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P Kellam

Publications and source records attributed to P Kellam.

At least 19 recordsLinked to original sources
PubMed2006

Current research priorities in chronic fatigue syndrome/myalgic encephalomyelitis: disease mechanisms, a diagnostic test and specific treatments.

Chronic fatigue syndrome (CFS) is an illness characterised by disabling fatigue of at least 6 months duration, which is accompanied by various rheumatological, infectious and neuropsychiatric symptoms. A collaborative study group has been formed to deal with the current areas for development in CFS research--namely, to develop an understanding of the molecular pathogenesis of CFS, to develop a diagnostic test and to develop specific and curative treatments. Various groups have studied the gene expression in peripheral blood of patients with CFS, and from those studies that have been confirmed using polymerase chain reaction (PCR), clearly, the most predominant functional theme is that of immunity and defence. However, we do not yet know the precise gene signature and metabolic pathways involved. Currently, this is being dealt with using a microarray representing 47,000 human genes and variants, massive parallel signature sequencing and real-time PCR. It will be important to ensure that once a gene signature has been identified, it is specific to CFS and does not occur in other diseases and infections. A diagnostic test is being developed using surface-enhanced, laser-desorption and ionisation-time-of-flight mass spectrometry based on a pilot study in which putative biomarkers were identified. Finally, clinical trials are being planned; novel treatments that we believe are important to trial in patients with CFS are interferon-beta and one of the anti-tumour necrosis factor-alpha drugs.

Fatigue Syndrome, Chronic↗
PubMed2005

A low temperature method of isolating normal human articular chondrocytes.

OBJECTIVE: Numerous methods for isolation of human chondrocytes are reported in the literature, most based on isolation from animal cartilage. Normal human articular cartilage (NHAC) poses particular problems for isolating chondrocytes when compared to animal or other types of human cartilage: a hardy matrix, combined with few and friable chondrocytes makes isolation difficult. Our objective was to develop an efficient method of isolating chondrocytes from NHAC without jeopardising the viability of these cells. DESIGN: In this study we demonstrate that lowering the enzymatic digestion temperature to 27 degrees C increases cell yield and chondrocyte viability. We then optimised this low temperature isolation of chondrocytes from NHAC by comparing the relative efficacies of trypsin and protease and hyaluronidase in combination with different types of collagenase (I, II and XI) at releasing chondrocytes from their surrounding cartilaginous matrix. Enzymes were tested at different concentrations and for differing times. Outcome measures included determining the amount of cartilage digested, the number of viable chondrocytes isolated per gram of cartilage and cell adherence rates. CONCLUSIONS: From these set of experiments, the method that maximised cell yield without jeopardising cell viability proved to be a two stage process: pre-digestion step using trypsin for 15 min; followed by overnight digestion with a combination of two types of collagenase (types I and II) and at a lower temperature of 27 degrees C. This has resulted in an efficient and robust method of releasing chondrocytes from cartilage, without jeopardising the viability of these cells.

Aged↗
PubMed2005

A statistical model for HIV-1 sequence classification using the subtype analyser (STAR).

MOTIVATION: HIV-1 antiretroviral drug resistance testing produces large amounts of HIV-1 protease and reverse transcriptase sequences. These provide an excellent resource to study the incidence, spread and clinical significance of HIV-1 subtypes. We have produced a program, Subtype Analyser (STAR) that rapidly and accurately subtypes HIV-1. Here we have determined a robust and statistically validated model for subtype assignment. RESULTS: We have significantly extended our HIV-1 subtyping tool (STAR), such that each query sequence when evaluated against subtype profile alignments, returns a discriminating score based on the ratio of subtype positive to negative amino acid positions. These scores were transformed into a Z-score distribution and evaluated. Of the 141 sequences used to define the subtype alignments, 98% were correctly reclassified. Inclusion of additional recombination detection within STAR increased the detection of known recombinant sequences to 95%. AVAILABILITY: STAR is available as compiled (Linux Fedora 3) or source code from http://pgv19.virol.ucl.ac.uk/download/star_linux.tar CONTACT: p.kellam@ucl.ac.uk SUPPLEMENTARY INFORMATION: http://pgv19.virol.ucl.ac.uk/download/star_supplement

Algorithms↗
PubMed2005

Gene expression in peripheral blood mononuclear cells from patients with chronic fatigue syndrome.

BACKGROUND: Chronic fatigue syndrome (CFS) is a multisystem disease, the pathogenesis of which remains undetermined. AIMS: To test the hypothesis that there are reproducible abnormalities of gene expression in patients with CFS compared with normal healthy persons. METHODS: To gain further insight into the pathogenesis of this disease, gene expression was analysed in peripheral blood mononuclear cells from 25 patients with CFS diagnosed according to the Centers for Disease Control criteria and 25 normal blood donors matched for age, sex, and geographical location, using a single colour microarray representing 9522 human genes. After normalisation, average difference values for each gene were compared between test and control groups using a cutoff fold difference of expression > or = 1.5 and a p value of 0.001. Genes showing differential expression were further analysed using Taqman real time polymerase chain reaction (PCR) in fresh samples. RESULTS: Analysis of microarray data revealed differential expression of 35 genes. Real time PCR confirmed differential expression in the same direction as array results for 16 of these genes, 15 of which were upregulated (ABCD4, PRKCL1, MRPL23, CD2BP2, GSN, NTE, POLR2G, PEX16, EIF2B4, EIF4G1, ANAPC11, PDCD2, KHSRP, BRMS1, and GABARAPL1) and one of which was downregulated (IL-10RA). This profile suggests T cell activation and perturbation of neuronal and mitochondrial function. Upregulation of neuropathy target esterase and eukaryotic translation initiation factor 4G1 may suggest links with organophosphate exposure and virus infection, respectively. CONCLUSION: These results suggest that patients with CFS have reproducible alterations in gene regulation.

Adolescent↗
PubMed2004

An experimental evaluation of a loop versus a reference design for two-channel microarrays.

MOTIVATION: Despite theoretical arguments that so-called 'loop designs' for two-channel DNA microarray experiments are more efficient, biologists continue to use 'reference designs'. We describe two sets of microarray experiments with RNA from two different biological systems (TPA-stimulated mammalian cells and Streptomyces coelicolor). In each case, both a loop and a reference design were used with the same RNA preparations with the aim of studying their relative efficiency. RESULTS: The results of these experiments show that (1) the loop design attains a much higher precision than the reference design, (2) multiplicative spot effects are a large source of variability, and if they are not accounted for in the mathematical model, for example, by taking log-ratios or including spot effects, then the model will perform poorly. The first result is reinforced by a simulation study. Practical recommendations are given on how simple loop designs can be extended to more realistic experimental designs and how standard statistical methods allow the experimentalist to use and interpret the results from loop designs in practice. AVAILABILITY: The data and R code are available at http://exgen.ma.umist.ac.uk CONTACT: veronica.vinciotti@brunel.ac.uk.

Algorithms↗
PubMed2001

VIDA: a virus database system for the organization of animal virus genome open reading frames.

VIDA is a new virus database that organizes open reading frames (ORFs) from partial and complete genomic sequences from animal viruses. Currently VIDA includes all sequences from GenBank for Herpesviridae, Coronaviridae and Arteriviridae. The ORFs are organized into homologous protein families, which are identified on the basis of sequence similarity relationships. Conserved sequence regions of potential functional importance are identified and can be retrieved as sequence alignments. We use a controlled taxonomical and functional classification for all the proteins and protein families in the database. When available, protein structures that are related to the families have also been included. The database is available for online search and sequence information retrieval at http://www.biochem.ucl.ac.uk/bsm/virus_database/ VIDA.html.

Animals↗
PubMed2001

Genomewide function conservation and phylogeny in the Herpesviridae.

The Herpesviridae are a large group of well-characterized double-stranded DNA viruses for which many complete genome sequences have been determined. We have extracted protein sequences from all predicted open reading frames of 19 herpesvirus genomes. Sequence comparison and protein sequence clustering methods have been used to construct herpesvirus protein homologous families. This resulted in 1692 proteins being clustered into 243 multiprotein families and 196 singleton proteins. Predicted functions were assigned to each homologous family based on genome annotation and published data and each family classified into seven broad functional groups. Phylogenetic profiles were constructed for each herpesvirus from the homologous protein families and used to determine conserved functions and genomewide phylogenetic trees. These trees agreed with molecular-sequence-derived trees and allowed greater insight into the phylogeny of ungulate and murine gammaherpesviruses.

Animals↗
PubMed2001

Kaposi's sarcoma-associated herpesvirus latent and lytic gene expression as revealed by DNA arrays.

Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is associated with three human tumors, Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. KSHV encodes a number of homologs of cellular proteins involved in the cell cycle, signal transduction, and modulation of the host immune response. Of the virus complement of over 85 open reading frames (ORFs), the expression of only a minority has been characterized individually. We have constructed a nylon membrane-based DNA array which allows the expression of almost every ORF of KSHV to be measured simultaneously. A PEL-derived cell line, BC-3, was used to study the expression of KSHV during latency and after the induction of lytic replication. Cluster analysis, which arranges genes according to their expression profile, revealed a correlation between expression and assigned gene function that is consistent with the known stages of the herpesvirus life cycle. Furthermore, latent and lytic genes thought to be functionally related cluster into groups. The correlation between gene expression and function also infers possible roles for KSHV genes yet to be characterized.

Amino Acid Sequence↗
PubMed2000

Host-pathogen studies in the post-genomic era.

Several studies are starting to show the power of DNA microarrays to identify interactions between animal hosts and their pathogens, and have revealed interesting correlations between host responses to different infectious agents.

Animals↗
PubMed2000

HHV-8 is associated with a plasmablastic variant of Castleman disease that is linked to HHV-8-positive plasmablastic lymphoma.

Castleman disease (CD) is a lymphoproliferative disorder of unknown etiology that is associated with the development of secondary tumors, including B-cell lymphoma. Human herpesvirus 8 (HHV-8) (Kaposi's sarcoma-associated herpesvirus) sequences have been described in some cases of multicentric Castleman disease (MCD). Using a monoclonal antibody against an HHV-8-latent nuclear antigen, we show that HHV-8 is specifically associated with a variant of MCD in which HHV-8-positive plasmablasts that show lambda light-chain restriction localize in the mantle zone of B-cell follicles and coalesce to form microscopic lymphomas in some cases. Furthermore, we show that the frank plasmablastic lymphoma that develops in patients with this plasmablastic variant of MCD is also positive for HHV-8 and lambda light chain. Plasmablastic lymphoma associated with MCD is a new disease entity associated with HHV-8 infection. (Blood. 2000;95:1406-1412)

Adolescent↗
PubMed2000

The latent nuclear antigen of Kaposi sarcoma-associated herpesvirus targets the retinoblastoma-E2F pathway and with the oncogene Hras transforms primary rat cells.

Kaposi sarcoma-associated herpesvirus (KSHV) is involved in the etiopathogenesis of Kaposi sar-coma and certain lymphoproliferative disorders. Open reading frame (ORF) 73 encodes the main immunogenic latent nuclear antigen (LNA-1) of KSHV. LNA-1 maintains the KSHV episome and tethers the viral genome to chromatin during mitosis. In addition, LNA-1 interacts with p53 and represses its transcriptional activity. Here we show that LNA-1 also interacts with the retinoblastoma protein. LNA-1 transactivated an artificial promoter carrying the cell cycle transcription factor E2F DNA-binding sequences and also upregulated the cyclin E (CCNEI) promoter, but not the B-myb (MYBL2) promoter. LNA-1 overcame the flat-cell phenotype induced by retinoblastoma protein in Saos2 cells. In cooperation with the cellular oncogene Harvey rat sarcoma viral oncogene homolog (Hras), LNA-1 transformed primary rat embryo fibroblasts and rendered them tumorigenic. These findings indicate that LNA-1 acts as a transcription co-factor and may contribute to KSHV-induced oncogenesis by targeting the retinoblastoma protein-E2F transcriptional regulatory pathway.

Animals↗
PubMed1999

Transcriptional analysis of human herpesvirus-8 open reading frames 71, 72, 73, K14, and 74 in a primary effusion lymphoma cell line.

We examined the transcription and splicing of open reading frames (ORFs) 71 (K13)-74 of human herpesvirus-8 (HHV-8) in the primary effusion lymphoma cell line BCP-1 (latently infected with HHV-8), using a combination of NORTHERN blot analysis, RT-PCR, and rapid amplification of cDNA ends (PCR-RACE). The three genes encoded by ORFs 71, 72, and 73 [viral FLICE inhibitory protein (v-FLIP), v-cyclin, latent nuclear antigen (LNA)] are transcribed from a common transcription start site in BCP-1 cells uninduced (latent) or induced (lytic) with n-butyrate. The resulting transcript is spliced to yield a 5.32-kb message encoding LNA, v-cyclin, and v-FLIP and a 1.7-kb bicistronic message encoding v-cyclin and v-FLIP. The two genes encoded by ORFs K14 and 74 (v-Ox2 and v-GPCR) are transcribed as a 2.7-kb bicistronic transcript that is induced with n-butyrate. A small (149-bp) intron is spliced from the intragenic noncoding region immediately before the v-GPCR initiating codon. Examination of sequence elements in the promoter of the LNA/v-cyclin/v-FLIP operon revealed TAATGARAT and Octamer binding motifs characteristic of herpesvirus immediate-early genes. Sequence elements in the v-Ox2/v-GPCR promoter included AP1 and Zta-like (EBV Zebra transactivator) binding motifs consistent with the n-butyrate induction of this operon.

Base Sequence↗
PubMed1999

Distribution of human herpesvirus-8 latently infected cells in Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma.

Human herpesvirus 8 (HHV-8, also called KSHV) is linked to the etiopathogenesis of Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL). The universal presence of HHV-8 in early KS has not yet been shown. We used a mAb (LN53) against latent nuclear antigen-1 (LNA-1) of HHV-8 encoded by ORF73 to study the distribution of the cell types latently infected by HHV-8 in patch, plaque, and nodular KS, MCD, and PEL. In early KS, HHV-8 is present in <10% of cells forming the walls of ectatic vessels. In nodular KS, HHV-8 is present in cells surrounding slit-like vessels and in >90% of spindle cells, but not in normal vascular endothelium. In addition, HHV-8 colocalizes with vascular endothelial growth factor receptor-3 (VEGFR-3), a marker of lymphatic and precursor endothelium. In early KS lesions, VEGFR-3 is more extensively expressed than LNA-1, indicating that HHV-8 is not inducing the proliferation of VEGFR-3-positive endothelium directly. In MCD, HHV-8 is present in mantle zone large immunoblastic B cells. No staining for LNA-1 is seen in samples from multiple myeloma, prostate cancer, and angiosarcoma, supporting the absence of any etiological link between these diseases and HHV-8.

Antigens, CD↗
PubMed1999

Characterization of monoclonal antibodies raised against the latent nuclear antigen of human herpesvirus 8.

Human herpesvirus 8 (HHV-8; also designated Kaposi's sarcoma-associated herpesvirus) is the likely etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes a latent nuclear antigen (LNA) which is the product of the viral gene orf 73. LNA is recognized by most infected patient sera and is the basis of current immunofluorescence assays used in epidemiological studies of HHV-8 infection. Here we describe the characterization of four monoclonal antibodies raised to the C-terminal third of LNA-glutathione S-transferase fusion proteins. These monoclonal antibodies recognized discrete linear epitopes within the C terminus and repetitive region of LNA, detected antigen in primary effusion lymphoma (PEL) cells, and precipitated a 220- to 230-kDa protein doublet corresponding to LNA from HHV-8-infected PEL cell lines. In situ immunocytochemistry of KS lesions with these antibodies show that LNA is extensively expressed in KS spindle cells.

Antibodies, Monoclonal↗
PubMed1998

Single amino acid substitutions disrupt tetramer formation in the dihydroneopterin aldolase enzyme of Pneumocystis carinii.

In the opportunistic pathogen Pneumocystis carinii, dihydroneopterin aldolase function is expressed as the N-terminal portion of the multifunctional folic acid synthesis protein (Fas). This region encompasses two domains, FasA and FasB, which are 27% amino acid identical. FasA and FasB also share significant amino acid sequence similarity with bacterial dihydroneopterin aldolases. In the present study, this enzyme function has been overproduced as an independent monofunctional activity in Escherichia coli. Recombinant FasAB-Met23 (amino acids 23-290 of the predicted open reading frame) was purified and shown to contain dihydroneopterin aldolase activity. The native FasAB-Met23 is a tetramer of the 30-kDa subunit, demonstrating characteristics of an associating-dissociating equilibrium system in which only the multimeric form of the enzyme is active. Multiple sequence alignment of FasA and FasB with other dihydroneopterin aldolases highlights only three positions where the amino acid is invariable between all the predicted proteins. The role of these conserved amino acid residues in enzyme function was investigated using site-directed mutagenesis. Mutant FasAB-Met23 species were overproduced and purified to near homogeneity. Three FasA domain mutants and two FasB domain mutants had little or no detectable dihydroneopterin aldolase activity, implicating both FasA and FasB in the catalytic mechanism. We show that each mutant protein containing an inactivating amino acid substitution has lost its ability to form stable tetramers.

Aldehyde-Lyases↗
PubMed1998

Molecular identification of novel viruses.

Viruses are responsible for many of the diseases caused by microbial infection. During the past two decades, approximately 20 new human viruses have been discovered. Many of these new viruses were initially identified using molecular biology techniques, a major advantage of which is the ability to search rapidly for new viruses, known viruses or related, but previously unidentified, members of established virus families in disease samples.

Humans↗
PubMed1998

Human herpesvirus type 8 and Kaposi's sarcoma.

Kaposi's sarcoma-associated herpesvirus or human herpesvirus type 8 (HHV-8) is present in all forms of Kaposi's sarcoma (KS) as well as in primary effusion lymphomas and some cases of Castleman's disease. In KS tissues, HHV-8 is present in endothelial and spindle cells. Current serologic tests suggest that HHV-8 is predominantly found in those at risk of KS and is not as widespread as most other human herpesviruses. HHV-8 encodes various proteins that may play a role in promotion of cellular growth, including cyclin- and G-coupled protein receptor homologues, and anti-apoptotic proteins, including Bcl-2, IL-6 (i.e., interleukin 6), and FLIP (i.e., FLICE inhibitory protein) homologues. In addition, HHV-8 encodes two macrophage inflammatory-like proteins with anti-human immunodeficiency virus and angiogenic potential.

Herpesvirus 8, Human↗