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Biomedical subjects

A Richards

Publications and source records attributed to A Richards.

At least 19 recordsLinked to original sources
PubMed2004

Fluoride-induced alterations of enamel structure: an experimental study in the miniature pig.

We studied the structural changes in the enamel of mandibular third molars of miniature pigs administered a daily oral dose of 2 mg NaF (approximately 0.9 mg of fluoride) per kg body weight (added to the feed) for 1 year. The treatment period covered most of the secretory stage and the entire post-secretory stage of amelogenesis of the M(3). The enamel of the molars from the fluoride-fed pigs appeared opaque and chalky, and the erupted portions were stained brown. The underlying histopathological change was a pronounced subsurface hypomineralization of the enamel beneath a thin surface rim of higher mineral content. This enamel hypomineralization was attributed to a fluoride-induced impairment of the process of enamel maturation. The most conspicuous finding in the fluorotic enamel was the presence of numerous pit-type hypoplastic defects, denoting a marked fluoride-induced disturbance also of the secretory stage of amelogenesis. Microradiography and scanning electron microscopy revealed an enhanced incremental pattern in the outer enamel of the fluorotic molars. Typically, the bottom of larger hypoplastic defects was underlain by a broad, grossly accentuated incremental line. Occurrence of larger hypoplasias was further associated with the presence of aprismatic enamel, the formation of which was attributed to a loss of the prism-forming (distal) portion of the Tomes' processes of secretory ameloblasts. The findings in the miniature pigs closely parallel earlier observations on fluorotic enamel of free-ranging deer and wild boar from fluoride-polluted areas.

Administration, Oral↗
PubMed2004

Complement regulatory proteins are expressed at low levels in embryonic human, wild type and transgenic porcine neural tissue.

Allotransplantation of human foetal neural tissue for neurodegenerative disorders has been shown to provide clinical benefit but is limited by a number of issues including donor supply. The use of porcine foetal tissue as an alternative source of cells is being investigated but xenotransplants survive poorly as a result of immunological rejection, which may involve complement. In this study we investigated the expression of the membrane-bound complement regulatory proteins--decay accelerating factor (DAF), membrane co-factor protein (MCP) and CD59 in embryonic neural tissue. Cells were derived from human foetuses, wild-type porcine foetuses and porcine foetuses transgenic for human complement regulatory proteins and analysed using flow cytometry and immunocytochemistry. Functional assessment of human complement regulatory protein expression in transgenic porcine tissue was assessed by C3b deposition and cell survival on exposure to human complement. Human and wild-type porcine foetal neural tissue expressed moderate levels of MCP and CD59 but low or no levels of DAF. Neural tissue from porcine foetuses transgenic for human MCP (E174) expressed the transgene but failed to significantly inhibit human C3b deposition compared with non-transgenic tissue. In contrast, foetal neural tissue from two different human DAF transgenic pig lines (A74 and E71) known to express high levels of human DAF on endothelial cells, failed to express significant levels of human DAF in foetal neural tissue. Complement regulatory proteins such as MCP and CD59 are expressed in the human and wild-type embryonic brain but in contrast, DAF is expressed at very low levels. Pigs transgenic for human DAF express very low levels of human DAF on embryonic neural tissue. In pigs transgenic for human MCP, the transgene is expressed at similar levels to that in human embryonic neural tissue but at an insufficient level to prevent activation of the complement cascade. Thus alternative approaches to reducing complement activation by xenografted neural foetal tissue will be required if this process proves to be important in the rejection process.

Animals↗
PubMed2002

Alpha Gal is widely expressed in embryonic porcine stem cells and neural tissue.

Fetal porcine neural xenografts are an alternative to human fetal tissue for cell based treatments of a number of neurodegenerative conditions but are currently limited by host immunological rejection. The expression of a major immunological epitope, Galalpha1-3Galbeta1-4GlcNAcbeta-R (alphaGal) was determined on stem cells and primary cells derived from E26 porcine fetal brains. alphaGal was detected on the majority of neural stem cells and cells from primary cell suspensions. The expression of this epitope paralleled the binding of human IgG and IgM to the cells, a binding that was significantly reduced with anti-alphaGal depleted human serum. This study demonstrates that alphaGal expression is extensive in embryonic porcine neural cells and will be of relevance to any clinical trials using this tissue.

Animals↗
PubMed2002

Simultaneous GeneGun immunisation with plasmids encoding antigen and GM-CSF: significant enhancement of murine antivenom IgG1 titres.

GeneGun DNA immunisation is a potent means of inducing antibody-dominant immune responses that we are exploiting to generate venom toxin-specific antibodies to improve the therapy of systemic envenoming by snakes. Here, we report that mice immunised with DNA encoding the carboxyl domain (JD9) of a haemorrhagic Zn metalloprotease (Jararhagin) in venom of the South American pit viper, Bothrops jararaca, and a plasmid expressing murine cytokine granulocyte/macrophage-colony stimulating factor (GM-CSF) raised significantly higher antigen-specific IgG1 titres than mice immunised with JD9 DNA alone. Serological responses to GeneGun JD9 DNA immunisation were shown to be dominated by IgG1, an IgG subclass associated with T lymphocyte helper 2 (Th2) immune responses. Further significant enhancement of JD9-specific IgG1 titres was achieved by increasing the number of immunisations. This report illustrates that DNA immunisation protocols to achieve high-titre, venom toxin-specific antibody production are well advanced and encourage the development of a DNA-based approach to antivenom production.

Animals↗
PubMed2002

Asbestos-related disease.

Until the 1980s, asbestos was widely used throughout the UK. The incidence of asbestos-related disease is still climbing because of the long delay in developing the disease from the initial exposure. The spectrum of diseases encompasses malignant mesothelioma, asbestosis, asbestos-related lung carcinoma and benign pleural disease, including pleural plaques.

Asbestos↗
PubMed2001

Expression of complement regulatory proteins on islets of Langerhans: a comparison between human islets and islets isolated from normal and hDAF transgenic pigs.

BACKGROUND: The expression of regulators of complement activity (RCAs) on islet cells may be of great importance for protecting them against complement-mediated lysis in the immediate posttransplant period after intraportal islet transplantation. We examined porcine and human islet cells for expression of RCA. We also examined to what extent human decay accelerating factor (hDAF) is expressed on adult and fetal islet cells isolated from hDAF transgenic (TG) pigs having a high transgene expression on endothelial cells. Moreover, the susceptibility of the various types of cells to lysis in human serum and blood was investigated. METHODS: Adult human islets (n=5), normal adult and fetal porcine islets (n=9 and n=8, respectively), and islets from adult and fetal hDAF TG pigs (n=5 and n=6, respectively) were examined. With islet single-cell suspensions and flow cytometry, adult human islet cells were examined for expression of hDAF (CD55), hCD59, and human membrane cofactor protein (hMCP; CD46), while porcine islet cells were examined for expression of pCD59 and pMCP. Islet cells from hDAF TG pigs were also examined for hDAF expression. Porcine peripheral blood lymphocytes, normal and hDAF TG porcine endothelial cell lines, a human endothelial cell line, and the human cell line U937 served as controls. Islet cytotoxicity was assayed after incubation of the islet cells with fresh human serum. Furthermore, adult islets from normal control pigs and hDAF TG pigs were exposed to fresh human blood in vitro for 60 min, and the inflammatory reaction elicited was compared between the different types of islets. RESULTS: All human islet cell preparations expressed hCD59, two of five expressed hMCP, but none expressed hDAF. Porcine islet cells expressed both pCD59 and pMCP. Normal adult porcine islet cells exposed to fresh human serum resulted in 74+/-5.4% cell lysis (mean+/-SEM, n=16). In comparison, only 1.3+/-2.8% (n=20, P<0.001) of human islet cells were lysed in the human serum. One islet cell preparation from an hDAF TG pig expressed small amounts of hDAF. This preparation from hDAF TG pigs bound significantly less C3c than did normal control islets (mean fluorescence ratio 16+/-2.2 and 58+/-4.3, respectively; P=0.046) and were partially protected from cell lysis in fresh human serum (47+/-10% and 78+/-18% cell lysis, respectively; P=0.046). The other four preparations from hDAF TG pigs were negative for hDAF and were equally susceptible to lysis as normal control islets. All fetal pancreatic islet cells from hDAF TG pigs analyzed were negative for hDAF expression. When exposed to fresh human blood in vitro, adult and fetal islets from hDAF TG pigs elicited equally strong inflammatory changes as did the normal control islets. The inflammatory changes were characterized by activation of the complement and coagulation systems, resulting in islet damage with "dumping" of insulin into the blood. CONCLUSIONS: Porcine and human islet cells express species-restricted complement regulatory proteins, with the human islet cells expressing mainly hCD59. A low expression of hDAF was detected on islet cells from one of five hDAF TG pigs. These islet cells displayed reduced islet cell cytotoxicity in fresh human serum. We conclude that protection from complement-mediated lysis will be important in the context of intraportal pig-to-human islet transplantation, and expression of a human RCA on islet cells should be beneficial in this context.

Animals↗
PubMed2001

Observation of high-energy neutrinos using Cerenkov detectors embedded deep in Antarctic ice.

Neutrinos are elementary particles that carry no electric charge and have little mass. As they interact only weakly with other particles, they can penetrate enormous amounts of matter, and therefore have the potential to directly convey astrophysical information from the edge of the Universe and from deep inside the most cataclysmic high-energy regions. The neutrino's great penetrating power, however, also makes this particle difficult to detect. Underground detectors have observed low-energy neutrinos from the Sun and a nearby supernova, as well as neutrinos generated in the Earth's atmosphere. But the very low fluxes of high-energy neutrinos from cosmic sources can be observed only by much larger, expandable detectors in, for example, deep water or ice. Here we report the detection of upwardly propagating atmospheric neutrinos by the ice-based Antarctic muon and neutrino detector array (AMANDA). These results establish a technology with which to build a kilometre-scale neutrino observatory necessary for astrophysical observations.

Journal Article↗
PubMed2001

Factor H mutations in hemolytic uremic syndrome cluster in exons 18-20, a domain important for host cell recognition.

Several recent studies have established an association between abnormalities of complement factor H (FH) and the development of hemolytic uremic syndrome (HUS). To identify the relative importance of mutations in FH as a cause of HUS, we have undertaken mutation screening of the FH gene in 19 familial and 31 sporadic patients with FH. Mutations were found in two familial and three sporadic patients, and these clustered in exons 18-20, a domain important for host recognition. Moreover, this study demonstrates that familial HUS is likely to be a heterogeneous condition.

Amino Acid Sequence↗
PubMed2001

A rapid method for the identification and differentiation of Helicoverpa nucleopolyhedroviruses (NPV Baculoviridae) isolated from the environment.

A diagnostic method is described for the identification and differentiation of nucleopolyhedrovirus (NPV) pathogens of Helicoverpa species (Lepidoptera: Noctuidae) isolated from the environment. The method is based on the polymerase chain reaction (PCR) used in conjunction with restriction fragment length polymorphism (RFLP) analysis and comprises three parts. The first part describes procedures for obtaining PCR quality viral DNA from individual diseased H. armigera cadavers recovered during bioassay analyses of soil and other types of environmental sample. These procedures were modified from standard techniques used for the routine purification and dissolution of NPV polyhedra and provided an overall PCR success rate of 95% (n=60). The second part describes the design of several sets of PCR primers for generating DNA amplification products from closely and distantly related NPVs. These PCR primers were designed from published DNA sequence data and from randomly cloned genomic DNA fragments isolated from a reference H. armigera SNPV (HaSNPV) isolate. The final part of the method describes how specific PCR products when digested with specific restriction endonuclease enzymes, can be used to generate diagnostic DNA profiles (haplotypes) that can be used both to identify heterologous NPVs e.g. Autographa californica MNPV and related viruses, and to differentiate genotypic variants of Helicoverpa SNPV. In the latter case, only two PCR products and four restriction digests were required to differentiate a reference set of 10 Helicoverpa SNPV isolates known to differ 0.1--3.5% at the nucleotide level. The diagnostic method described below marks the second part of a two-phase quantitative-diagnostic protocol that is now being applied to a variety of ecological investigations. In particular, its application should lead to a significant improvement in our understanding of the distribution and population genetics of Helicoverpa SNPVs in the Australian environment, as well as providing a sound basis for the design of pre- and post-release monitoring systems for genetically enhanced bioinsecticides. It is also likely that this method can be adapted readily to the study of other insect pathogen associations important economically.

Animals↗
PubMed2001

Porcine neural xenografts in the immunocompetent rat: immune response following grafting of expanded neural precursor cells.

Intracerebral neural xenografts elicit a host immune response that results in their rapid rejection. This forms a key barrier to the therapeutic use of xenogeneic tissue transplantation for conditions such as Parkinson's disease. The current study sought to provide insight into the cellular components of donor cell suspensions that are important in stimulating the host rejection response and thereby to suggest rational manipulations of xenogeneic donor tissue that might ultimately enhance its clinical utility. The neural stem cell mitogens, epidermal growth factor and fibroblast growth factor-2, have been used to isolate and expand populations of primordial neural precursor cells from the embryonic pig brain. The immune response elicited by these cells on transplantation into the non-immunosuppressed rat has been fully characterised. In the first experiments, expanded neural precursors were grafted into the hemi-parkinsonian, non-immunosuppressed Sprague-Dawley rat and graft status and host response examined 10, 21, 35 and 60 days post-transplantation. While equivalent primary tissue grafts were completely eliminated at 35 days, grafts of expanded neural precursors with healthy neurofilament-positive projections were present at all time-points, and two large grafts remained even at 60 days. Some grafts appeared to elicit minimal host immune responses at the time-points they were examined, although most did appear to be undergoing a rejection process since a co-ordinated response involving host cytotoxic T-lymphocytes, microglia/macrophages, immunoglobulin M and complement could be demonstrated to varying degrees. Subsequent experiments went on to demonstrate further that expanded precursor populations and primary tissue suspensions differed in their immunogenic profile. Firstly, when primary tissue was injected intraperitoneally into immunocompetent rats a vigorous primary humoral response was generated. No such response was detected following injection of expanded neural precursors. Secondly, flow cytometric analysis revealed small but significant levels of class II porcine major histocompatibility complex expression in primary cell suspensions but no such expression in expanded precursor populations.The results of this study therefore demonstrate that the immunogenicity of porcine neural cell suspensions used for intracerebral grafting is reduced when neural stem cell mitogens are used to expand precursor cells. The implications of these findings in the development of novel xenogeneic cellular therapies for neurodegenerative conditions such as Parkinson's disease are discussed.

Animals↗
PubMed2001

Performance anxiety: loss of the spoken edge.

The study aimed to assess whether a specific training program in vocal and physical skills could reduce the level of perceived performance anxiety. Eighteen undergraduate performing arts students were divided into two even groups. The experimental group experienced 10 two-hour sessions in a specialized vocal and physical skills training program. The control group received the same number of sessions in text analysis by the same teacher. Ten weeks after the training period, both groups were videotaped delivering a short speech of general interest. The videotaped material was assessed by four judges, using a visual analog scale (VAS). Perceptual variables considered were eight vocal and physical features: physical ease, physical presence, effective gesture use, effective eye contact, correct breath use, suitable pace, vocal variety, speech clarity, and one performance feature: perceived performance anxiety. Results indicated that the particular vocal and physical skills training program used showed positive results in effectively reducing the level of perceived performance anxiety. The experimental group scored significantly higher on each of the eight vocal and physical features and significantly lower on the perceived anxiety feature than the control group. This research has implications for those affected by performance anxiety in vocal presentation.

Adolescent↗
PubMed2001

Inhibitors of caspase homologues suppress an apoptotic phenotype in cultured rabbit corpora lutea.

Apoptosis is a morphologically defined type of cell death initiated by various stimuli that results in the activation of caspases (cysteine-containing aspartate-specific proteases). In the present study, it was determined that caspases are present during, and play a role in, corpus luteum (CL) apoptosis in vitro. Pseudopregnancy was induced in rabbits with 100 IU human chorionic gonadotrophin. On Day 11 of pseudopregnancy, CL were isolated and cultured for 0, 2, 4, 6, and 8 h in the absence of trophic support to induce spontaneous apoptosis. Total RNA was extracted and analysed for caspase-I expression by Northern blot analysis. The results demonstrated caspase-I expression from 4 h. In the second part of the study, CL were incubated without trophic support for 4 h with increasing concentrations of three general caspase inhibitors, sodium aurothiomalate (SAM), iodoacetic acid (IAA) and N-tosyl-L-phenylalanylchloromethylketone (TPCK), and two specific caspase inhibitors, N-acetyl (Ac)-Tyr-Val-Ala-Asp (YVAD)-chloromethylketone (CMK) (Ac-YVAD-CMK) and Ac-Asp-Glu-Val-Asp (DEVD)-aldehyde (CHO) (Ac-DEVD-CHO). At completion, DNA was isolated and integrity assessed. Treatment of CL with SAM, IAA or Ac-DEVD-CHO effectively suppressed apoptotic DNA fragmentation. The final component of the study was to examine caspase-3 protein expression. Western blot analysis revealed a significant increase in caspase-3 expression over the experimental time-course. The results of the present study clearly demonstrate a time-dependent link between the caspases, specifically caspase-3 and spontaneous apoptosis in the rabbit CL.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Characterization and functional analysis of laminin isoforms in human bone marrow.

Laminins are a family of disulfide-linked heterotrimeric proteins consisting of 3 different subunits termed alpha, beta, and gamma chains. Combinations of 11 characterized laminin subunits (alpha 1-alpha 5, beta 1-beta 3, and gamma 1-gamma 3) generate at least 12 laminin isoforms, which can serve different functions. Although expression of laminin in the hematopoietic microenvironment has been known for many years, the nature of the laminin isoforms present in the human bone marrow is poorly characterized. The present study attempts to clarify this issue. Reverse transcriptase-polymerase chain reaction analysis of human bone marrow stromal cells suggested the expression of many laminin isoforms in the marrow. Northern blot and immunoblot analysis, however, showed that laminin-8/9 and laminin-10/11 are the most abundant laminin isoforms synthesized by human bone marrow stromal cells. Other isoforms, if present, certainly play a minor role in the hematopoietic microenvironment. Functionally, laminin-10/11 preparations showed strong adhesive interactions with human CD34(+) cell lines. Antibodies against the beta 1 integrin subunit inhibited these interactions. Other laminin isoforms, especially laminin-1 and laminin-2/4, showed only weak or no adhesive interactions with the hematopoietic cell lines tested, explaining former negative results. In addition to its adhesion-mediating properties, laminin-10/11 preparations also showed a mitogenic activity for human hematopoietic progenitor cells. Taken together, these data suggest that laminin in the bone marrow plays a hitherto unexpected important function in the development of hematopoietic progenitor cells. (Blood. 2000;96:4194-4203)

Blotting, Western↗
PubMed2000

HLA-DR-promiscuous T cell epitopes from Plasmodium falciparum pre-erythrocytic-stage antigens restricted by multiple HLA class II alleles.

Previously, we identified and established the antigenicity of 17 CD8+ T cell epitopes from five P. falciparum Ags that are restricted by multiple common HLA class I alleles. Here, we report the identification of 11 peptides from the same Ags, cicumsporozoite protein, sporozoite surface protein 2, exported protein-1, and liver-stage Ag-1, that bind between at least five and up to 11 different HLA-DR molecules representative of the most common HLA-DR Ags worldwide. These peptides recall lymphoproliferative and cytokine responses in immune individuals experimentally immunized with radiation-attenuated Plasmodium falciparum sporozoites (irradiated sporozoites) or semi-immune individuals naturally exposed to malaria in Irian Jaya or Kenya. We establish that all peptides are recognized by individuals of each of the three populations, and that the frequency and magnitude of helper T lymphocyte responses to each peptide is influenced by the intensity of exposure to P. falciparum sporozoites. Mean frequencies of lymphoproliferative responses are 53.2% (irradiated sporozoites) vs 22.4% (Kenyan) vs 5.8% (Javanese), and mean frequencies of IFN-gamma responses are 66.3% (irradiated sporozoites) vs 27.3% (Kenyan) vs 8. 7% (Javanese). The identification of HLA class II degenerate T cell epitopes from P. falciparum validates our predictive strategy in a biologically relevant system and supports the potential for developing a broadly efficacious epitope-based vaccine against malaria focused on a limited number of peptide specificities.

Adolescent↗
PubMed2000

Disturbed enamel formation in wild boars (Sus scrofa L.) from fluoride polluted areas in Central Europe.

The pathological alterations of enamel structure in the teeth of wild boars from fluoride polluted areas in N-Bohemia (Czech Republic) and S-Saxony (Germany) were studied on a macroscopic and a microscopic level. Mandibular bone fluoride concentration (mg F(-)/kg, dry wt; mean +/-SD, individuals <24 months of age) in the specimens from N-Bohemia (754.3+/-149.6) and S-Saxony (490.8+/-135.1) was significantly higher than that of controls (free of dental fluorosis), originating from the western part of Germany (304.7+/-91.0). Fluoride content in bulk enamel (mg F(-)/kg, ash wt) of fluorotic permanent teeth from N-Bohemia (382.1+/-165.2) and S-Saxony (125.0+/-38.3) was likewise significantly increased over that of non-fluorotic control teeth from W-Germany (33.6+/-26.7). Macroscopically, fluorosed wild boar enamel exhibited opacity and discoloration of varying extent, accentuated perikymata as well as hypoplastic and posteruptive surface defects. Microradiographic and scanning electron microscopic analyses revealed enamel subsurface hypomineralization, accentuated Retzius lines and occurrence of broad, hypomineralized incremental bands of abnormal structure underlying hypoplastic enamel surface defects. The presence of zones of aprismatic enamel was associated with these bands. Incremental bands with altered enamel structure and enamel surface hypoplasias, both denoting a severe disturbance during the secretory stage of amelogenesis, have previously been observed in rodents following acute parenteral fluoride dosing. It is concluded that in the chronically fluoride exposed wild boars periods of especially elevated plasma fluoride levels exerted an acute toxic effect on the secretory ameloblasts. A feature not previously reported from fluorosed enamel was the occurrence of canal-like structures that originated at the broad incremental bands and extended into the external enamel. The presence of these canals presumably results from a delay in the resumption of secretory activity by groups of ameloblasts following a fluoride insult. Based on experimental evidence in domestic pigs and in sheep, the overall subsurface hypomineralization of fluorosed wild boar enamel is attributed to a disturbance of enamel maturation. The distribution of fluorotic enamel changes within the dentition of the wild boars could be related to the developmental sequence of tooth formation in the species. Teeth whose crown formation took place prenatally (deciduous teeth) or largely pre-weaning (permanent first molars) exhibited no or only moderate fluorotic enamel alterations. Based on the extension of enamel surface hypoplasias along the coronoapical axes of the tooth crowns, the timing of excess fluoride exposure that caused a marked disruption of enamel matrix secretion was estimated in specimens with a known date of death. The results indicate that the wild boars had been exposed to a particularly severe fluoride impact during autumn and winter of their first year of life.

Age Factors↗