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Biomedical subjects

G M Smith

Publications and source records attributed to G M Smith.

At least 55 records · Page 3Linked to original sources
PubMed1998

Activity of amoxicillin-clavulanate against penicillin-resistant Streptococcus pneumoniae in an experimental respiratory infection model in rats.

High doses of amoxicillin, equivalent to those produced by 500- and 750-mg oral doses in humans (area under the plasma concentration-time curve), were effective against a penicillin-resistant strain of Streptococcus pneumoniae in an experimental respiratory tract infection in immunocompromised rats; this superior activity confirms the results of previous studies. An unexpected enhancement of amoxicillin's antibacterial activity in vivo against penicillin-resistant and -susceptible S. pneumoniae strains was observed when subtherapeutic doses of amoxicillin were coadministered with the beta-lactamase inhibitor potassium clavulanate. The reason for this enhancement was unclear since these organisms do not produce beta-lactamase. The differential binding of clavulanic acid and amoxicillin to penicillin-binding proteins may have contributed to the observed effects.

Amoxicillin-Potassium Clavulanate Combination↗
PubMed1998

1H-nuclear magnetic resonance studies on the conformational changes related to the foaming properties of beta-lactoglobulin.

beta-Lactoglobulin (beta-LG) is a whey protein with foaming ability that can be used as a food ingredient. The structural changes that occur during foaming cannot be easily assessed. In combination with deuterium exchange, 1H-NMR allows new insight into the structural features of beta-LG during foaming. beta-Lactoglobulin was dissolved in D2O and forced to foam. During foaming, the amide protons of exposed residues were exchanged for deuterium atoms, which do not appear in the 1H-NMR spectrum. Protein in solutions that had produced unstable foams showed no exchange beyond that found in unfoamed controls, indicating that the structure had remained intact. Protein in solutions that had produced stable foams showed extensive exchange. Protons of both Trp19 and Met107 exchanged with deuterium, indicating that most of the interior had been exposed to solvent. Most of the beta-LG structure was recovered after collapse of the foam. In the early steps of foaming, apparently only random coil or other exposed regions are involved in foam stabilization. More vigorous shear stress forces induce further unfolding of the beta-strands. This unfolding is suggested to be a dynamic, reversible equilibrium between an open and closed conformation of beta-strands that allows not only for interaction of the protein with the air phase but also for some secondary structure to be retained and the original structure to be recovered. Freeze-dry foaming is also discussed.

Chemical Phenomena↗
PubMed1998

Preclinical antitumor activity of an antibody against the leukocyte antigen CD48.

We have evaluated the antitumor activity of a murine antibody (IgG2a) against the leukocyte antigen CD48. CD48 is expressed on T and B lymphocytes, monocytes, and a wide range of lymphoid malignancies. To assess the therapeutic potential of an anti-CD48 antibody, we established a reproducible model of human B-cell (Raji) leukemia/lymphoma in C.B17/scid mice, where untreated mice develop hind leg paralysis due to tumor engraftment. Using this model, the murine anti-CD48 antibody HuLy-m3 was shown to mediate a strong in vivo antitumor effect. Long-term survival (>1 year) of scid mice was obtained after treatment with three 200-microg i.v. doses of anti-CD48 antibody on days 0, 2, and 4 after i.v. injection of tumor cells. In contrast, mice treated with an isotype control antibody developed hind leg paralysis after 34 +/- 3 days. A strong antitumor response was still observed when a dose of 20 microg of HuLy-m3 antibody was used. During preclinical investigations, we also examined a number of properties of the CD48 antigen. CD48 is present at high levels on the surface of T and B cells, but most (>95%) CD34-positive cells do not express CD48. Anti-CD48 antibodies are maintained on the surface of antigen-positive cells for extended periods (>24 h). These properties suggest that anti-CD48 antibodies may be useful in the treatment of a number of diseases including lymphoid leukemias and lymphomas.

Animals↗
PubMed1997

Macrophage/Microglia regulation of astrocytic tenascin: synergistic action of transforming growth factor-beta and basic fibroblast growth factor.

After injury to the CNS, extracellular matrix molecules such as tenascin are upregulated around the injury site and may be involved in inhibition of axon growth. In the present study, astrocytes were investigated to determine which cell types, growth factors, or cytokines are responsible for the injury-induced regulation of tenascin. The addition of activated macrophage- or microglial-conditioned medium increased astrocytic expression of tenascin 2.5-fold, as determined by Northern and Western blot analysis and ELISA. Of the cytokines and growth factors examined, only transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) significantly induced an increase in the production of astrocytic tenascin. Examination of macrophage and microglial supernatants showed the presence of TGF-beta1 but not bFGF; however, the TGF-beta1 concentration in supernatants was lower than that expected to induce an increase in astrocytic tenascin similar to that seen with recombinant TGF-beta1. Western blot analysis of astrocytes showed only the presence of bFGF. Compared with the responses of the individual growth factors, tenascin production by astrocytes was dramatically potentiated when grown in the presence of a combination of both TGF-beta1 and bFGF. A similar synergistic effect was observed after the addition of either TGF-beta1 or bFGF to macrophage-conditioned medium. Northern analysis also showed concomitant increases in TGF-beta1, bFGF, and tenascin after CNS injury to animals 14 d of age or older. These results show that the regulation of astrocytic tenascin is mediated by the synergistic action of TGF-beta1 and bFGF in vitro and after injury in vivo.

Age Factors↗
PubMed1997

Altered apolipoprotein E secretion in cytokine treated human astrocyte cultures.

Apolipoprotein E (ApoE), postulated to be a major lipid carrier protein in brain, is synthesized and secreted primarily by astrocytes and is involved in brain development and repair. We have analyzed its secretion in primary cultures of older (high passage) slowly dividing and younger (lower passage) rapidly dividing fetal human astrocytes exposed to various inflammatory and anti-inflammatory cytokines, alone and in combination. ApoE secretion was reduced in high passage astrocytes when compared to lower passage astrocytes. A further reduction in ApoE secretion in high passage cells was consistently produced by the combination of cytokines interleukin 1 (IL-1) alpha and beta and interferon (IFN-gamma) cytokines or by the basic fibroblast growth factor (basic-FGF) alone. Epidermal growth factor (EGF) increased ApoE secretion. The combination of these cytokine effects in chronically degenerating brain regions of Alzheimer's disease and other neurodegenerative diseases could reduce the amount of ApoE available for neuronal regeneration. EGF, or agents inducing EGF, could ameliorate these ApoE deficiencies.

Apolipoproteins E↗
PubMed1997

Detection of a soluble form of the leukocyte surface antigen CD48 in plasma and its elevation in patients with lymphoid leukemias and arthritis.

Proteins with glycosylphosphatidylinositol (GPI) anchors exhibit a range of activities and some of these proteins exist in both a membrane-associated and a soluble form. CD48 is a 47-kd GPI-linked glycoprotein which is expressed on T and B lymphocytes, monocytes, and many lymphoid malignancies. The biological function of CD48 is unknown. We describe the detection of a soluble form of CD48 in plasma and serum. Its level was quantified by an immunoenzymometric assay (IEMA) specific for soluble CD48. While soluble CD48 was detected in the plasma of healthy individuals (median = 29 ng/ml; range, 15-48 ng/ml), elevated levels were detected in some patients with lymphoproliferative disease (median = 41 ng/ml; range, 9-213 ng/ml, arthritis (median = 42 ng/ml; range, 13-67 ng/ml), and acute EBV infection (174 ng/ml). Soluble CD48 was also detectable in tissue culture supernatants from the Raji lymphoid cell line. The mechanism of CD48 release from cells is unclear. The finding of significant levels of soluble CD48 in plasma and the development of a sensitive IEMA for its measurement will facilitate further studies on its normal function and its role in disease.

Adult↗
PubMed1997

A recombinant GM-CSF-PE40 ligand toxin is functionally active but not cytotoxic to cells.

A granulocyte/macrophage colony-stimulating factor (GM-CSF)-Pseudomonas exotoxin (PE) 40 fusion protein was constructed for potential use in the treatment of myeloid leukaemias, as a conditioning agent prior to allogeneic bone marrow transplantation or for ex vivo purging of malignant cells prior to autologous bone marrow transplantation. The GM-CSF-PE40 fusion protein successfully binds to the GM-CSF receptor and is capable of initiating a mitogenic signal similar to native GM-CSF in the GM-CSF-dependent TF1 cell line. The toxin component also appears to be fully functional as determined by an in vitro adenosine diphosphate-ribosylation assay. The GM-CSF-PE40 fusion protein, however, was not cytotoxic to a number of myeloid leukaemia cell lines. It is suggested that the mechanism of internalization of the GM-CSF receptor is not appropriate for the translocation of PE to the cytosol where it can fulfil its cytotoxic potential.

ADP Ribose Transferases↗
PubMed1997

Semaphorin III can repulse and inhibit adult sensory afferents in vivo.

During development, semaphorins (collapsin, fasciclin) mediate repulsive and inhibitory guidance of neurons. Semaphorin III, a secretable member of this family, is expressed by the ventral spinal cord at the time corresponding to projection of sensory afferents from the dorsal root ganglion (DRG) into the spinal cord. The inhibitory effect of E14 ventral cord is active only on nerve growth factor (NGF)-responsive sensory afferents (small-diameter A-delta and C fibers subserving sensations of temperature and pain). Similarly, COS cells secreting recombinant semaphorin III are able to selectively repel DRG afferents whose growth is stimulated by NGF and not NT-3. However, it is not known whether these molecules can exert a functional role in the fully developed adult peripheral nervous system. In this study, we demonstrated that gene gun transfection and production of semaphorin III in corneal epithelial cells in adult rabbits in vivo can cause repulsion of established A-delta and C fiber trigeminal sensory afferents. In addition, it is shown that, following epithelial wounding and denervation, semaphorin III is able to inhibit collateral nerve sprouts from innervating the reepithelialized tissue. These findings are significant in that they provide direct evidence that small-diameter adult sensory neurons retain the ability to respond to semaphorin III. In addition, the corneal gene gun technique may be generally used to study the in vivo effects of neural growth modulators by quantifying the amount of sensory nerve growth.

Animals↗
PubMed1997

G-CSF after peripheral blood stem cell transplantation in lymphoma patients significantly accelerated neutrophil recovery and shortened time in hospital: results of a randomized BNLI trial.

We have undertaken a prospective randomized study in 90 patients with relapsed or resistant lymphomas to assess the value of G-CSF (lenograstim) in the acceleration of myeloid recovery after peripheral blood stem cell transplantation (PBSCT). A common regimen of cyclophosphamide 1.5 g/m2 on day 1 and lenograstim 263 microg s.c. on days 2-10 with two aphereses on days 10 and 11 was used for stem cell mobilization. 77% of patients achieved an adequate PBSC collection in two harvests (> 2 x 10(8) MNC/kg or > 2 x 10(6) CD34+ cells/kg). 65 patients went on to receive high-dose BEAM chemotherapy and engraftment data was available for 62. 34 patients had been randomized to receive lenograstim 263 microg/d s.c. and 28 to no growth factor. The median time to ANC > 0.5 x 10(9)/l was 9 d in the lenograstim arm versus 12.5 d in the no-lenograstim arm (P=0.0001). This was associated with a median duration of time in hospital post PBSCT of 13 d in the lenograstim arm versus 15.5 d in the no-lenograstim arm (P=0.0002). Median days to platelet independence, platelet transfusions, incidence of infection and red cell transfusion were the same in both arms. These data indicate that lenograstim significantly accelerated myeloid recovery after PBSCT and shortened the duration of hospital stay.

Adult↗
PubMed1997

Circulating plasma cells in multiple myeloma: characterization and correlation with disease stage.

The aim of this study was to develop a flow cytometric test to quantitate low levels of circulating myeloma plasma cells, and to determine the relationship of these cells with disease stage. Cells were characterized using five-parameter flow cytometric analysis with a panel of antibodies, and results were evaluated by comparison with fluorescent consensus-primer IgH-PCR. Bone marrow myeloma plasma cells, defined by high CD38 and Syndecan-1 expression, did not express CD10, 23, 30, 34 or 45RO, and demonstrated weak expression of CD37 and CD45. 65% of patients had CD19- 56+ plasma cells, 30% CD19- 56(low), and 5% CD19+ 56+, and these two antigens discriminated myeloma from normal plasma cells, which were all CD19+ 56(low). Peripheral blood myeloma plasma cells had the same composite phenotype, but expressed significantly lower levels of CD56 and Syndecan-1, and were detected in 75% (38/51) of patients at presentation, 92% (11/12) of patients in relapse, and 40% (4/10) of stem cell harvests. Circulating plasma cells were not detectable in patients in CR (n = 9) or normals (n = 10), at a sensitivity of up to 1 in 10,000 cells. There was good correlation between the flow cytometric test and IgH-PCR results: myeloma plasma cells were detectable by flow cytometry in all PCR positive samples, and samples with no detectable myeloma plasma cells were PCR negative. Absolute numbers decreased in patients responding to treatment, remained elevated in patients with refractory disease, and increased in patients undergoing relapse. We conclude that flow cytometry can provide an effective aternative to IgH-PCR that will allow quantitative assessment of low levels of residual disease.

Adult↗
PubMed1997

Effectiveness of alpha-tocopherol and selenium supplements in preventing lupinosis-associated myopathy in sheep.

OBJECTIVE: To compare the effectiveness of combined selenium and alpha-tocopherol acetate treatments in preventing lupinosis-associated myopathy in sheep. DESIGN: Measurement of plasma muscle and liver enzymes, and histopathological examination of muscle and liver in the treatment groups. PROCEDURE: The treatments were: subcutaneous injections of selenomethionine and vitamin E (sc(SeM+E)), an intraruminal selenium pellet and oral doses of vitamin E and intramuscular injections of selenomethionine combined with either oral doses of vitamin E (imSeM+orE) or intramuscular injections of vitamin E in an oily carrier. Another group received no supplements, while a control group was given selenium pellets on day 0 and fortnightly oral doses of vitamin E from day 0 to 72. To produce lupinosis-associated myopathy, the sheep were fed a diet low in vitamin E and given repeated injections of a crude extract of Diaphorthe toxica. Groups sc(SeM+E) and imSeM+orE were stressed by dosing with protected polyunsaturated fatty acids from day 56 onwards. RESULTS: Lupinosis-associated myopathy was induced in all unsupplemented sheep. In these sheep the storage of Se increased and that of vitamin E decreased. The subcutaneous treatment was highly effective in preventing lupinosis-associated myopathy and also produced the highest vitamin E concentrations in plasma and liver. Supplemental vitamin E was more efficacious than supplemental Se. Concentrations of vitamin E in the livers of sheep given intramuscular vitamin E were higher than expected based on plasma concentrations. Oral doses of vitamin E proved the least effective method of increasing concentrations in liver. Lupinosis did not affect Se concentrations in liver or muscle. CONCLUSION: The sc(SeM+E) treatment is highly effective in preventing lupinosis-associated myopathy but needs to be further assessed when selenium and vitamin E are both limiting in the diet.

Administration, Oral↗
PubMed1997

Identification of D motif epitopes in Staphylococcus aureus fibronectin-binding protein for the production of antibody inhibitors of fibronectin binding.

A fibronectin-binding protein (FnBP) adhesin of Staphylococcus aureus possesses three 37- or 38-amino-acid motifs (D1, D2, and D3) that can each bind fibronectin (Fn) with low affinity and that in tandem comprise D1-3, a high-affinity Fn-binding domain. To identify epitopes for the generation of adhesion-blocking antibodies, rabbits were immunized with recombinant D1-3 or with a glutathione S-transferase fusion protein, GSTD1-3. Affinity-purified antibodies from the D1-3 immunization were poor inhibitors of Fn binding to S. aureus and recognized several different epitopes, with a preference for clusters of acidic amino acids that do not contribute to Fn binding. Antibodies generated with GSTD1-3 as an immunogen were more effective inhibitors, but concentrations in excess of 20 microg x ml-1 did not promote more than 50% inhibition. These antibodies were highly specific for amino acids 21 to 34 of D1 (D1(21-34)), which contain a sequence that is essential for Fn binding and are identical to D2 at 12 of 14 residues. Neither antibody preparation recognized D3(20-33) of the D3 motif, where the only homology to D1(21-34) and D2(21-34) comprises a sequence motif, GG(X3,4)(I/V)DF, that is critical to Fn binding. However, antibodies specific for both D1(21-34) and D3(20-33) could be obtained by using synthetic peptides corresponding to these sequences as immunogens. F(ab')2 fragments derived from these antibodies each caused 40 to 50% inhibition of Fn binding to S. aureus, and their ability to bind to purified FnBP was eliminated by competing Fn. However, mixtures of the two F(ab')2 preparations did not provide additive or synergistic inhibition of Fn binding. Therefore, inhibition of Fn binding to S. aureus requires antibodies specific for D1(21-34) and D3(20-33), but a mixture of antibodies specific for both sequences did not provide complete inhibition.

Adhesins, Bacterial↗
PubMed1997

Electrophysiological analysis of dorsal root ganglion neurons pre- and post-coexpression of green fluorescent protein and functional 5-HT3 receptor.

Aequorea green fluorescent protein (GFP) is an excellent marker to examine genetically altered live cells in whole animals or culture. Its potential use in identifying genetically modified neurons, however, has not been investigated extensively. To examine the usefulness, toxicity, and potential electrophyiological effects of GFP expression in neurons, we generated adenovirus containing the mGFP4 cDNA. One week after virus transfection of dorsal root ganglion neurons (DRG), 10% of postnatal DRG neurons appeared brightly fluorescent, labelling the soma and neurites. Temporal examination of these neurons demonstrated no toxicity to DRG neurons even after several weeks in culture with repeated daily epifluorescent exposure. Electrophysiological analysis and comparison of control and viral exposed (GFP- and GFP+) DRG neurons did not demonstrate any differences in whole cell resistance, resting potential, action potential (AP) threshold, AP duration, AP amplitude, or whole cell capacitance. To investigate the usefulness of GFP as a marker for identifying neurons genetically altered to express a novel neurotransmitter receptor, a second adenovirus construct was generated containing both GFP and serotonin type 3 (5-HT3) receptor cDNAs. Transfection of DRG neurons with this virus produced an inward current in the presence of serotonin only in DRG neurons that were GFP-positive. It is concluded that adenoviral transfection of neurons with GFP, for cellular labeling, and coexpression of GFP-neurotransmitter constructs are safe, nontoxic, methods for electrophysiologically investigating neurons over several weeks. The uniqueness of the vector used in these experiments is that it was constructed to express GFP in a second cassette so that it would label the transduced cells, but have no potential for interfering with the function of the foreign 5-HT3 receptor.

Animals↗
PubMed1997

Electroconvulsive therapy for treatment of intractable seizures. Initial findings in two children.

We treated two children with intractable epilepsy with electroconvulsive therapy (ECT) for seizure control. One child showed a change in seizure pattern with treatment, which at greater intensity was also effective in stopping nonconvulsive status epilepticus. The other child showed a decrease in spontaneous seizure frequency during short-term treatment. These findings suggest a possible role for ECT in the management of intractable epilepsy in children who are not candidates for epileptic surgery.

Adolescent↗
PubMed1997

Mobilisation and reinfusion of Philadelphia negative peripheral blood mononuclear cells in chronic myeloid leukaemia with hydroxyurea and G-CSF.

Unmanipulated autologous transplantation of marrow of peripheral blood stem cells has been performed in small numbers of patients with CML for many years. More recently there has been interest in attempting to 'purge' the autograft of clonal cells as defined by the presence of the Philadelphia chromosome or BCR-ABL rearrangement. One method by which this might be achieved in vivo has been developed in Genoa and involves the administration of high dose chemotherapy and G-CSF followed by peripheral blood stem cell collection. These collections are frequently devoid of Philadelphia positive cells and the hope is that this will enhance the effects of subsequent autograft. We have investigated the use of a less toxic regimen for this procedure using oral hydroxyurea and G-CSF. In this review we describe the background to autografting in CML and the development of strategies to mobilise Philadelphia negative cells into the peripheral blood. We go on to present an update of our data using hydroxyurea and discuss some of the practical and theoretical issues behind the procedure.

Adult↗
PubMed1996

A cis-trans interaction at the 3'-untranslated region of ribonucleotide reductase mRNA is regulated by TGF-beta 1, TGF-beta 2, and TGF-beta 3.

The R2 component of ribonucleotide reductase is rate-limiting for DNA synthesis in proliferating cells, and recently, it has been shown that aberrant expression of R2 directly alters the malignant potential of tumor cells. We show that R2 gene expression is elevated in BALB/c 3T3 cells treated with transforming growth factor (TGF)-beta 1, TGF-beta 2, or TGF-beta 3, as determined by Northern blot analysis. Gel shift assays and UV crosslinking studies demonstrated similar post-transcriptional regulation at the 3'-untranslated region (3'-UTR) of the R2 mRNA, by TGF-beta 1, TGF-beta 2, and TGF-beta 3. The three growth factors induced a common 75 kDa RNA-protein complex. A 9 nucleotide sequence, GAGUUUGAG, previously shown to be responsive to TGF-beta 1-mediated R2 message stability changes, effectively competed out the formation of the R2 3'-UTR complex. We propose that these three different members of the TGF-beta family work through a common mechanism to control an important component of cell proliferation and a potential determinant of malignant progression.

3T3 Cells↗