Search PubMed⌕ Search

Biomedical subjects

M Clark

Publications and source records attributed to M Clark.

At least 145 records · Page 8Linked to original sources
PubMed1993

The generation of a humanized, non-mitogenic CD3 monoclonal antibody which retains in vitro immunosuppressive properties.

CD3 antibodies are proven immunosuppressants capable of reversing transplant rejection episodes. Their general application has been limited both by their immunogenicity and, in particular, by the "first-dose" cytokine-release syndrome experienced by patients after the initial administration of antibody. We have produced a set of variants of the humanized YTH 12.5 CD3 monoclonal antibody (mAb) (Routledge et al., Eur. J. Immunol. 1991. 21: 2717) bearing different human heavy (H) chain constant regions, with the intention of finding a form of the antibody that is not able to activate T cells. Comparison of the variants having gamma 1, gamma 2, gamma 3 and gamma 4 H chains in a competitive binding assay showed that antibody avidity was not affected by IgG subclass. Using a sensitive indicator of FcR binding activity (the capacity of the CD3 mAb to redirect cytotoxic T cells to kill the monocytic cell line U-937) we demonstrated a functional hierarchy of gamma 1 = gamma 4 > alpha 2 =/> gamma 3 mb >> gamma 2. An aglycosyl version of the gamma 1 CD3 mAb, produced by site-directed mutagenesis (Asn297 to Ala), still had considerable activity in this assay (intermediate to the gamma 1 and alpha 2 CD3 mAb), albeit at a level approximately 10-fold lower than that of the parental gamma 1 form. When we tested their ability to stimulate T cell proliferation in vitro in the presence of 5% human serum, all of the wild-type immunoglobulin isotypes were found to be active, although there were T cell donor-dependent variations in the extent of the responses. The aglycosyl gamma 1 mAb was, however, completely non-mitogenic in all of ten donors tested, unless the assay was performed in IgG-free medium. Despite being non-stimulatory, this mAb was also able to inhibit the mixed lymphocyte reaction responses of both naive and primed T cells. Comparison of the gamma 1 and aglycosyl gamma 1 mAb in an experimental mouse model for CD3 mAb-induced cytokine release indicated that removal of the carbohydrate moiety from the gamma 1 constant region reduced the in vivo tumor necrosis factor-alpha response by a factor of at least 16-fold. These data suggest that the aglycosyl gamma 1 CD3 mAb is a promising candidate for immunosuppressive therapy without "first dose" side effects.

Animals↗
PubMed1993

Structural motifs involved in human IgG antibody effector functions.

A humanized IgG antibody to CAMPATH-1 antigen (CDw52) is known to be lympholytic both in vitro and in vivo. So as to improve therapeutic potency through protein engineering strategies, we wish to define the structural motifs underlying some of the documented differences in function between human (h) IgG1 and IgG4 forms of the antibody. By the creation of heavy chain domain-switch and intra-domain recombinant antibodies we have established an important role for the carboxy-terminal half of the CH2 domain in determining differential behaviour in antibody-dependent cytotoxicity (ADCC) and in complement lysis. If this same region were necessary for the effector mechanisms that operate in vivo, then it might be possible to improve antibody effector functions by construction of novel antibodies that possess within the one molecule multiple copies of the crucial hinge-CH2 associated structures. Although our previous work suggested that the hIgG4 CAMPATH-1 antibody was ineffective at ADCC, we found this to be so only in some individuals. In others, IgG4, and indeed all the IgG subclasses were able to mediate ADCC. Overall, though, hIgG1 remains the best choice isotype for lytic therapy in vivo.

Amino Acid Sequence↗
PubMed1993

Purification and properties of Alligator mississipiensis cytochrome c.

Cytochrome c has been purified to homogeneity from alligator liver (Alligator mississipiensis) using aluminum sulfate precipitation, CM-cellulose and gel-filtration chromatography, and reverse-phase HPLC. The protein exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an approximate molecular weight of 12,000 Da. Oxidized and reduced visible spectra yielded maxima at 408 (tau) nm and 315 (delta), 415 (tau), 520 (beta), and 550 (alpha) nm, respectively, while potentiometric titrations in the presence of dye-mediators yielded an Eo of +265 mV. N-terminal analysis of the protein yielded no sequence which indicates a blocked residue. A combination of amino acid sequencing, using peptides obtained from Staphylococcus aureus V8 protease, endoproteinase Lys-C, and CNBr digests of the protein and total amino acid analyses, using equine and avian cytochromes c as standards yielded the primary sequence GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLIGRKTGQAPGFSYTEANKNKGITWGEETLMEYLE NPKKYIPGTKMIFAGIKKKPERADLIAYLKEATSN. Comparison with sequences of other cytochromes c indicated the closest similarity to cytochrome c from snapping turtle (Chelydra serpentina) with substitutions at five positions corresponding to residues 32 (His-->Asn), 44 (Glu-->Pro), 89 (Ala-->Pro), 100 (Asp-->Glu), and 104 (Lys-->Asn), respectively. The presence of Pro and Asn residues at positions 89 and 104, respectively, are unique to alligator cytochrome c.

Alligators and Crocodiles↗
PubMed1993

Chronic changes in thyroid hormones do not affect brain adenosine receptors.

1. In this study, the authors examined the effects of chronic (14 days) changes in thyroid function on a major neuromodulatory receptor system in the brain- the adenosinergic system. While previous investigators have examined the effects of alteration in thyroid function on adenosine receptors in peripheral tissues (adipocytes), this is the first study to examine such effects in brain. 2. Three groups of male Sprague-Dawley rats were treated for 14 days with either a) oral PTU (0.00625%), iodine-free diet, and i.p. saline injections, b) i.p. saline injections, or c) i.p. triiodothyronine (25 micrograms/100 g) injections. 3. These manipulations reliably resulted in the production of hypothyroidism (TSH 30.2 +/- 8.6 ng/ml), euthyroidism (TSH 2.1 +/- 0.9), and hyperthyroidism (TSH < 0.4). 4. Treatment had no significant effect on the Bmax or Kd of [3H]DPCPX (A1-antagonist) binding to homogenates from cerebral cortex, cerebellum or hippocampus; similarly, no effect on [3H]CGS-21680 (A2-agonist) binding to striatal homogenates was noted. 5. Similarly, quantitative autoradiographic studies failed to reveal consistent regional alterations unique to either hypo- or hyperthyroidism. 6. Incubation of sections with GppNHp resulted in the expected reduction (approximately 40%) in agonist binding, but there was no differential effect seen for either the hypo- or hyperthyroid tissues. 7. These preliminary findings suggest that alterations in brain adenosine receptors or G-protein-receptor coupling are unlikely to be requisite correlates of abnormal thyroid hormone levels.

Adenosine↗
PubMed1993

Correlation between the DNA-binding affinity of topoisomerase inhibiting drugs and their capacity to induce hematopoetic cell differentiation.

There is accumulating evidence that both type I and type II DNA-topoisomerases play a key role in cellular differentiation. Human HL-60 leukemia cells can be induced to monocytic or granulocytic differentiation with various compounds; amongst them camptothecin, a topoisomerase I inhibitor and VP-16, VM-26 and mitoxantrone, all potent topoisomerase II inhibitors. During HL-60 cell differentiation topoisomerase I activity increases and topoisomerase II activity decreases. The two isoenzymes topoisomerase II alpha and topoisomerase II beta seem to have different physiological functions in highly proliferating cells, G0 cells and differentiated cells as their expression is regulated differently. In concentrations sublethal to undifferentiated cells, m-AMSA, also a potent topoisomerase II inhibitor, is able to prevent DMSO-induced granulocytic HL-60 cell differentiation. In drug-sensitive cells derived from several sources (mouse erythroleukemia, human gastric carcinoma, human leukemia), we found a functional heterogeneity of topoisomerase activity which is altered specifically during cellular differentiation. The isoactivities can be separated by their different pH and salt requirements and they exhibit different sensitivity to topoisomerase II inhibiting drugs. Functional heterogeneity of topoisomerases seems to be a prerequisite to high drug sensitivity of the cells, since drug-resistant sublines in our experiments do not exhibit this heterogeneity. We propose that the topoisomerase II inhibiting drugs which are able to induce differentiation, namely the epipodophyllotoxines VP-16 and VM-26, inhibit subfractions of the topoisomerase II pool which are necessary to maintain the undifferentiated status of the cells. These drugs induce differentiation in concentrations 10-100-fold below the lethal dose, the concentration must be sufficient to inhibit topoisomerase II but well below the concentration to induce the cleavable complex. This might be the reason that anthracyclines with a high DNA binding affinity have low differentiation-inducing capacity.

Amsacrine↗
PubMed1993

The basis of the histamine assay in human skin: II. Wheal formation.

1. Subepidermal injection of histamine solutions in isotonic saline in four subjects evoked whealing after a mean delay of 2.6 min, this estimate being derived by regression modelling. 2. Wheal growth was better modelled by the logarithm of time than by time, suggesting that wheal size depends upon assisted diffusion of histamine in the dermis. 3. Wheal growth was piecemeal, not continuous, consistent with the successive involvement of neighbouring vascular territories. 4. Wheal growth was completed by 9 min after injection.

Adult↗
PubMed1993

The use of mouse/human chimaeric antibodies to investigate the roles of different antibody isotypes, including IgA2, in the killing of Schistosoma mansoni schistosomula by eosinophils.

We report the use of a matched set of mice/human chimaeric antibodies, directed against the 5-iodo-4-hydroxyl-3-nitrophenacetyl (NIP) hapten, to investigate the roles of different human isotypes in antibody-mediated eosinophil-dependent killing of schistosomula. The chimaeric antibodies consist of mouse VH, VL and CL regions with human gamma 1, gamma 2, gamma 3 (2 allotypes), gamma 4, alpha 2, mu or epsilon CH regions and were used in in vitro assays with human eosinophils and NIP-coated S. mansoni schistosomula. Some anti-NIP isotypes mediated high levels of killing, which was specific for NIP-coated larvae, and we suggest that these antibodies will be a valuable tool for studies on the role of antibody isotypes in anti-schistosome immune effector mechanisms. In particular, this method directly demonstrated, for the first time, that IgA is highly effective in mediating the killing of metazoan parasites by human eosinophils.

Animals↗
PubMed1993

The association between hypertension and sleep apnea in obese patients.

Strong associations have been demonstrated between hypertension (HTN) and obesity, and obesity and sleep apnea (SA). However, it is unclear whether previously suggested relationships between HTN and SA were based on causal mechanisms, or rather, were confounded by obesity. To investigate whether SA may be an independent predictor of HTN, we measured respiration during sleep and BP in 19 participants in a hospital-based weight control program. Additionally, subjects underwent cephalometry, measurement of weight and hip circumferences, and pulmonary function tests. Studies were repeated in 14 subjects following weight loss. Subjects consisted of 9 men and 10 women, ages 43 +/- 11 years (mean +/- SD), and weight of 114 +/- 20 kg. Hypertension (diastolic BP > 95 mm Hg, systolic BP > 160 mm Hg, or a report of current use of an antihypertensive medication) was present in 6 (32 percent) subjects. Sleep apnea was more prevalent among the hypertensive (5/6; 83 percent) than normotensive (2/13; 15 percent) subjects (p < 0.01); respiratory disturbance index (RDI) was 42.4 +/- 16.0 vs 6.2 +/- 2.3 (p < 0.01) in these groups. Compared with normotensive subjects, hypertensive subjects were of similar weight, but had greater central obesity and had lower levels of vital capacity. Regression analyses demonstrated that RDI was the strongest independent predictor of BP level, accounting for approximately 60 percent of the variability in this measurement. Following weight loss, BP and RDI both significantly declined, soft palate width decreased, and vital capacity increased. Thus, in this group of obese subjects, BP level was associated most significantly with the magnitude of sleep-related respiratory disturbances. The obese subjects who are most likely to have SA and HTN are male and/or subjects with greater central obesity and lower vital capacity.

Adult↗
PubMed1993

Can melatonin improve shift workers' tolerance of the night shift? Some preliminary findings.

The pineal hormone melatonin is potentially useful in the treatment of disorders, especially sleep disorders, associated with circadian rhythm disturbance. We have examined its effects on sleep, mood, and behaviour in a double-blind, placebo-controlled study of a small group of police officers working spans of seven successive night shifts. Compared to placebo, and to no treatment, melatonin (5 mg) taken at the desired bedtime improved problems related to sleep and increased alertness during working hours, especially during the early morning. In letter-target performance tests visual search speed and accuracy were either unchanged or slightly improved. Memory scanning speed and perception of mental load were adversely affected. This preliminary study suggests that melatonin has beneficial effects on sleep and alertness, but that its effects on performance need careful evaluation.

Adult↗
PubMed1992

Expression of c-fos mRNA in acute and kindled cocaine seizures in rats.

In situ hybridization for c-fos mRNA was performed on brain sections (a) from rats after an acute cocaine-induced seizure or from saline-injected controls and (b) from rats after their first cocaine-kindled seizure, as well as from rats that had not yet developed cocaine-kindled seizures (but were exposed to the same amount of cocaine as those that did exhibit convulsions) and from saline-injected controls. Increased expression of c-fos mRNA was observed in animals demonstrating cocaine-induced seizures acutely or following pharmacological kindling. Rats that experienced acute seizures after cocaine (65 mg/kg) showed pronounced increase in expression of c-fos mRNA in the dentate gyrus of the hippocampus and olfactory bulb. Increases were also observed in several other limbic cortical regions, as well as the striatum and ventromedial hypothalamic nucleus (VMH). In rats that were injected daily with an initially subconvulsive dose of cocaine-HCl (40 mg/kg), the cocaine-kindled seizures induced elevations in c-fos mRNA in the same brain regions as with an acute cocaine-induced seizure with the single exception of the VMH. These findings not only suggest the involvement of limbic, cortical and striatal structures in the cocaine-induced seizure, but also raise the possibility that alterations in the proto-oncogene c-fos and its subsequent impact on gene expression could play a role in the changes in neural excitability associated with cocaine-induced kindling.

Animals↗
PubMed1992

Cholesterol-related counseling by registered dietitians in northern California.

BACKGROUND: An estimated 40 million Americans have serum cholesterol levels that warrant medically supervised dietary intervention. Although registered dietitians are expected to play an important role in treating these patients, current treatment practices in the community are largely unknown. METHODS: A questionnaire concerning treatment practices was mailed to all 377 registered dietitians listed in the directories of the American Dietetic Association for two large California districts. Number of patients seen and length and content of dietary counseling were ascertained for three types of patients: (a) hypercholesterolemic outpatients without heart disease, (b) hypercholesterolemic outpatients who have heart disease, and (c) inpatients with myocardial infarction. RESULTS: A return rate of 59% (n = 252) was obtained for the questionnaire. A total of 44% of the registered dietitians counseled hypercholesterolemic patients in any of the categories surveyed. About 30% of the respondents counseled hypercholesterolemic outpatients without heart disease. They saw an average of 4.9 such patients a week, spent an average of 53 min in an initial session, and usually did not see the patient again in follow-up. Fewer than 10% of patients had as many as four sessions. About 27% of the respondents saw hypercholesterolemic outpatients with heart disease, averaging 3.5 such contacts per week. The reported practices were similar to those provided to noncardiac outpatients. About 22% of registered dietitians worked with hospitalized myocardial infarction patients. They spent an average of a total of 41 min over 2.5 visits with each patient. CONCLUSION: Currently, outpatient registered dietitian counseling for hypercholesterolemia appears to be limited in both the number of patients reached and the duration of the counseling. Further research into the impact of, barriers to, and efficacy of alternative delivery methods of dietary counseling is needed.

Adult↗
PubMed1992

Quantitative autoradiography of 3H-paroxetine binding sites in rat brain.

The use of 3H-paroxetine as a ligand for quantitative autoradiography of serotonin (5-HT) transport sites was optimized, and the distribution of 3H-paroxetine binding sites in rat brain was studied. Under the conditions described, 3H-paroxetine binding in forebrain sections was of high affinity and saturable, with a Kd of 0.18 +/- 0.02 nM (mean +/- SEM) and Bmax of 268 +/- 12 fmol/mg of protein (n = 3). Nonspecific binding was 10.7% +/- 1.0 of the total binding (n = 8). The distribution of 3H-paroxetine binding sites closely matched the regional distribution of 5-HT nerve terminals and cell bodies. The highest concentrations of 3H-paroxetine binding sites were found in the dorsal raphe nucleus (563 +/- 55 fmol/mg tissue, n = 4), and high densities of binding were also found in the locus coeruleus, medial forebrain bundle, substantia nigra, several limbic structures (amygdala, hippocampus, hypothalamus, olfactory tubercle, septum, and thalamus), and components of the visual relay system (superior colliculus and the lateral geniculate body). Although lesioning of 5-HT neurons with p-chloroamphetamine (PCA) drastically eliminated 3H-paroxetine binding in most regions of the rat brain, significant binding remained in the raphe nuclei and medial forebrain bundle suggesting that 3H-paroxetine binding in these regions was to presynaptic sites on cell bodies or axons relatively resistant to PCA.

Animals↗
PubMed1992

Killing of human leukaemia/lymphoma B cells by activated cytotoxic T lymphocytes in the presence of a bispecific monoclonal antibody (alpha CD3/alpha CD19).

Bispecific antibodies (BsAb) can be used to retarget T cells irrespective of their specificity to certain target cells inducing target cell lysis. We have tested the efficacy of the BsAb SHR-1, directed against the T cell antigen CD3 and the B cell antigen CD19 to induce (malignant) B cell kill by T cells as measured in a 51Cr-release assay. Two cytotoxic T cell clones (CTL), expressing TCR alpha beta or TCR gamma delta, were effective in killing CD19 expressing B cell lines at different stages of differentiation in the presence, but not in the absence, of the BsAb. CD19- target cells were not killed. Fresh CD19+ leukaemia/lymphoma cells were also efficiently killed by SHR-1 preincubated CTL clones. In addition, phytohaemagglutinin (PHA) or CD3-activated IL-2 expanded peripheral blood mononuclear cells (PBMC) of normal donors did so after 2 weeks of stimulation. A concentration of 100 ng/ml of the BsAb was sufficient to obtain optimal lysis of all target cells tested. These results show that fresh human leukaemia/lymphoma cells, freshly derived from active lymphoblastic leukaemia (ALL) as well as non-Hodgkin's lymphoma (NHL) patients, can be effectively killed in the presence of this BsAb by activated T cells.

Animals↗
PubMed1992

Matching patient need for pressure sore prevention with the supply of pressure redistributing mattresses.

The authors have conducted a series of pressure sore prevalence surveys within one health district over a 4-year period. Additional surveys of the availability and deployment of pressure redistributing (PR) bed mattresses have also been made within the same district. The prevalence of pressure sores increased from 6.8% (1986) to 14.2% (1989) while the available stocks of PR mattresses increased from 69 (1987) to 186 (1989). Our results appear to question the common assumption that successful pressure sore prevention can be achieved through expanding the stocks of PR mattresses. Five possible reasons why the increase in mattress provision was matched with an increased prevalence of pressure sores are forwarded. The use of measures of prevalence rather than incidence rates to monitor the effects of clinical initiatives is discussed.

Beds↗