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Biomedical subjects

M R Howard

Publications and source records attributed to M R Howard.

At least 19 recordsLinked to original sources
PubMed2006

Regulation and role of REST and REST4 variants in modulation of gene expression in in vivo and in vitro in epilepsy models.

Repressor element-1 silencing transcription factor (REST) is a candidate modulator of gene expression during status epilepticus in the rodent. In such models, full-length REST and the truncated REST4 variant are induced and can potentially direct differential gene expression patterns. We have addressed the regulation of these REST variants in rodent hippocampal seizure models and correlated this with expression of the proconvulsant, substance P encoding, PPT-A gene. REST and REST4 were differentially regulated following kainic acid stimulus both in in vitro and in vivo models. REST4 was more tightly regulated than REST in both models and its transient expression correlated with that of the differential regulation of PPT-A. Consistent with this, overexpression of a truncated REST protein (HZ4, lacking the C-terminal repression domain) increased expression of the endogenous PPT-A gene. Similarly the proximal PPT-A promoter reporter gene construct was differentially regulated by the distinct REST isoforms in hippocampal cells with HZ4 being the major inducer of increased reporter expression. Furthermore, REST and REST4 proteins were differentially expressed and compartmentalized within rat hippocampal cells in vitro following noxious stimuli. This differential localization of the REST isoforms was confirmed in the CA1 region following perforant path and kainic acid induction of status epilepticus in vivo. We propose that the interplay between REST and REST4 alter the expression of proconvulsant genes, as exemplified by the PPT-A gene, and may therefore regulate the progression of epileptogenesis.

Animals↗
PubMed2003

The development of targeted chemotherapy for CNS lymphoma-a pilot study of the IDARAM regimen.

PURPOSE: We have developed and evaluated a CNS-targeted chemotherapy regimen based on the pharmacokinetic properties of the individual drugs in the combination. PATIENTS AND METHODS: In a twin-track study, 16 patients with secondary CNS lymphoma (SCNSL) and 8 with primary CNS lymphoma (PCNSL) were treated with IDARAM which comprised idarubicin 10 mg/m(2) i.v., days 1 and 2; dexamethasone 100 mg, 12-h infusion, days 1, 2 and 3; cytosine arabinoside (ARA-C) 1.0 g/m(2), 1-h infusion, days 1 and 2; methotrexate 2.0 g/m(2), 6-h infusion, day 3 (with folinic acid rescue); and cytosine arabinoside 70 mg plus methotrexate 12 mg, intrathecally, days 1 and 8. Two cycles were delivered at 3-weekly intervals. After response assessment, patients received adjuvant cranial radiotherapy (40 Gy over 20 fractions). RESULTS: The series comprised 24 patients, 11 male and 13 female. Their median age was 53 years (range 21 to 73 years). Grade 4 neutropenia and thrombocytopenia occurred in the majority of patients treated. Of the eight PCNSL patients, seven achieved complete remission (CR). Four remained in CR at the time of this report with a median duration of follow-up of 25 months (range 11 to 42 months). Of the 16 SCNSL patients, 12 achieved CR. Seven patients remained in CR at the time of this report with a median duration of follow-up of 24 months (range 18 to 57 months). CONCLUSION: This study suggests that IDARAM is an effective regimen in both PCNSL and SCNSL and is suitable for further development and evaluation.

Adult↗
PubMed2003

No barrier to diffusion between cell soma and neurite membranes in sympathetic neurons for a GPI-anchored glycoprotein.

As neurons extend their axons, it is thought that newly synthesised membrane components travel in vesicles along the axon, fuse with the growth cone membrane, and diffuse back along the axonal membrane. However, it is difficult to explain how axons continue to be populated with membrane proteins as they extend in length. To investigate this problem, we have used a CEPU-green fluorescent protein (GFP) chimeric protein to study the site of insertion of new glycosyl phosphatidyl inositol (GPI)-anchored glycoproteins and their subsequent behaviour in chick dorsal root ganglia (DRG) neurons. Infection of cultures grown for 24 h revealed rapid expression of CEPU-GFP over the whole surface of the neuron, more rapidly than could be accounted for by diffusion from the growth cone, and fluorescence intensity was uniform along the length of the neurite. Photobleaching experiments of neurite membrane revealed that recovery of fluorescence was due to diffusion from adjacent membranes and there was no evidence for membrane flow in either direction. Photobleaching of membrane adjacent to the cell body also showed rapid recovery, with chimera diffusing both from cell body membrane and the distal neurite membrane into the bleached area. These results suggest there is no barrier to diffusion between the cell body and neurite membrane in DRG and sympathetic neurons cultured for 1 or 2 days in vitro. We propose that the neurite is populated by newly synthesised chimera by diffusion from both regions. This situation may also occur in neurons in the early stages of extending axons in vivo prior to polarisation and the development of the dendritic field.

Adrenergic Fibers↗
PubMed2002

A prospective study of the prevalence of undiagnosed coeliac disease in laboratory defined iron and folate deficiency.

AIMS: To determine the prevalence of coeliac disease in a group of patients in the community who have been shown in the laboratory to have iron and/or folate deficiency. To assess the cost efficiency of this laboratory based case finding strategy. METHODS: The study was undertaken in a large general hospital in the UK serving a population of 300 000. Three hundred and thirty three eligible patients with iron and/or folate deficiency were identified and contacted over an 18 month period. Case finding was by testing for coeliac disease using serological methods and subsequent histological confirmation. RESULTS: Of the 333 eligible and contactable patients with iron and/or folate deficiency, 258 (77%) consented to coeliac disease antibody testing. Twenty eight patients (10.9%) were positive for coeliac disease antibodies. Of these, 24 patients proceeded to endoscopy and biopsy, resulting in 12 cases of histologically confirmed coeliac disease (4.7% (95% confidence interval, 2.1% to 6.8%) of patients tested for coeliac disease antibodies). CONCLUSIONS: This laboratory based methodology detected a considerable number of new coeliac disease cases in the community. Many of these patients did not present with clinical findings suggestive of malabsorption and might not otherwise have been diagnosed. Laboratory based methodologies should be considered in conjunction with other strategies for the early identification and treatment of coeliac disease.

Adolescent↗
PubMed2002

High-level expression of recombinant Fc chimeric proteins in suspension cultures of stably transfected J558L cells.

Recombinant Fc chimeric proteins are useful tools for studying protein function, including the analysis of molecular interactions by techniques such as expression cloning. Here we describe a method we have used to express the IgLON family proteins, CEPU1 and OBCAM, as recombinant Fc chimeric proteins in stably transfected mouse J558L myeloma cells. The use of this cell line provided the opportunity to maximize protein production, as it secretes antibodies in large quantities and can be grown to high density in small volumes of culture medium. Isolation of recombinant OBCAMFc from the adherent COS7 cell line suggested a minimum level of expression of 0.07 mg OBCAMFc/100 mL culture medium, while the J558L cell line expressed OBCAMFc at approximately 11.4 mg/100 mL culture medium. Purification of IgLON-Fc expressed by J558L cells was simpler than purification from COS7 cells because of the lower volume of culture medium generated. Furthermore, contamination of J558L expressed IgLONFc with bovine IgG from the culture medium was negligible. The method presented, which utilizes a commercially available small-scale bioreactor, provides the nonspecialist protein expression laboratory with the means to produce recombinant proteins quickly and easily in milligram quantities.

Animals↗
PubMed2001

Identification and characterization of CEPU-Se-A secreted isoform of the IgLON family protein, CEPU-1.

CEPU-1/Neurotrimin is a neuronal glycoprotein thought to play a role in axon guidance and cell-cell recognition. It is a member of the IgLON family, has three C2 domains, and is attached to the plasma membrane by a GPI-anchor. We report here the characterisation of an alternatively-spliced isoform of CEPU-1 that is secreted. This isoform, termed CEPU-Se, is coexpressed with CEPU-1 in retina, cerebellum, and DRG neurons. In the cerebellum CEPU-1/CEPU-Se is expressed predominantly on granule cells and in the molecular layer. Divalent but not monovalent CEPU-Se interacts with CEPU-1 and other IgLONs, suggesting that the ability of CEPU-Se to modify the activity of the IgLON family may require an additional cofactor. CEPU-Se does not support the outgrowth of DRG neurons or the extension of established growth cones; however, neurite outgrowth on laminin is unaffected by CEPU-Se. Our data suggest that CEPU-Se may act to modulate the ability of CEPU-1, LAMP, and OBCAM to influence neurite outgrowth.

Alternative Splicing↗
PubMed2000

Co-localisation, heterophilic interactions and regulated expression of IgLON family proteins in the chick nervous system.

The chick glycoprotein GP55 has been shown to inhibit the growth and adhesion of DRG and forebrain neurons. GP55 consists of several members of the IgLON family, a group of glycoproteins including LAMP, OBCAM, CEPU-1 (chick)/neurotrimin (rat) and neurotractin (chick)/kilon (rat) thought to play a role in the guidance of growing axons. IgLONs belong to the Ig superfamily and have three C2 domains and a glycosyl phosphatidylinositol anchor which tethers them to the neuronal plasma membrane. We have now completed the deduced amino acid sequence for two isoforms of chicken OBCAM and used recombinant LAMP, OBCAM and CEPU-1 to raise antisera specific to these three IgLONs. LAMP and CEPU-1 are co-expressed on DRG and sympathetic neurons, while both overlapping and distinct expression patterns for LAMP, OBCAM and CEPU-1 are observed in retina. Analysis of IgLON mRNA expression reveals that alternatively spliced forms of LAMP and CEPU-1 are developmentally regulated. In an attempt to understand how the IgLONs function, we have begun to characterise their molecular interactions. LAMP and CEPU-1 have already been shown to interact homophilically. We now confirm that OBCAM will bind homophilically and also that LAMP, OBCAM and CEPU-1 will interact heterophilically with each other. We propose that IgLON activity will depend on the complement of IgLONs expressed by each neuron.

Amino Acid Sequence↗
PubMed2000

Artefactual serum hyperkalaemia and hypercalcaemia in essential thrombocythaemia.

AIM: To investigate possible abnormalities of serum potassium and calcium levels in patients with essential thrombocythaemia and significant thrombocytosis. METHODS: 24 cases of essential thrombocythaemia with significant thrombocytosis (platelet count > 700 x 10(9)/litre) had serum potassium and calcium estimations performed at the time of maximum thrombocytosis before treatment, and at the time of low platelet count after treatment with cytoreductive drugs. Selected patients were further investigated with plasma sampling and estimation of ionised calcium and parathyroid hormone. RESULTS: At the time of maximum thrombocytosis six patients had serum hyperkalaemia (> 5.5 mmol/litre) and five had serum hypercalcaemia (> 2.6 mmol/litre). Following treatment and reduction of the platelet count, hyperkalaemia resolved in all cases and hypercalcaemia in four of the five cases. Mean serum potassium and calcium concentrations were raised (p < 0.0001) at maximum thrombocytosis compared with the values when the platelet count was low. Serum potassium and calcium values were significantly correlated at all stages. Measurements on plasma consistently corrected the hyperkalaemia but not the hypercalcaemia. Serum hypercalcaemia was associated with raised ionised calcium and normal parathyroid hormone concentrations. CONCLUSIONS: Essential thrombocythaemia with significant thrombocytosis is associated with serum hyperkalaemia and hypercalcaemia. The probable mechanism of hypercalcaemia is the secretion of calcium in vitro from an excessive number of abnormally activated platelets. It is thus likely that the hypercalcaemia is an artefact, as is the hyperkalaemia.

Adolescent↗
PubMed2000

Herpesvirus infection of eye and brain in HIV infected patients.

OBJECTIVES: To compare histological with genome detection methods for diagnosis of herpesvirus infection in eye and brain of HIV infected patients undergoing necropsy and to correlate these findings with both antemortem clinical findings and postmortem evidence of extraocular herpesvirus infection, especially in the CNS. METHODS: A prospective study of 31 consecutive HIV infected patients undergoing necropsy. In life 11 patients had been assessed by an ophthalmologist because of ocular symptoms. Ocular and brain samples were examined for herpesviruses by conventional histological methods and by nested polymerase chain reaction (nPCR) for all eight human herpesviruses; evidence of extraneural herpesvirus infection was sought by histological methods. RESULTS: Although only 12 out of 31 patients (39%) had antemortem clinical evidence of ocular or CNS herpesvirus associated disease, herpesviruses were detected by nPCR in eye and brain from 26 (84%) patients; six patients had more than one herpesvirus infection. There was concordance between ocular and CNS findings in 15 of 19 patients (79%) with CMV infection. 17 of 31 patients (55%) had extraocular or CNS CMV infection at necropsy. Genome detection using nPCR was superior to histological methods for diagnosis of ocular and CNS herpesvirus infection. CONCLUSION: Herpesvirus infection of eye and brain was a frequent finding at necropsy in this group of HIV infected patients; almost a fifth were co-infected by more than one herpesvirus. This was more than twice the incidence predicted from clinical evidence before death. Genome detection using nPCR was superior to histological methods for diagnosis of ocular and CNS herpesvirus infection.

Adult↗
PubMed1999

Early diagnosis of septicaemia in preterm infants from examination of peripheral blood films.

Clinically significant staphylococcal septicaemia associated with the presence of bacteria in neutrophils in a routine peripheral blood film is described in four preterm infants. In all cases this allowed the diagnosis of infection prior to the result of microbiological cultures. It is likely that this finding is relatively common in this subgroup of patients resulting from a combination of severe infection associated with indwelling catheters and defective neutrophil bacteriacidal mechanisms. The findings in these cases suggest that careful blood film inspection in selected premature neonates may allow the early identification of sepsis.

Female↗
PubMed1999

Review of the clinical activity of medical microbiologists in a teaching hospital.

BACKGROUND: The clinical interactive role of medical microbiologists has been underestimated and the discipline is perceived as being confined to the laboratory. Previous studies have shown that most microbiology interaction takes place over the telephone. AIM: To determine the proportion of clinical ward based and laboratory based telephone interactions and specialties using a microbiology service. METHODS: Clinical microbiology activity that took place during November 1996 was prospectively analysed to determine the distribution of interactions and specialties using the service. RESULTS: In all, 1177 interactions were recorded, of which nearly one third (29%) took place at the bedside and 23% took place on call. Interactions involving the intensive treatment unit, general ward visits, and communication of positive blood cultures and antibiotic assays were the main areas of activity identified. There were 147 visits to 86 patients on the general wards during the study, with the number of visits to each individual varying from one to eight. The need for repeated visits reflected the severity of the underlying condition of the patients. Ward visits were regarded as essential to obtain missing clinical information, to assess response to treatment, and to make an appropriate entry in a patient's notes. CONCLUSIONS: Ward visits comprise a significant proportion of clinical microbiology interactions and have potential benefits for patient management, service utilisation, and education.

England↗
PubMed1998

Detection of Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus DNA in CSF from persons infected with HIV who had neurological disease.

OBJECTIVES: To determine the frequency and clinical relevance of Epstein-Barr virus (EBV) and Kaposi's sarcoma associated herpesvirus (KSHV) DNA detection in the CSF from patients infected with HIV. METHODS: Cerebrospinal fluid was obtained prospectively from 115 consecutive patients infected with HIV undergoing diagnostic lumbar puncture for investigation of neurological disease. Amplification of DNA was performed using a nested polymerase chain reaction (PCR) for detection of EBV internal repeat and KSHV minor capsid sequences. RESULTS: EBV DNA was detected in the CSF supernatant of 18 patients. This included all patients with primary CNS lymphoma (seven patients) or a combination of systemic and CNS lymphoma (two patients). By contrast EBV DNA was not detected in the CSF supernatant of any patient with systemic, but not CNS, lymphoma (10 patients). EBV DNA was also detected in the supernatant of nine further patients without a diagnosis of lymphoma at the time of lumbar puncture, two of whom subsequently developed CNS lymphoma. No EBV DNA was detected in CSF supernatant from the remaining 87 samples (two of these patients subsequently developed lymphoma). KSHV DNA was detected in the CSF of two patients, one had systemic (but not CNS) lymphoma and the other did not have lymphoma. CONCLUSION: A diagnosis of CNS lymphoma is strongly associated with the presence of EBV DNA in CSF. In the absence of clinical and radiological features of CNS lymphoma, the presence of detectable CSF EBV DNA may predict subsequent tumour development. KSHV DNA is rarely detected in CSF in this patient group and shows no correlation with lymphoma or other neurological disease.

AIDS Dementia Complex↗
PubMed1998

Association of Kaposi's sarcoma associated herpesvirus (KSHV) DNA in bronchoalveolar lavage fluid of HIV infected individuals with bronchoscopically diagnosed tracheobronchial Kaposi's sarcoma.

OBJECTIVES: To determine the frequency of detection of Kaposi's sarcoma associated herpesvirus (KSHV), also known as human herpesvirus (HHV) type 8, DNA in bronchoalveolar lavage (BAL) fluid from HIV infected individuals with and without KS and to compare this with the detection rate in peripheral blood. Also to identify whether KSHV was associated with specific cell types in lavage fluid. METHODS: Nested PCR was used to detect KSHV DNA in BAL fluid from 41 consecutive individuals with Kaposi's sarcoma (KS) and in 41 controls with similar CD4 lymphocyte counts. Semiquantification of viral DNA was by end point titration. A positive cell sorting selection procedure was used to isolate specific BAL fluid cell types. RESULTS: KSHV DNA was detected in BAL fluid from 24 of 29 (83%) individuals with a bronchoscopic diagnosis of tracheobronchial KS. None was detected in 12 individuals with only cutaneous KS, or in 41 matched controls without KS. In five, KSHV DNA was detected in the cell depleted and cellular fractions of BAL fluid and in 1/5 in the CD14 (macrophage) fractions. None was detected in the CD19 (B lymphocyte) or CD4/CD8 (T lymphocyte) fractions. CONCLUSIONS: There was a clear association between the diagnosis of tracheobronchial KS and detection of KSHV DNA in BAL fluid. The cell type supporting KSHV in the respiratory tract is not CD 19 positive and has yet to be conclusively identified.

Bronchial Neoplasms↗