Biomedical subjects
M E Martin
Publications and source records attributed to M E Martin.
Conformational changes in rodent and human alpha-fetoprotein: influence of fatty acids.
Binding, spectral and immunological studies were performed to demonstrate the conformational changes in rodent and human alpha-fetoprotein (AFP) induced by a free fatty acid environment. Scatchard analysis of estradiol (E2) binding to purified rat AFP indicated that unsaturated fatty acids changed the number of binding E2 sites and the apparent E2 equilibrium dissociation constant which varied non-linearly with docosahexaenoic acid concentration. UV spectral analysis of rodent and human AFPs showed that the absorbance minimum of AFP incubated with unsaturated fatty acid (L-AFP) was red-shifted, broadened and less pronounced than that of purified native AFP (N-AFP). Immunochemical studies with specific polyclonal antibodies to purified rodent and human AFPs (N-AFP antibodies) showed that these proteins lost immunoreactivity after incubation with unsaturated fatty acid. N-AFP antibodies recognized fewer epitopes on L-AFP than on N-AFP, whatever the species. Specific anti-rat L-AFP antibodies were used to demonstrate specific epitopes on rat L-AFP. Rat L-AFP antibodies did not recognize rat N-AFP. Saturated fatty acids were without effect on the binding, spectral and immunological properties of rodent and human AFPs. RIA or ELISA values for human AFP from fetal serum, hepatoma serum, and cord serum, were reduced 80, 50 and 5%, respectively, by unsaturated fatty acids. This decrease correlated with the relative percentage of polyunsaturated fatty acid in each biological fluid. Such results indicate that an unsaturated fatty acid environment induces conformational changes in AFP which may modulate the endocrine and immune functions of this protein.
Polyvalent human immunodeficiency virus synthetic immunogen comprised of envelope gp120 T helper cell sites and B cell neutralization epitopes.
In previous studies, we have used antisera raised to envelope (env)-gene-encoded synthetic peptides to identify a region of (HIV) glycoprotein (gp) 120 env protein designated SP10 that contains a type-specific neutralizing determinant. To develop a polyvalent, synthetic peptide inoculum that can evoke both neutralizing antibodies and T cell proliferative responses to more than one HIV isolate, synthetic peptides containing type-specific neutralizing determinants of gp120 from HIV isolates HTLV-IIIB (IIIB), HTLV-IIIMN (MN) and HTLV-IIIRF (RF) were coupled to a 16 amino acid T cell epitope (T1) of HIV-IIIB gp120 and used to immunize goats. Goat antisera to each T1-SP10 peptide derived from the SP10 region of gp120 of IIIB, MN, and RF neutralized HIV isolates IIIB, MN and RF in a type-specific manner. Moreover, peripheral blood T cells from immunized goats also proliferated in a type-specific manner to peptides derived from gp120 of IIIB, MN, and RF. When combined in a trivalent inoculum, T1-SP10 peptides from HIV-1 isolates IIIB, MN, and RF evoked a high titered neutralizing antibody response to isolates IIIB, MN, and RF in goats and as well induced immune T cells to undergo blast transformation in the presence of peptides derived from gp120 of all three HIV isolates. The T1 portion of the T1-SP10 construct was shown to induce a B cell antibody response against determinants within the T1 peptide in addition to inducing T cell proliferative responses in immune goat T cells. Moreover, the SP10 portion of the T1-SP10 constructs not only induced B cell antibody production but also induced type-specific T cell proliferative responses localized to the C-terminal variable sequences of the SP10 peptides. Finally, the T1-SP10 peptide construct induced memory T cell proliferative responses to native gp120 env protein. Thus, combinations of homologous SP10 region synthetic peptides containing type-specific neutralizing determinants and T cell epitopes of HIV gp120 may be useful in man to elicit high titered neutralizing B cell responses and, as well, T cell responses to more than one HIV isolate.
Immunohistologic analysis of the distribution of cell adhesion molecules within the inflammatory synovial microenvironment.
Antigen-independent binding of T lymphocytes to a variety of cell types has been shown to be mediated by receptor-ligand pairs of adhesion molecules. In forms of inflammatory synovitis (including rheumatoid arthritis), T cells home to synovium, become activated, and participate in the generation of chronic synovitis. Using indirect immunofluorescence assays on synovial frozen tissue sections and on synovial fibroblast cell lines, we studied the distribution of cell adhesion molecules on components of the synovial microenvironment in inflammatory synovitis. We reasoned that analysis of the cell types within synovium that express adhesion molecules might provide clues to lymphocyte-stromal interactions that occur in inflammatory synovitis. We found that antibodies against the lymphocyte function-associated antigen 3 (LFA-3) molecule and the intercellular adhesion molecule 1 (ICAM-1) both reacted with macrophage-like type A synovial cells and synovial fibroblasts, as well as with tissue macrophages and vessel endothelium. Using flow cytometry, we found that anti-LFA-3 and anti-ICAM-1 (but not antibodies against their ligands CD2 and LFA-1) reacted with synovial fibroblast cells cultured in vitro. Thus, these data demonstrate that the ligands for lymphocyte LFA-1 molecules (ICAM-1) and for T cell CD2 molecules (LFA-3) are widely distributed among cell types of the synovial microenvironment and provide numerous cell types with which lymphocytes can interact via these 2 adhesion pathways during the course of inflammatory synovitis.
Prolongation of simple and choice reaction times in a double-blind comparison of twice-daily hydroxyzine versus terfenadine.
Newer, nonsedating antihistamines provide a therapeutic alternative for the patient with allergy whose work is impaired by the side effects of traditional H1 antihistamines. To assess the differential effect of these antihistamines on reaction times and subjective symptoms, we compared terfenadine, 60 mg twice daily, to hydroxyzine, 25 mg twice daily, in a double-blind, placebo-controlled, crossover study of 16 healthy, asymptomatic adults. Simple reaction time and choice reaction time were measured with a computer-based, eye-hand, reaction-time testing apparatus. Reaction times and symptom scores were assessed 90 minutes after the fourth and tenth doses of each drug. Hydroxyzine, but not terfenadine, significantly prolonged both simple and choice reaction time (p less than or equal to 0.0001). However, decision time, the time to process one bit of spatial information, was not prolonged by either antihistamine. Therefore, hydroxyzine prolonged the interpretation and response to stimuli of the central nervous system without increasing single-bit processing time. Although terfenadine was not different from placebo for any symptom assessed, hydroxyzine produced significant drowsiness (p = 0.001), dry mouth (p = 0.022), and irritability (p = 0.021). During the 5 days of hydroxyzine administration, neither objective nor subjective symptoms demonstrated the development of tolerance. No correlation was found between subjective symptoms and prolongation of reaction times by hydroxyzine, suggesting that side effect symptoms of traditional antihistamines are unreliable predictors of objective performance. Terfenadine provides a promising therapeutic alternative to traditional antihistamines for individuals performing critical tasks.
Suppression of the late cutaneous response by immunotherapy.
In a prospective, double-blind, placebo-controlled study, we examined the effect of mountain cedar (MC) immunotherapy on the MC-induced late cutaneous response (LCR). Fourteen MC-sensitive patients were intradermally skin tested before and after immunotherapy with MC extract. We measured the size of the wheal at 15 minutes and the area of tissue swelling at 6 hours. Patients were matched by the size of the LCR and started receiving either MC immunotherapy or placebo immunotherapy. MC-specific immunoglobulins (MC sIgG, MC sIgG1, MC sIgG4, and MC sIgE) were measured by ELISA. Symptom-medication scores (SMSs) were recorded on a daily basis during the MC season and tabulated at the end of the study. Comparison of the 14 paired patients revealed no significant differences between MC-treated and placebo-treated groups in preimmunotherapy MC sIgG1 and SIgG4. However, when MC immunotherapy was compared to placebo immunotherapy, patients receiving MC immunotherapy developed significantly higher MC sIgG1 (p less than 0.04) and MC sIgG4 (p less than 0.01) after immunotherapy. Patients receiving MC immunotherapy also demonstrated significantly greater suppression of the LCR after immunotherapy (p less than 0.005) with the postimmunotherapy LCR correlating significantly with both MC sIgG4 (rs = 0.715; p = 0.008) and cumulative dose of MC received (rs = 0.808; p = 0.004). MC sIgE was similar in both groups after immunotherapy. The reduction in SMSs in the MC-treated group did not reach significance, nor was there a correlation of SMSs with MC sIgE, sIgG, sIgG1, or sIgG4.(ABSTRACT TRUNCATED AT 250 WORDS)
Cultural relativity and poverty.
The nurse who practices from a perspective of cultural relativity attempts to understand client behaviors within the context of the clients' culture. Viewing customs (behaviors) as a reflection of client beliefs and values can enhance the nurse's effectiveness with clients in poverty. This paper presents a case study in which a culturally relativistic perspective was used to assess and intervene with a family living in poverty.
Transmission of disease in dogs by toothbrushing.
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Two rapid methods for determination of serum theophylline concentrations: a comparison.
Two methods for determining serum theophylline concentrations were compared against a standard laboratory assay. Each method was evaluated for ease of use, accuracy, and precision. Although both methods were found to be easy to use, there were major differences between the assays in both accuracy and precision.
Analysis of expression of CD2, CD3, and T cell antigen receptor molecules during early human fetal thymic development.
To define early stages of T cell maturation during human fetal thymic development, we have used mAb reactive with CD2, CD3, and TCR molecules in indirect immunofluorescence assays on a series of early human fetal thymic specimens. Using a technique of quantitating the relative proportions of fluorescent-positive cells present in tissue sections, we found at 8.5 wk of gestational age after arrival of CD7+ T cell precursors into the thymic rudiment, 60% of thymic CD7+ cells were CD2+, 4% were CD3+ and none was TCR-delta+ or TCR beta+. Moreover, cells reactive with anti-CD2 antibodies against T11(2) and T11(3) epitopes of CD2 as well as thymic stromal cells expressing the CD2 ligand, lymphocyte function associated Ag-3, were also present at 8.5 wk. From 9.5 wk to birth TCR beta+ cells increased to include greater than 90% of all CD7+ cells while TCR-delta+ cells fell from a peak of 11% of CD7+ cells at 9.5 wk to 1% of CD7+ cells at birth. These data suggest that epitopes of CD2 molecules are expressed early on during fetal thymic development. Moreover, these data suggest that CD7+, CD2+, cytoplasmic CD3+ T cell precursors in man give rise to both TCR-delta+ T cells as well as to T cells expressing TCR-alpha beta.
Early events in human T cell ontogeny. Phenotypic characterization and immunohistologic localization of T cell precursors in early human fetal tissues.
During early fetal development, T cell precursors home from fetal yolk sac and liver to the epithelial thymic rudiment. From cells that initially colonize the thymus arise mature T cells that populate T cell zones of the peripheral lymphoid system. Whereas colonization of the thymus occurs late in the final third of gestation in the mouse, in birds and humans the thymus is colonized by hematopoietic stem cell precursors during the first third of gestation. Using a large series of early human fetal tissues and a panel of monoclonal antibodies that includes markers of early T cells (CD7, CD45), we have studied the immunohistologic location and differentiation capacity of CD45+, CD7+ cells in human fetal tissues. We found that before T cell precursor colonization of the thymus (7-8 wk of gestation), CD7+ cells were present in yolk sac, neck, upper thorax, and fetal liver, and were concentrated in mesenchyme throughout the upper thorax and neck areas. By 9.5 wk of gestation, CD7+ cells were no longer present in upper thorax mesenchyme but rather were localized in the lymphoid thymus and scattered throughout fetal liver. CD7+, CD2-, CD3-, CD8-, CD4-, WT31- cells in thorax and fetal liver, when stimulated for 10-15 d with T cell-conditioned media and rIL-2, expressed CD2, CD3, CD4, CD8, and WT31 markers of the T cell lineage. Moreover, CD7+ cells isolated from fetal liver contained all cells in this tissue capable of forming CFU-T colonies in vitro. These data demonstrate that T cell precursors in early human fetal tissues can be identified using a mAb against the CD7 antigen. Moreover, the localization of CD7+ T cell precursors to fetal upper thorax and neck areas at 7-8.5 wk of fetal gestation provides strong evidence for a developmentally regulated period in man in which T cell precursors migrate to the epithelial thymic rudiment.
Human transcortin (CBG): fatty acids induce selective steroid binding changes associated with immunological modifications.
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Polymerized serum albumin beads possessing slow release properties for use in vaccines.
Experimental vaccines have been made by co-polymerizing virus particles and virus subunits into rabbit serum albumin beads. When injected into rabbits these model vaccines induced specific humoral antibody production and mimicked vaccines prepared using Freund's incomplete adjuvant. No adverse reactions occurred in experimental rabbits and no antibodies reacting with polymerized rabbit serum albumin beads were found. Immunostimulation is attributed to a slow release of antigen coinciding with the gradual breakdown of bead structure.
Activation of the polyomavirus enhancer by a murine activator protein 1 (AP1) homolog and two contiguous proteins.
The polyomavirus enhancer is composed of multiple DNA sequence elements serving as binding sites for proteins present in mouse nuclear extracts that activate transcription and DNA replication. We have identified three such proteins and their binding sites and correlate them with enhancer function. Mutation of nucleotide (nt) 5140 in the enhancer alters the binding site (TGACTAA, nt 5139-5145) for polyomavirus enhancer A binding protein 1 (PEA1), a murine homolog of the human transcription factor activator protein 1 (AP1). This mutation simultaneously reduces polyomavirus transcription and DNA replication. Reversion of this mutation simultaneously restores binding of PEA1 and both DNA replication and transcription. Binding of a second protein, PEA2, adjacent to the PEA1 site at nt 5147-5155 is enhanced by PEA1 binding, suggesting that these proteins interact. A third protein, PEA3, binds to the sequence AGGAAG (nt 5133-5138) adjacent to the PEA1 binding site; integrity of this late-proximal PEA3 binding site or an additional early-proximal site (nt 5228-5233) is important for enhancer function. We correlate binding of PEA1 and PEA2 with the induction of a DNase I-hypersensitive site in polyomavirus minichromosomes isolated from mouse fibroblasts.
Multiple-dose pharmacokinetics of intravenously administered cefoperazone and sulbactam when given in combination to infected, seriously ill, elderly patients.
The pharmacokinetics of cefoperazone and sulbactam in combination were evaluated in six, elderly, seriously ill patients treated with the drug combination for intra-abdominal infections. After giving informed consent, three males and three females aged 63.5 to 77.5 (mean 67.9) years and weighing 54.5 to 86.8 (mean, 67.6) kg were treated with cefoperazone (2.0 g) and sulbactam (1.0 g) infused intravenously every 12 h for at least 5 days. Cefoperazone and sulbactam pharmacokinetics were characterized on both days 1 and 5 of treatment. Eleven serial blood samples were obtained just prior to and following dose 1 on days 1 and 5 of treatment. Mean estimates of cefoperazone maximal concentration in plasma (Cmax), area under the curve of drug concentration in plasma versus time (AUC), half-life (t 1/2), apparent volume of distribution by the area method (Varea), apparent volume of distribution at steady state (Vss), and total body clearance (CL) for day 1 (day 5) were 297.5 237.5) micrograms/ml, 1,247 (1,063) micrograms.h/ml, 7.0 (4.9) h, 16.1 (13.4) liter, 13.1 (14.4) liter, and 28.9 (34.2) ml/min, respectively. Day 1 (day 5) mean values for sulbactam Cmax, AUC, t 1/2, Varea, Vss, and CL were 110.3 (78.0) micrograms/ml, 228 (217) micrograms.h/ml, 3.4 (2.5) h, 26.1 (18.5) liter, 18.9 (15.4) liter, and 97 (94) ml/min, respectively. Both drugs evidenced slower elimination and greater pharmacokinetic variability in these patients compared with values previously reported for normal volunteers. As patients improved during the course of therapy, the only pharmacokinetic parameter significantly changed between days 1 and 5 was a shortened sulbactam t 1/2. Our inability to find substantial evidence of pharmacokinetic normalization may have been related to sample size and study duration. Both drugs were present in potentially therapeutic concentrations for the entire 12-h dosing interval, but without undue accumulation from days 1 to 5.
Selective changes in binding and immunological properties of human corticosteroid binding globulin by free fatty acids.
The steroid hormones, progesterone (P4) and cortisol (F), have different biological activities but are both bound to human corticosteroid binding globulin (CBG) with similar affinity. This study examines the effect of physiological concentrations of FFA on the binding of these steroids to purified CBG and to the serum of pregnant women. It also analyzes the influence of the FFA environment on the immunological behavior of CBG. Unsaturated fatty acids (UFA) had a dose-dependent inhibitory effect (P less than 0.001) on steroid binding to CBG which was offset by saturated fatty acid-induced potentiation of binding (P less than 0.01) when both were present with CBG. UFAs inhibited P4 binding more than F binding. Comparable results were obtained with pregnant serum or with pure CBG. UFAs seemed able, depending on their concentration, to promote different molecular states of CBG, some with enhanced F binding and significantly reduced P4 binding, and others in which both P4 and F binding was markedly reduced. Scatchard analysis of steroid binding to purified CBG indicated that the UFAs influenced the association constant (Ka) and the number of binding sites (n) for F and P4 binding differently. Low concentrations (less than 16 microM) of arachidonic acid (C20:4) slightly potentiated F binding, with no change in Ka and a 1.6-fold increase in n; this concentration of C20:4 reduced n for P4 binding by 40% and did not affect Ka. Higher C20:4 concentrations (greater than 32 microM), reduced the Ka for F binding but did not apparently change n; for P4 binding, Ka was sharply reduced and n increased. The apparent equilibrium dissociation constant (Kd) for both F and P4 binding varied nonlinearly and differently with increasing C20:4 concentration. Immunoelectrophoresis and immunoautoradiography showed a reduction, or loss, of CBG immunoreactivity in the presence of UFA. The extent of these changes varied with the concentration and class of the UFA. These results indicate that FFA induce conformational changes in CBG which may modulate its activity and so influence the role of this protein in both the endocrine and immune systems.
A Streptococcus mutans superoxide dismutase that is active with either manganese or iron as a cofactor.
The superoxide dismutase produced by Streptococcus mutans OMZ176 during aerobic growth in a chemically defined medium (modified FMC) that was treated with Chelex 100 (to lower trace metal contamination) and supplemented with high purity manganese was purified (162-fold) by heat treatment, ammonium sulfate precipitation, and chromatofocusing chromatography. The superoxide dismutase produced during aerobic growth in the same medium, but without manganese and supplemented with high purity iron, was similarly purified (220-fold). The molecular masses of each holoenzyme were approximately 43,000 with a subunit mass of 20,700, indicating that the enzymes were dimers of two equally sized subunits. The superoxide dismutase from manganese-grown cells was a manganese enzyme (MnSOD) containing 1.2 atoms of manganese and 0.25 atoms of iron/subunit. The superoxide dismutase from iron-grown cells was an iron enzyme (FeSOD) containing 0.07 atoms of manganese and 0.78 atoms of iron/subunit. The amino acid compositions of the MnSOD and the FeSOD were virtually identical, and their amino-terminal sequences were identical through the first 22 amino acids. Dialysis of the FeSOD with o-phenanthroline and sodium ascorbate generated aposuperoxide dismutase with 94% loss of activity; subsequent dialysis of apoenzyme with either manganese sulfate or ferrous sulfate reconstituted activity (recoveries of 37 and 30%, respectively). Electrophoretic determination of cytoplasmic radioiron distribution indicated that (during aerobic growth) manganese prevented insertion of iron into superoxide dismutase, although the iron levels of at least two other cytoplasmic fractions were not altered by manganese. Therefore, S. mutans used the same aposuperoxide dismutase to form either FeSOD or MnSOD, depending upon which metal was available in the culture medium. Such "cambialistic" enzymes (those capable of making a cofactor substitution) may represent a previously unrecognized family of superoxide dismutases.
Modifications of the properties of human sex steroid-binding protein by nonesterified fatty acids.
The effect of unsaturated and saturated nonesterified fatty acids (NEFAs) on the electrophoretic, immunological, and steroid-binding properties of human sex hormone-binding protein (SBP) were investigated. Tests were carried out on whole serum from pregnant women and on purified SBP using polyacrylamide gel electrophoresis, crossed immunoelectrophoresis with autoradiography, and equilibrium dialysis. All three methods showed that NEFAs influence the binding of sex steroids to SBP both in whole serum and with the purified protein. Saturated NEFAs caused a 1.5-2-fold increase in binding of dehydrotestosterone, testosterone, and estradiol to SBP, while unsaturated NEFAs, such as oleic (18:1) and docosahexaenoic (22:6) acids inhibited the binding of these steroids to SBP. Thus, unsaturated NEFAs in the concentration range 1-100 microM are more inhibitory for estradiol binding than for testosterone or dehydrotestosterone binding. In addition to these binding changes, polyacrylamide gel electrophoresis and immunoelectrophoretic studies revealed a shift in SBP from the slow-moving active native form to a fast-moving inactive one. There was also a reduction in the apparent SBP concentration by Laurell immunoelectrophoresis in the presence of unsaturated NEFA (5.5 nmol of NEFA/pmol of protein). These studies indicate that unsaturated NEFAs induce conformational changes in human SBP which are reflected in its electrophoretic, immunological, and steroid-binding properties. They suggest that the fatty acid content of the SBP environment may result in lower steroid hormone binding and thus increased free hormone levels.