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Biomedical subjects

P G Kennedy

Publications and source records attributed to P G Kennedy.

At least 19 recordsLinked to original sources
PubMed2006

The effect of different pine hosts on the sampling of Rhizopogon spore banks in five Eastern Sierra Nevada forests.

Our primary goal was to determine whether detection of Rhizopogon internal transcribed spacer (ITS) groups is affected by the pine species used in seedling bioassays. Our secondary goal was to investigate composition of Rhizopogon spore banks in the Eastern Sierra Nevada of California, a previously unsampled region. We used seedlings of Pinus contorta, Pinus jeffreyi, Pinus lambertiana, and Pinus muricata as bioassay plants and identified the Rhizopogon retrieved by internal transcribed spacer (ITS) sequence analysis. We found that each of the pine species retrieved all of the abundant Rhizopogon ITS groups, but there were significant differences among pines in the richness of Rhizopogon ITS groups recovered. Pinus muricata recovered all ITS groups found in this study and was significantly better than P. lambertiana. Rhizopogon communities from the five sampled sites contained six to eight ITS groups per site, with two unique sequence groups and a higher abundance of the Rhizopogon ellenae and Rhizopogon arctostaphyli groups than at previously sampled sites. These results show high cross-receptivity between Rhizopogon and pine species, and regional patterns in spore bank composition.

Basidiomycota↗
PubMed2004

The importance of seed reserves for seedling performance: an integrated approach using morphological, physiological, and stable isotope techniques.

To investigate how seed reserves affect early seedling performance, we conducted a factorial greenhouse experiment using Lithocarpus densiflora (Tanoak). Seedlings were grown from large (5.8+/-0.7 g) and small (3.2+/-0.4 g) seeds and, following shoot emergence, seeds were either removed or left attached. Seedlings were harvested for quantification of biomass and delta13C at seven time periods following seed removal (2, 4, 8, 16, 32, 64, 128 days) and seedling photosynthesis was measured three separate time periods (2-4, 49-82, 95-128 days after seed removal). Biomass increased for all seedlings, but the increase was significantly larger for seedlings with attached seeds than with removed seeds. Seed removal just after shoot emergence significantly decreased seedling biomass, but seed removal 64 days after shoot emergence had no effect on seedling biomass. Seedling photosynthesis per unit leaf area varied by time and seed presence, but not by seed size. At the first period, seedlings with attached seeds had significantly higher photosynthetic rates than seedlings with removed seeds, at the second period there was no effect of seed removal, and at the third time period seedlings with attached seeds had significantly lower photosynthetic rates than seedlings with removed seeds. Despite temporal variation in photosynthesis per unit leaf area, seedlings with attached seeds always had significantly greater leaf area than seedlings with removed seeds, resulting in significantly higher total plant photosynthesis at all three time periods. The delta13C values of both the leaves and roots were more similar to that of the seed for seedlings with attached seeds than for seedlings with removed seeds, however, seed removal and seed size strongly affected root delta13C. This study demonstrates that seed reserves have important effects on the early growth, physiology, and delta13C of L. densiflora seedlings.

Analysis of Variance↗
PubMed2001

Varicella-Zoster virus gene expression in latently infected rat dorsal root ganglia.

Latent infection of human ganglia with Varicella-Zoster virus (VZV) is characterized by a highly restricted pattern of viral gene expression. To enhance understanding of this process we used in situ hybridization (ISH) in a rat model of VZV latency to examine expression of RNA corresponding to eight different VZV genes in rat dorsal root ganglia (DRG) at various times after footpad inoculation with wild-type VZV. PCR in situ amplification was also used to determine the cell specificity of latent VZV DNA. It was found that the pattern of viral gene expression at 1 week after infection was different from that observed at the later times of 1 and 18 months after infection. Whereas multiple genes were expressed at 1 week after infection, gene expression was restricted at the later time points when latency had been established. At the later time points after infection the RNA transcripts expressed most frequently were those for VZV genes 21, 62, and 63. Gene 63 was expressed more than any other gene studied. While VZV DNA was detected almost exclusively in 5-10% of neurons, VZV RNA was detected in both neurons and nonneuronal cells at an approximate ratio of 3:1. A newly described monoclonal antibody to VZV gene 63-encoded protein was used to detect this protein in neuronal nuclei and cytoplasm in almost half of the DRG studied. These results demonstrate that (1) this rat model of latency has close similarities in terms of viral gene expression to human VZV latency which makes it a useful tool for studying this process and its experimental modulation and (2) expression of VZV gene 63 appears to be the single most consistent feature of VZV latency.

Animals↗
PubMed2001

Involvement of apolipoprotein E in herpes simplex encephalitis.

APOE polymorphism may influence risk for cold sores and, by interacting with latent HSV-1, risk for Alzheimer's disease (AD). APOE genotype also influences outcome after brain injury. We sought to determine whether APOE genotype influences risk for herpes simplex encephalitis (HSE), whether apoE is involved in the response to HSE and if APOE genotype influences outcome from HSE. There was increased immunoreactivity of neurons, neuropil and glia for apoE areas of brain damaged by HSE. APOE genotypes for cases of HSE (n = 57) were similar to those of controls (n = 41). APOE genotypes for survivors of HSE were similar to those of patients who died. We conclude that apoE is involved in the response to damage associated with HSE, as in other forms of brain injury. However, APOE genotype does not appear to influence either the risk of developing HSE or subsequent mortality.

Adolescent↗
PubMed2001

Cytokines and the acute phase response in post-treatment reactive encephalopathy of Trypanosoma brucei brucei infected mice.

Stimulation of the acute phase response during infection of mice with Trypanosoma brucei brucei (T. b. brucei) was investigated in an experimental model of the post-treatment reactive encephalopathy (PTRE), a common side-effect of anti-trypanosome therapy. Plasma levels of the acute phase proteins (APP), haptoglobin (Hp) and serum amyloid P (SAP) increased by day 7 post-infection, but by day 20 had fallen to an intermediate level. This was accompanied by induction of the cytokines, interleukin (IL)-6 and tumour necrosis factor-alpha (TNFalpha) in both liver and brain. Treatment of mice on day 21 with a subcurative dose of diminazene aceturate (Berenil), a procedure known to induce a mild PTRE, cleared the parasite from the circulation with plasma APP and liver expression of mRNA for IL-6 and TNFalpha returning to the levels in the controls. Cytokine mRNA for both IL-6 and TNFalpha was detected in the brains of animals with developing PTRE although TNFalpha was not significantly greater than in the control group. A further subcurative dose of Berenil, leading to a more severe PTRE, was associated with elevated serum concentrations of Hp and SAP, increased TNFalpha mRNA in the liver and detectable IL-6 and TNFalpha mRNA in the brain. mRNA for IL-1alpha was expressed in brain and liver samples from all animals. A severe PTRE caused a systemic acute phase response which was not apparent with a mild PTRE. The pattern of cytokine mRNA induction was similar following both drug treatments. However, the difference in APP production could be caused by a breakdown in the blood-brain barrier during severe PTRE allowing cytokine synthesised in the brain to enter the circulation and maintain a systemic response.

Acute-Phase Proteins↗
PubMed2001

Adeno-associated virus-mediated delivery of glial cell line-derived neurotrophic factor protects motor neuron-like cells from apoptosis.

Motor neuron disorders including amyotrophic lateral sclerosis may benefit from the induction of neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) that are known to be trophic and protective for motor neurons. However, the application of such factors is limited by an inability to successfully target their expression in the nervous system. In this study we investigate the potential of using adeno-associated virus (AAV) as a vector for gene delivery into motor neuron-like cells. In initial experiments on the motor neuron cell line NSC-19 using a recombinant AAV vector expressing the reporter gene beta-galactosidase (AAV-LacZ), we successfully demonstrate the utility of AAV for gene transfer. In addition, a recombinant AAV vector expressing GDNF was shown to express and secrete high levels of the neurotrophic factor into the surrounding media of NSC-19 infected cells. Finally, the AAV-GDNF vector is demonstrated to act in a neuroprotective fashion. Withdrawal of trophic support from NSC-19 cells through serum deprivation results in a subsequent increase in the number of cells entering apoptosis. However, the percentage of apoptotic cells are significantly reduced in cells infected with the AAV-GDNF vector, as compared to AAV-LacZ or uninfected controls. This work demonstrates the potential of using AAV as a vector in motor neuron-like cells and should prove important in devising future gene therapy strategies for the treatment of in vivo motor neuron disorders.

Animals↗
PubMed2000

Replication of the herpes simplex virus type 1 RL1 mutant 1716 in primary neuronal cell cultures--possible relevance to use as a viral vector.

The herpes simplex virus type 1 (HSV-1) RL1 deletion mutant 1716 has properties that make it a promising candidate as a viral vector for gene therapy in the human nervous system. These properties include its ability to spread along neural pathways and establish a latent infection in post-mitotic neurons, while retaining a non-virulent phenotype in vivo and an inability to cause a lytic infection in stationary or fully differentiated cells. In this study, we used viral replication assays and indirect immunofluorescence to investigate the ability of 1716 to bind to, enter, express genes and produce progeny virus in dissociated neuronal cell cultures prepared from rat hippocampal, medial septal and dorsal root ganglion (DRG) tissues and in primary rat astrocyte cultures. Both heterogeneous cultures and those that had been enriched for neurons were employed. Following both low and high multiplicities of virus infection, the behaviour of 1716 was compared with its wild-type parent HSV-1 strain 17 in these cultures. It was found that the growth of 1716 was significantly impaired compared to wild type HSV-1, with these differences being magnified at lower multiplicities of viral infection as well as in neuron-enriched cultures: this impairment is likely to be due to decreased replication, as immunofluorescence assays showed that 1716 bound to, entered and expressed genes in all neuronal cell types and astrocytes with similar efficiency to the wild type virus. This ability of 1716 to enter and express genes in different neuronal populations demonstrates its potential suitability as a viral vector.

Animals↗
PubMed2000

Identification of a human olfactory ensheathing cell that can effect transplant-mediated remyelination of demyelinated CNS axons.

The olfactory ensheathing cell (OEC) has attracted much interest recently because of its potential for transplantation-based therapy of CNS disease. Rat OECs are able to remyelinate demyelinated axons and support regeneration of damaged axons. Although OECs can be grown readily from the rat, a macrosmatic species, it has been uncertain whether it would be similarly straightforward to obtain these cells from the human, a microsmatic species with a relatively poorly developed olfactory system. In this study, we have identified a human OEC which shares many properties with its rat counterpart, including expression of the low-affinity nerve growth factor receptor (L-NGFr) and similar growth factor requirements. Purified populations of human OECs obtained by selection with L-NGFr antibodies have extremely high viability in tissue culture, and are capable of remyelinating persistently demyelinated CNS axons following transplantation into experimentally induced demyelinating lesions in the rat spinal cord. Thus, the human OEC represents an important new cell for the development of transplant therapy of CNS diseases.

Animals↗
PubMed2000

Varicella-zoster virus gene expression in latently infected and explanted human ganglia.

A consistent feature of varicella-zoster virus (VZV) latency is the restricted pattern of viral gene expression in human ganglionic tissues. To understand further the significance of this gene restriction, we used in situ hybridization (ISH) to detect the frequency of RNA expression for nine VZV genes in trigeminal ganglia (TG) from 35 human subjects, including 18 who were human immunodeficiency virus (HIV) positive. RNA for VZV gene 21 was detected in 7 of 11 normal and 6 of 10 HIV-positive subjects, RNA for gene 29 was detected in 5 of 14 normal and 11 of 11 HIV-positive subjects, RNA for gene 62 was detected in 4 of 10 normal and 6 of 9 HIV-positive subjects, and RNA for gene 63 was detected in 8 of 17 normal and 12 of 15 HIV-positive subjects. RNA for VZV gene 4 was detected in 2 of 13 normal and 4 of 9 HIV-positive subjects, and RNA for gene 18 was detected in 4 of 15 normal and 5 of 15 HIV-positive subjects. By contrast, RNAs for VZV genes 28, 40, and 61 were rarely or never detected. In addition, immunocytochemical analysis detected the presence of VZV gene 63-encoded protein in five normal and four HIV-positive subjects. VZV RNA was also analyzed in explanted fresh human TG and dorsal root ganglia from five normal human subjects over a period of up to 11 days in culture. We found a very different pattern of gene expression in these explants, with transcripts for VZV genes 18, 28, 29, 40, and 63 all frequently detected, presumably as a result of viral reactivation. Taken together, these data provide further support for the notion of significant and restricted viral gene expression in VZV latency.

Adult↗
PubMed1999

Latent Varicella-zoster virus in human dorsal root ganglia.

To understand further the molecular events underlying the process of Varicella-zoster virus (VZV) latency in human ganglionic tissues, in situ hybridisation (ISH) for VZV RNA and DNA, and PCR in situ amplification for VZV DNA were used in human dorsal root ganglia from 12 individuals (3 normal and 9 who had died with AIDS). The results showed that (a) two separate regions of the VZV genome, represented by genes 4 and 40, were detected in neurons in two normal and three AIDS ganglia, (b) evidence of transcription of VZV genes 4, 21, 29, and 63 was found in normal and AIDS cases, and (c) VZV DNA and RNA for the same gene (gene 29) was detected in neurons in serial tissue sections in three cases. Thus more than one region of the VZV genome is present in neurons during VZV ganglionic latency, and the presence of both a VZV gene and its corresponding RNA transcript can be shown to occur in the same localised region of DRG tissue.

AIDS-Related Opportunistic Infections↗
PubMed1999

The pathogenesis and modulation of the post-treatment reactive encephalopathy in a mouse model of Human African Trypanosomiasis.

Drug treatment of late-stage human African Trypanosomiasis (HAT) in which the central nervous system (CNS) is involved may be complicated by a severe post-treatment reactive encephalopathy (PTRE) which can be fatal in up to 10% of cases. In order to understand the immunopathogenesis of this complication, an experimental mouse model has been developed that mirrors many of the pathological features of the PTRE in humans, and which allows various anti-inflammatory therapeutic regimes to be evaluated. Following the development of the PTRE in this model a number of cytokines are increased within the CNS including tumour necrosis factor (TNF) alpha, interleukins 1, 4 and 6, and macrophage inflammatory protein (MIP)-1. These cytokines appear at the same time as astrocyte activation which is an early event occurring before the development of the marked meningoencephalitic inflammatory response. The immunosuppressant drug azathioprine prevents but does not reduce the severity of an established PTRE and has a minimal effect on astrocyte activation. The ornithine decarboxylase inhibitor eflornithine prevents the induction, and ameliorates the severity, of the PTRE, and also reduces the degree of astrocyte activation. The Substance P antagonist RP-67,580 ameliorates the severity of an established PTRE, and also reduces astrocyte activation, indicating an important role of SP in the generation of the inflammatory response. Continued use of this mouse model should lead to further enhancement of our understanding of the pathogenesis of the PTRE and to improved drug regimes to prevent and/or treat it.

Analgesics↗
PubMed1999

Reduced levels of a specific myelin-associated oligodendrocytic basic protein isoform in shiverer myelin.

Myelin-associated oligodendrocytic basic protein (MOBP) and myelin basic protein (MBP) share many structural similarities. MOBP is synthesised by mature oligodendrocytes and localised at the major dense line (MDL), suggesting a role in the myelin compaction process. The shiverer mouse, a deletion mutant of the myelin basic protein (Mbp) gene, has poorly compacted myelin with essentially no MDL. In this study we compare the developmental expression of the Mobp gene in wild-type and shiverer mice. The significant finding is that one of the two abundant MOBP isoforms, the approximately 20-kD species, is poorly incorporated into shiverer myelin. The absence is specific to shiverer and is not a feature of dysmyelinating mutants with an abnormal intraperiod line. Our data suggest that incorporation of this MOBP isoform into shiverer myelin may be influenced by the presence of MBP or be a consequence of a disrupted MDL.

Aging↗
PubMed1998

Latent varicella-zoster virus is located predominantly in neurons in human trigeminal ganglia.

Varicella-zoster virus (VZV) is a human herpesvirus that causes varicella (chicken pox) as a primary infection and, after a variable period of latency in trigeminal and dorsal root ganglia, reactivates to cause herpes zoster (shingles). Both of these conditions may be followed by a variety of neurological complications, especially in immunocompromised individuals such as those with human immunodeficiency virus (HIV) infection. There have been a number of conflicting reports regarding the cellular location of latent VZV within human ganglia. To address this controversy we examined fixed wax-embedded trigeminal ganglia from 30 individuals obtained at autopsy, including 11 with HIV infection, 2 neonates, and 17 immunocompetent individuals, for the presence of latent VZV. Polymerase chain reaction (PCR), in situ hybridization, and PCR in situ amplification techniques with oligonucleotide probes and primer sequences to VZV genes 18, 21, 29, and 63 were used. VZV DNA in ganglia was detected in 15 individuals by using PCR alone, and in 12 individuals (6 normal non-HIV and 6 positive HIV individuals, but not neonatal ganglia) by using PCR in situ amplification. When in situ hybridization alone was used, 5 HIV-positive individuals and only 1 non-HIV individual showed VZV nucleic acid signals in ganglia. In all of the VZV-positive ganglia examined, VZV nucleic acid was detected in neuronal nuclei. Only occasional nonneuronal cells contained VZV DNA. We conclude from these studies that the neuron is the predominant site of latent VZV in human trigeminal ganglia.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Cytoskeletal and nuclear localization of myelin oligodendrocytic basic protein isoforms.

The recently described single copy myelin-associated oligodendrocytic basic protein (Mobp) gene is expressed exclusively in the central nervous system (CNS). The gene encodes a family of small highly basic polypeptides with predicted amino acid lengths of 69, 71, 81, 99 and 170, all of which share a 68 residue amino terminal. Here we report on the subcellular distribution of two of these polypeptides termed MOBP81 and MOBP170 in transiently transfected Cos7 cells using an antibody raised against a region common to all isoforms of MOBP. Additionally, we describe MOBP trafficking in cultured mouse spinal cord oligodendrocytes. Immunostaining for MOBP81 is intense in the perinuclear region and extends throughout the cytoplasm colocalizing with the microtubular cytoskeletal network. Consistent with this we demonstrate that MOBP partitions with the cytoskeletal fraction prepared from myelin. In contrast, although MOBP170 is present in the cytoplasm it does not colocalize with the cytoskeleton and displays a greater variation in distribution. In the majority of transfectants immunostaining is present throughout the karyoplasm but with increased intensity around the nucleolus. Within mouse primary oligodendrocytes endogenous MOBP is present in the cell body and processes colocalizing with the microtubular network. Immunoreactivity is not detectable in the nucleus in these mature oligodendrocytes. These significant differences in MOBP81 and MOBP170 protein kinesis coupled to different expression profiles of their respective message populations may be indicative of both myelin structural and cellular/regulatory functions, respectively, for these polypeptides.

Animals↗
PubMed1998

Functional analysis of the herpes simplex virus type 2 strain HG52 RL1 gene: the intron plays no role in virulence.

Sequence analysis predicts that herpes simplex virus type 2 (HSV-2) strain HG52 contains an open reading frame, RL1, encoding a polypeptide equivalent to ICP34.5 of HSV-1. Similarly to HSV-1, deletion of the region spanning RL1 abolishes the virulence of HSV-2 strain HG52 and its ability to grow in stationary 3T6 cells. In contrast to HSV-1, the HSV-2 strain HG52 RL1 gene is predicted to contain a 154 bp intron. Previously, we have demonstrated that this intron is spliced from RL1 poly(A)+ mRNA at the predicted splice donor/ acceptor sites. To determine if the intron affects the function of ICP34.5 of HSV-2 strain HG52, we have constructed a virus, 2624, in which the RL1 intron is deleted: 2624 retains wild-type growth both in vivo and in 3T6 cells, indicating that the presence of an intron does not affect the function of RL1 in HSV-2 strain HG52. 2624 has wild-type growth kinetics in BHK21/C13 cells.

Animals↗