclide imaging is now widely used whenever functional information is required. We present a new approach to dynamic SPECT imaging (dSPECT method) that uses a single slow rotation of a conventional camera and allows us to reconstruct a series of 3D images corresponding to the radiotracer distribution in the body at various times. Using simulations of various camera configurations and acquisition protocols, we have shown that this method is able to reconstruct washout half-lives with an accuracy greater than 90% when used with triple-head SPECT cameras. Accuracy decreases when using fewer camera heads, but dual-head geometries still give an accuracy greater than 80% for short and 90% for long half-lives and about 50-75% for single-head systems. Dynamic phantom experiments have yielded similar results. Presence of attenuation and background activity does not affect the accuracy of the dSPECT reconstructions. In all situations investigated satisfactory dynamic images were produced. A preliminary normal volunteer study measuring renal function was performed. The reconstructed dynamic images may be presented as a three-dimensional movie showing movement of the tracer through the kidneys and the measurement of the regional renal function can be performed. The time-activity curves determined from this dSPECT data are very similar to those obtained from dynamic planar scans.

Heart↗

PubMed2000

Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein.

The E2 protein of hepatitis C virus (HCV) is believed to be a virion surface glycoprotein that is a candidate for inclusion in an antiviral vaccine. A truncated soluble version of E2 has recently been shown to interact with CD81, suggesting that this protein may be a component of the receptor for HCV. When expressed in eukaryotic cells, a significant proportion of E2 forms misfolded aggregates. To analyze the specificity of interaction between E2 and CD81, the aggregated and monomeric forms of a truncated E2 glycoprotein (E2(661)) were separated by high-pressure liquid chromatography and analyzed for CD81 binding. Nonaggregated forms of E2 preferentially bound CD81 and a number of conformation-dependent monoclonal antibodies (MAbs). Furthermore, intracellular forms of E2(661) were found to bind CD81 with greater affinity than the extracellular forms. Intracellular and secreted forms of E2(661) were also found to differ in reactivity with MAbs and human sera, consistent with differences in antigenicity. Together, these data indicate that proper folding of E2 is important for its interaction with CD81 and that modifications of glycans can modulate this interaction. Identification of the biologically active forms of E2 will assist in the future design of vaccines to protect against HCV infection.

Amino Acid Sequence↗

PubMed1999

Characterization, cloning and immunogenicity of antigens released by lung-stage larvae of Schistosoma mansoni.

Lung-stage schistosomula are the target of protective immunity in mice vaccinated with attenuated cercariae of Schistosoma mansoni. Therefore, proteins present at this developmental stage, and in particular those which are secreted, are a potential source of novel vaccine candidates. However, little information is available about such molecules. Here we describe the cDNA clones identified by screening expression libraries with serum raised against proteins released by lung-stage schistosomula. In total, 11 different cDNA species were identified, 6 of which have been described previously in S. mansoni; these included fructose 1,6-bisphosphate aldolase and Sm21.7 which together accounted for two-thirds of all positive clones. Of the 5 newly described schistosome genes, 1 cDNA had a high degree of homology to the s5a subunit of 26S proteasomes, most significant being with the human protein. The remaining 4 clones showed no significant homologies to any genes sequenced previously. Fructose 1,6-bisphosphate aldolase, Sm21.7, the proteasome homologue and 1 unknown clone (A26) have been expressed in a bacterial expression system and serum produced against each recombinant protein. Immunolocalization showed fructose 1,6-bisphosphate aldolase, Sm21.7 and the proteasome homologue to be most abundant in muscle cells whilst clone A26 was distributed throughout many tissues, but was most abundant in the tegument. Analysis of the cellular immune responses of vaccinated mice showed 3 of the 4 expressed clones to be highly immunogenic, inducing the secretion of large quantities of the Th1-type cytokine interferon gamma.

Amino Acid Sequence↗

PubMed1998

Multiple line source array for SPECT transmission scans: simulation, phantom and patient studies.

UNLABELLED: Accurate attenuation and scatter corrections in quantitative SPECT studies require attenuation maps of the density distribution in the scanned object. These can be obtained from simultaneous emission/transmission scans. METHODS: A new method has been developed using a multiple line source array (MLA) for transmission scans, and its performance has been investigated using computer simulations and experimental data. The activity in the central lines of the MLA was higher than at the edges of the system, so that more transmission photons would be directed toward the thicker parts of the human body. A series of transmission-only and simultaneous emission/transmission studies were performed for different phantom configurations and human subjects. Attenuation maps were generated and used in reconstruction of attenuation-corrected emission images. RESULTS: The mu coefficients for attenuation maps obtained using the MLA system and simulated and experimental data display no artifacts and are qualitatively and quantitatively correct. For phantoms, the agreement between the measured and the true value of mu for water was found to be better than 4%. The attenuation-corrected emission images for the phantom studies demonstrate that the activity in the heart can be accurately reconstructed. A significant qualitative improvement was also obtained when the attenuation correction was used on patient data. CONCLUSION: Our results indicate that the MLA transmission source can be used in simultaneous transmission/emission imaging to generate accurate attenuation maps. These maps allow for performing an object-specific, attenuation correction of the emission images.

Computer Simulation↗

PubMed1997

Evaluation of cDNA libraries from different developmental stages of Schistosoma mansoni for production of expressed sequence tags (ESTs).

A comparative study of the gene expression profile in different developmental stages of Schistosoma mansoni has been initiated based on the expressed sequence tag (EST) approach. A total of 1401 ESTs were generated from seven different cDNA libraries constructed from four distinct stages of the parasite life cycle. The libraries were first evaluated for their quality for a large-scale cDNA sequencing program. Most of them were shown to have less than 20% useless clones and more than 50% new genes. The redundancy of each library was also analyzed, showing that one adult worm cDNA library was composed of a small number of highly frequent genes. When comparing ESTs from distinct libraries, we could detect that most genes were present only in a single library, but others were expressed in more than one developmental stage and may represent housekeeping genes in the parasite. When considering only once the genes present in more than one library, a total of 466 unique genes were obtained, corresponding to 427 new S. mansoni genes. From the total of unique genes, 20.2% were identified based on homology with genes from other organisms, 8.3% matched S. mansoni characterized genes and 71.5% represent unknown genes.

Animals↗

PubMed1997

Minilibraries constructed from cDNA generated by arbitrarily primed RT-PCR: an alternative to normalized libraries for the generation of ESTs from nanogram quantities of mRNA.

The generation of expressed sequenced tags (ESTs) depends on the arbitrary selection of individual cDNA clones from libraries. The efficiency of this process reflects the clonal structure of the library used and can be significantly increased using size selected, directional, normalized cDNA libraries. This strategy, however, is not readily applicable when mRNA is limiting, as is the case in the study of complex microorganisms such as parasites, fetal tissues or tumor biopsies. We show here that the construction and systematic sequencing of minilibraries of cDNAs produced by arbitrarily primed PCR provides an alternative means of efficiently generating ESTs in situations where only nanogram quantities of RNA are available. This methodology greatly compensates for unequal message abundance, avoids the need for complex library construction, is equally applicable to the analysis of abundant or rare biological material and is ideally suited to multicenter programmes.

Animals↗

PubMed1996

Potential allergens stimulate the release of mediators of the allergic response from cells of mast cell lineage in the absence of sensitization with antigen-specific IgE.

A number of structurally diverse antigens preferentially stimulate the synthesis of IgE antibodies, but no unifying principle has been proposed that explains the nature of isotype selection. In the present study, we show that common allergens present in bee venom, house dust mite emanations and parasite proteins induce mast cell and basophil degranulation and stimulate interleukin-4 synthesis, and secretion in the absence of antigen-specific IgE. These data point to a linkage between the initial activation of cells of the innate immune system and subsequent adaptive immune responses. They suggest that IgE-independent mast cell and basophil degranulation is predictive of potential allergenicity and can be evaluated by means of a cellular assay. Our study indicates that non-immunological degranulation by prototypic allergens, such as bee venom phospholipase A2 or proteases associated with house dust mite emanations, is critically dependent on enzymatic activity. These findings have potentially important implications for vaccine design in allergic and parasitic disease.

Allergens↗

PubMed1996

Characterization of dose distribution in radiation therapy plans.

As a method of considering only significant radiation doses to different tissues, the ICRU Report 50 recommends taking the dose given to a significant tissue volume (minimum diameter greater then 15 mm) instead of choosing a single, potentially insignificant, voxel value. In order to find this significant volume, we have adapted an emission imaging analysis method to radiation therapy planning. The resulting method finds and characterizes the dose distribution in the volumes of interest in a way that includes spatial arrangement. The data can be used to signal significant hot or cold volumes in the dose plan and to score the plans based on significant dose to the tissues.

Humans↗

PubMed1995

Antigens derived from lung-stage larvae of Schistosoma mansoni are efficient stimulators of proliferation and gamma interferon secretion by lymphocytes from mice vaccinated with attenuated larvae.

Protective immunity in C57BL/6 mice exposed to optimally irradiated larvae of Schistosoma mansoni operates against challenge parasites in the lungs and is dependent upon T-helper 1 (Th1) lymphocytes which secrete abundant gamma interferon (IFN-gamma). As an initial step in the identification of the molecules which mediate this immunity, antigenic materials released by larvae at various times during in vitro culture were compared for the ability to induce proliferation of lymph node cells recovered from mice 4 to 6 days after exposure to attenuated parasites. Cells from mice vaccinated with cercariae proliferated most strongly to larval antigens released soon after transformation. In contrast, cells from mice immunized with lung-stage schistosomula responded poorly to these early secretions but proliferated vigorously to antigens released by older larvae. In further studies on the cytokine profile of the responding lymphocytes, it was observed that the balance between IFN-gamma and interleukin-4 (IL-4) secretion depended on the source of antigen used for restimulation. Thus, material released between days 6 and 8 by in vitro-cultured larvae, and the soluble extracts of whole lung-stage larvae, induced abundant IFN-gamma but little IL-4. This finding implies that an overwhelming proportion of the lymphocytes responsive to lung-stage antigens had the Th1 phenotype. In contrast, antigens from cercariae and skin-stage larvae induced the lowest levels of IFN-gamma but higher levels of IL-4. It appears that a proportion of the cells with specificities for early antigens had the Th2 or Th0 phenotype. Our results emphasize that antigens from lung-stage larvae are an important source of potentially protective molecules.

Age Factors↗

PubMed1993

Protein synthesis and release by cultured schistosomula of Schistosoma mansoni.

The lung schistosomulum of Schistosoma mansoni is the target of protective immunity in mice singly vaccinated with irradiated cercariae. Since the effector responses are T cell-mediated, their initiation requires the release of antigens from the intact parasite. We have used the technique of biosynthetic labelling with [35S]methionine, before and after transformation of the cercariae, to analyse the kinetics of protein synthesis and release by the schistosomulum. In addition, the proteins present in the soluble fraction of the parasite and those released during in vitro culture have been characterized. During a 7-day culture period schistosomula derived from labelled cercariae lost proteins most rapidly within the first 3 h after transformation. Two proteins of molecular weight 61 and 20 kDa were dominant and may correspond to areas of proteolytic activity. Analysis of the rate of protein synthesis of schistosomula labelled after transformation revealed four different phases, which may relate to the developmental processes occurring in vivo. During the first 24 h, synthesis was very low, increasing to a plateau and then rising to a peak at day 8; therefore the rate declined rapidly. Whilst some stage-specific synthesis of proteins was detected in the soluble fractions of the parasite bodies, the pattern of proteins released by cultured larvae was remarkably uniform. At least 15 proteins were detected by autoradiography with bands at 61, 45 and 20 kDa being particularly prominent. These proteins merit further study as potential mediators of the protective immune response.

Animals↗

PubMed1993

Irradiation of Schistosoma mansoni cercariae impairs neuromuscular function in developing schistosomula.

Optimally attenuated larvae of Schistosoma mansoni (20 krad) are incapable of migrating farther than the lungs, and they induce high levels of resistance in mice to a subsequent challenge infection. The effect of gamma radiation on the parasite is described and related to the migratory pattern of attenuated larvae. Scanning electron microscopy revealed marked morphological differences between normal and irradiated larvae at the lung stage of development. The latter exhibited random constrictions, probably resulting from contraction of circular muscle fibers, at intervals along the length of the body and showed subtle differences in motility. We suggest these abnormalities account for persistence of attenuated larvae in the skin-draining lymph nodes and lungs, 2 events that are instrumental to the induction of protective immunity in this vaccine model.

Animals↗

PubMed1992

Full data utilization in PVI using the 3D radon transform.

An algorithm is described for three-dimensional (3D) image reconstruction in positron volume imaging (PVI) using the inversion of the 3D radon transform (RT) for a truncated cylindrical detector geometry. A discrete version of the 3D RT is formed in a 3D histogram from the event-line coordinates of the detected events. The histogram entries represent the plane integrals of the activity in the field of view. Conventional inversion of the x-ray transform (XT) excludes oblique events in non-iterative reconstruction methods. by employing a spatially varying angular acceptance of events into the histogrammed plane integrals of the 3D RT, it is possible to include most of the detected oblique events in the reconstruction of the image using the standard 3D RT inversion formula. the oblique events are added to the histogram with a partial weight compared to those in complete (XT) projections. This single-pass reconstruction image has better statistical noise properties than images formed by RT inversion from complete XT projections, but only for some detector geometries is it significantly better. Monte Carlo simulations were used to study the statistical noise in images reconstructed using the new algorithm. The inherent difference in the axial versus the transaxial statistical noise in images reconstructed from truncated detectors is noted and is found to increase by including oblique events with this new algorithm.

Algorithms↗

PubMed1990

Correlations of regional postmortem enzyme activities with premortem local glucose metabolic rates in Alzheimer's disease.

Correlations were sought between local cerebral metabolic rates (LCMRs) for glucose in various regions of the cortex, determined in premortem PET scans, with the regional activities of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), beta-glucuronidase (Gluc, a probable index of reactive gliosis), and phosphate-activated glutaminase (PAG, a possible indice of the large pyramidal neurons) measured on postmortem tissue. Significant negative correlations between LCMRs and Gluc activities were found in 6 PET-scanned cases of Alzheimer disease (AD), and positive correlations of LCMRs with PAG were found in 5. By contrast, a positive correlation with ChAT and AChE was found in only 1. The results are consistent with the metabolic deficits in AD being primarily a reflection of local neuronal loss and gliosis. Similar data on two cases of Huntington's disease showed no significant correlations, while 1 patient with Parkinson dementia showed a significant (negative) correlation only with Gluc.

Aged↗

PubMed1990

18Fluorodeoxyglucose positron emission tomography studies in presumed Alzheimer cases, including 13 serial scans.

Positron emission tomographic (PET) data on local cerebral metabolic rates for glucose (LCMR) are reported for 32 regions of interest (ROI)s in cross-sectional studies on 57 patients with clinically diagnosed Alzheimer's disease (AD) and 20 neurologically normal controls, and in serial studies on 13 of the AD cases, including a familial, young-onset case where the diagnosis has been confirmed at autopsy. Extensive psychological testing was done on all the AD cases. Almost all cortical regions showed a significant decline in LCMR with age in the control subjects. There were the expected cortical metabolic deficits in AD and the serial studies showed a general increase in such deficits over time in 12 of the 13 cases. The regions showing the greatest declines with time in serial studies are the same as those showing the most severe deficiencies in cross-sectional studies. The young-onset case did not show a greater rate of metabolic decline than many of the older cases studied. Results on individual psychological tests tended to correlate with metabolic rates in multiple, rather than single, cortical regions, suggesting intact neuronal networks are required for good performance. The correlations with cortical metabolic activity found were of a sign indicating that the higher the metabolic rates and the better the left:right asymmetry index, the better was the performance.

Aged↗

PubMed1989

Cortical glutaminase, beta-glucuronidase and glucose utilization in Alzheimer's disease.

Large pyramidal neurons of rat and human neocortex stain immunohistochemically for phosphate-activated glutaminase (PAG). In a limited number of postmortem brains, we find large reductions in cortical PAG activity in Alzheimer's disease (AD). This finding is consistent with histological evidence that pyramidal neurons are affected in AD. The reductions are greater than those found in the same samples in choline acetyltransferase (ChAT) but the possible deleterious effects of coma and similar premortem factors on human PAG activity have yet to be assessed. The activity of beta-glucuronidase, a lysosomal enzyme which occurs in reactive astrocytes, is elevated in the same samples. Positron emission tomography (PET) studies, using 18F-fluorodeoxyglucose (FDG), have demonstrated significant deficiencies in glucose metabolism in the cortex in AD, with the parietal, temporal and some frontal areas being particularly affected. We found in serial scans of 13 AD cases, including one relatively young (44-46 year old) familial case, an exacerbation of the defect over time in most cases. We have found a negative correlation between the regional metabolic rates for glucose (LCMR(s] measured premortem and the beta-glucuronidase activities measured postmortem on a few AD cases that have come to autopsy. The correlations between LCMR(s) and PAG and ChAT activities tend to be positive. The results are consistent with previous suggestions that decreased LCMR(s) in AD reflect local neuronal loss and gliosis.

Adult↗

PubMed1989

Crossed cerebellar and uncrossed basal ganglia and thalamic diaschisis in Alzheimer's disease.

We detected crossed cerebellar as well as uncrossed basal ganglia and thalamic diaschisis in Alzheimer's disease by positron emission tomography (PET) using 18F-fluorodeoxyglucose. We studied a series of 26 consecutive, clinically diagnosed Alzheimer cases, including 6 proven by later autopsy, and compared them with 9 age-matched controls. We calculated asymmetry indices (AIs) of cerebral metabolic rate for matched left-right regions of interest (ROIs) and determined the extent of diaschisis by correlative analyses. For the Alzheimer group, we found cerebellar AIs correlated negatively, and thalamic AIs positively, with those of the cerebral hemisphere and frontal, temporal, parietal, and angular cortices, while basal ganglia AIs correlated positively with frontal cortical AIs. The only significant correlation of AIs for normal subjects was between the thalamus and cerebral hemisphere. These data indicate that PET is a sensitive technique for detecting diaschisis.

Alzheimer Disease↗