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Biomedical subjects

M Philpott

Publications and source records attributed to M Philpott.

At least 19 recordsLinked to original sources
PubMed2001

Effects of the serotonin receptor antagonist cyproheptadine on the activity and pharmacokinetics of 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

BACKGROUND: DMXAA (5,6-dimethylxanthenone-4-acetic acid) is a new drug synthesized in this laboratory and currently in phase I clinical trial. In mice it acts as an antivascular drug, selectively inhibiting tumour blood flow and inducing tumour haemorrhagic necrosis with resultant tumour regression. It also induces the synthesis of tumour necrosis factor (TNF), nitric oxide and serotonin. Cyproheptadine, a type 2 serotonin receptor antagonist, is known to reduce the degree of tumour necrosis-induced TNF in mice. We investigated the pharmacological interaction between a suboptimal dose of DMXAA (20 mg/kg) and cyproheptadine (20 mg/ kg) using mice with Colon 38 tumours that are sensitive to DMXAA. METHODS: Mice with or without tumours were treated with DMXAA and/or cyproheptadine. Concentrations of plasma and tissue DMXAA and the serotonin metabolite 5-hydroxyindoleacetic acid were measured by high performance liquid chromatography. TNF concentrations were measured by ELISA. RESULTS: While DMXAA alone (20 mg/kg) showed little or no antitumour activity, coadministration with cyproheptadine was curative in four of five mice. DMXAA half-lives in plasma and tumour tissue were increased 5.1- and 5.6-fold, respectively, and the appearance of DMXAA glucuronides in bile was almost completely inhibited for up to 4 h. Serum TNF was low and unchanged by cyproheptadine, and plasma concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were also not substantially changed. CONCLUSION: The augmentation by cyproheptadine of the induction of tumour response to DMXAA reflects a pharmacological interaction, leading to increased plasma and tumour half-lives, and to reduced excretion. However, serum TNF concentrations were not increased, suggesting that the increased anti-tumour effects are mediated by an increased local tumour response, arising from the extended tumour DMXAA concentrations.

Animals↗
PubMed2001

The antitumour agent 5,6-dimethylxanthenone-4-acetic acid acts in vitro on human mononuclear cells as a co-stimulator with other inducers of tumour necrosis factor.

5,6-dimethylxanthenone-4-acetic acid (DMXAA), currently in phase I trials, demonstrates excellent activity against transplantable murine tumours with established vasculature. The induction of cytokines, particularly of tumour necrosis factor (TNF), appears to be critical to its action. We investigated TNF induction by DMXAA in cultured human peripheral blood leucocytes (HPBL). TNF was measured by an enzyme-linked immunosorbent assay after 8 h, and NF-kappaB induction by electrophoretic mobility shift assays (EMSA) after 2 h. DMXAA (800 microg/ml) had no effect alone on TNF production but augmented, by up to 4-fold, the ability of bacterial lipopolysaccharide (LPS) to induce TNF. Previously reported results showing TNF production by DMXAA alone were traced to the presence in an earlier batch of DMXAA of a small amount of LPS, the action of which could be blocked by polymyxin B. DMXAA stimulated TNF production by deacylated LPS, which alone had little effect. An antibody (MEM-18) to the CD14 receptor, while blocking the induction of TNF by LPS, enabled DMXAA to both synthesise TNF and induce NF-kappaB. The structurally related drug, flavone acetic acid (FAA), did not induce TNF or synergise with anti-CD14 antibody. DMXAA strongly augmented the ability of suboptimal concentrations of interleukin-1 (IL-1) (25 ng/ml), okadaic acid (OA) (20 ng/ml) and phorbol-12-myristate-13-acetate (PMA) (5 ng/ml) to induce TNF production, suggesting that it affects multiple pathways converging on NF-kappaB activation. Sodium salicylate, a drug reported to inhibit the beta-subunit of IkappaB kinase (IKK), appeared to competitively inhibit TNF production by DMXAA in the presence of anti-CD14 antibody. Taken together, the results indicate DMXAA acts in vitro on HPBL to co-stimulate TNF production by a wide variety of agents, and suggests that IKK is the target that mediates this action.

Antineoplastic Agents↗
PubMed2000

Visualization of Taylor-Couette and spiral Poiseuille flows using a snapshot FLASH spatial tagging sequence.

A new magnetic resonance imaging technique was applied to the Taylor-Couette and spiral Poiseuille (Taylor-Couette with superposed mean axial flux) flows for the first time. The experimental technique is a combination of spatial tagging methods and a snapshot FLASH imaging sequence, which allows the full-field visualization of 2-D slices of the flow field, with image acquisition times approximately half a second. By acquiring images every few seconds, direct visualization of flow patterns can be obtained in the form of cinematography. Tagged images of the Taylor-Couette flow were acquired in both the axial and transverse planes and confirmed previously reported numerical predictions of Taylor cell size. Tagged images of the spiral Poiseuille flows verified that the cells in this flow propagate at a higher velocity than the mean axial flow. In addition, intermittent cell formation was observed as the axial flow was increased.

Equipment Design↗
PubMed1997

The regulatory biology of the human pilosebaceous unit.

The last few years have witnessed an acceleration in our understanding of the regulation of the human pilosebaceous unit. Recombination and histochemical experiments are beginning to elucidate the role of homeotic genes, transcription factors, growth factors and adhesion molecules in pilosebaceous embryology. Histochemical studies, experiments in gene-modified animals, and in vitro studies on growing human hairs, have identified a number of growth factors that are central to normal hair growth. Thus epidermal growth factor and transforming growth factor-alpha appear to be involved in the triggering of both anagen and catagen. Insulin-like growth factor-I appears to sustain normal anagen growth, transforming growth factor-beta will inhibit anagen growth, while interleukin-1-alpha and tumour necrosis factor-alpha will induce matrix cell death. These complex growth factor effects are beginning to be moulded into an integrated model of pilosebaceous regulation. The role of steroid hormones in modulating these growth factor effects is also beginning to be understood.

Cytokines↗
PubMed1997

Production of tumour necrosis factor-alpha by cultured human peripheral blood leucocytes in response to the anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (NSC 640488).

The investigative anti-tumour agent 5,6-dimethylxanthenonone-4-acetic acid (DMXAA, NSC 640488), developed in this laboratory as an improved analogue of flavone acetic acid (FAA, NSC 347512), is currently in clinical trial. The ability of DMXAA to up-regulate tumour necrosis factor (TNF) mRNA and protein synthesis in cultured human peripheral blood leucocytes (HPBLs) has been investigated and compared with that of flavone acetic acid (FAA) and of bacterial lipopolysaccharide (LPS). Human peripheral blood leucocytes were isolated from buffy coats obtained from a blood transfusion centre and also from blood samples from laboratory volunteers. At a concentration of 400 microg ml(-1) and an incubation time of 2 h, DMXAA up-regulated mRNA synthesis in six of eight individuals tested, as measured by Northern blotting. The degree of up-regulation varied in different individuals from one to nine times that of control levels. In contrast, FAA caused no induction above that of control levels and in some cases suppressed expression relative to controls, extending previous data that DMXAA but not FAA up-regulates TNF mRNA in the human HL-60 tumour cell line. At the same concentration but with longer incubation times (6-12 h), DMXAA induced increases in TNF protein in 11 of 15 samples of HPBLs from buffy coats and also in 11 of 15 samples of HPBLs from volunteers, as measured by cytotoxicity assays with L929 cells. FAA caused no increase in TNF protein, while LPS induced TNF to approximately 20-fold higher levels than did DMXAA. Considerable heterogeneity of response was observed with both sources of HPBLs, and there was little or no correlation between the extent of TNF induction by DMXAA and LPS in individual samples. In vitro analysis of the response of human peripheral blood leucocytes to DMXAA may be a useful test in clinical trials of agents such as DMXAA.

Antineoplastic Agents↗
PubMed1995

Induction of tumour necrosis factor-alpha by single and repeated doses of the antitumour agent 5,6-dimethylxanthenone-4-acetic acid.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a low-molecular-weight biological response modifier scheduled for clinical evaluation, induced synthesis of tumour necrosis factor-alpha (TNF-alpha) in serum of mice, with maximal activity being observed at 2-3 h after administration. At a dose of 27.5 mg/kg, DMXAA induced similar TNF-alpha concentrations as did flavone-8-acetic acid given at its maximum tolerated dose (MTD; 330 mg/kg), whereas 8-methylxanthenone-4-acetic acid, which has no antitumour activity, did not induce serum TNF-alpha at its MTD (440 mg/kg). The dependence of schedule on TNF-alpha induction was studied by giving DMXAA to mice in two doses of 27.5 mg/kg each separated by different intervals. An interval of 0 (i.e. 55 mg/kg given in a single dose) produced a TNF-alpha concentration 9-fold that produced by a single dose of 27.5 mg/kg. This dose, although higher than the MTD of 30 mg/kg, did not affect the health of mice at the time of assay (3 h). An interval of 1 day produced very low levels of serum TNF-alpha after the second injection. An interval of 3 days produced high levels of serum TNF-alpha after the second injection (9-fold that detected in mice receiving 27.5 mg/kg in a single dose) but no long-term toxicity, whereas an interval of 7 days produced an intermediate response. Thus, the first dose can either potentiate or suppress the TNF-alpha response to a second dose. Mice with advanced subcutaneous colon 38 tumours were treated either with a single dose of DMXAA (27.5 mg/kg) or with a divided dose (two doses of 27.5 mg/kg given 3 days apart). Both the cure rate and the tumour-growth delay were enhanced by the divided-dose schedule. The results are relevant to the design of clinical administration schedules of DMXAA and emphasise the importance of TNF-alpha induction in the antitumour response.

Aging↗
PubMed1990

Overlapping cytotoxic T-lymphocyte and B-cell antigenic sites on the influenza virus H5 hemagglutinin.

To define the recognition site of cytotoxic T lymphocytes (CTLs) on influenza virus H5 hemagglutinin (HA), an H5 HA-specific CTL clone was examined for the ability to recognize monoclonal antibody-selected HA variants of influenza virus A/Turkey/Ontario/7732/66 (H5N9). On the basis of 51Cr release assays with the variants, a CTL epitope was located near residue 168 of H5 HA. To define the epitope more precisely, a series of overlapping peptides corresponding to this region was synthesized and tested for CTL recognition. The minimum peptide recognized by the CTL clone encompassed residues 158 to 169 of H5 HA. Relative to the H3 HA three-dimensional structure, this CTL epitope is located near the distal tip of the HA molecule, also known as a major B-cell epitope on H3 HA. A single mutation at residue 168 (Lys to Glu) in the H5 HA variants abolished CTL recognition; this same amino acid was shown previously to be critical for B-cell recognition (M. Philpott, C. Hioe, M. Sheerar, and V. S. Hinshaw, J. Virol. 64:2941-2947, 1990). Additionally, mutations within this region of the HA molecule were associated with attenuation of the highly virulent A/Turkey/Ontario/7732/66 (H5N9) (M. Philpott, B. C. Easterday, and V.S. Hinshaw, J. Virol. 63:3453-3458, 1989). When tested for recognition of other H5 viruses, the CTL clone recognized the HA of A/Turkey/Ireland/1378/83 (H5N8) but not that of A/Chicken/Pennsylvania/1370/83 (H5N2), even though these viruses contain identical HA amino acid 158-to-169 sequences. These results suggest that differences outside the CTL epitope affected CTL recognition of the intact HA molecule. The H5 HA site defined in these studies is, therefore, important in both CTL and B-cell recognition, as well as the pathogenesis of the virus.

Amino Acid Sequence↗
PubMed1990

Hemagglutinin mutations related to attenuation and altered cell tropism of a virulent avian influenza A virus.

The H5 hemagglutinin (HA) of a highly virulent avian influenza virus, A/Turkey Ontario/7732/66 (H5N9), was previously shown to have five neutralizing epitopes, and escape mutants within one epitope (group 1) were markedly attenuated (M. Philpott, B. C. Easterday, and V. S. Hinshaw, J. Virol. 63:3453-3458, 1989). To define the genetic changes related to these antigenic and biologic properties, the HA genes of mutants within each of the epitope groups were sequenced by using the polymerase chain reaction. The mutations in the attenuated group 1 mutants were located near the distal tip of the HA molecule in close proximity to the receptor-binding site, on the basis of alignment with the three-dimensional structure of the H3 HA. All group 1 mutations involved charged amino acids. The group 1 mutants, similar to the wild-type virus, spread systemically and were recovered from the spleens of infected chickens but, unlike the wild-type virus, failed to produce severe necrosis in the spleens. Viral replication in the spleens was investigated by in situ hybridization of spleen sections from chickens infected with the wild-type or attenuated mutants. Wild-type virus replication was demonstrated in large, mononuclear, macrophagelike cells; however, group 1 mutant virus was detected attached only to erythrocytes within the red pulp. These results suggest that the attenuated mutants differ in their cell tropism within the spleen.

Amino Acid Sequence↗
PubMed1989

Neutralizing epitopes of the H5 hemagglutinin from a virulent avian influenza virus and their relationship to pathogenicity.

To define and characterize the major neutralizing epitopes of the H5 hemagglutinin, a panel of monoclonal antibodies specific for the H5 hemagglutinin of the virulent avian influenza virus A/Turkey/Ontario/7732/66 (H5N9) was prepared. Antibodies which neutralized infectivity of the virus were used to select a panel of escape mutants. Reactivity patterns of the panel of monoclonal antibodies against the panel of mutants by both enzyme-linked immunosorbent assay serology and hemagglutination inhibition operationally defined five distinct epitopes on the H5 molecule. The mutants were analyzed in vivo for virulence in chickens, and the findings indicate that viruses with mutations in four of five epitopes were no less virulent than the wild type, producing a rapidly fatal disease, while all viruses with mutations in the fifth epitope (group 1 mutants) were attenuated. These group 1 mutants were unaltered in the cleavage properties of the hemagglutinin, suggesting that the mechanism of attenuation is unrelated to processing of the hemagglutinin. One of the group 1 mutants, 77B1v, was characterized for its ability to produce necrosis of the spleen and was found to produce none of the lesions in the spleen which are characteristic of the wild-type virus, although virus was present in this organ. The results suggest an altered tissue tropism, perhaps sparing a population of cells critical to an effective immune response.

Animals↗
PubMed1989

Studies on the biochemistry and morphology of freshly isolated and maintained rat hair follicles.

We have already shown that shearing can be used to yield large numbers of viable intact hair follicles. We now show that these follicles can be viably maintained on permeable supports for 7 days in vitro as determined by their adenine nucleotide contents, rates of [methyl-3H]thymidine and [U-14C]leucine uptake, [methyl-3H]thymidine autoradiography, patterns of keratin synthesis and light and electron microscopy. These studies, however, show that after 7 days maintenance the morphology of maintained follicles shows a closer resemblance to the telogen rather than the anagen follicle. We therefore conclude that the failure of previous attempts at maintaining hair growth in culture is due to hair follicles prematurely entering the resting stage of their hair growth cycle, possibly as a response to isolation.

Adenosine Diphosphate↗