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Biomedical subjects

E Hardy

Publications and source records attributed to E Hardy.

At least 19 recordsLinked to original sources
PubMed1999

Structural characterization, stability and fatty acid-binding properties of two French genetic variants of human serum albumin.

Four bisalbuminemic, unrelated persons were found in Bretagne, France, and their variant and normal albumins were isolated by DEAE ion exchange chromatography, reduced, carboxymethylated and treated with CNBr. Comparative two-dimensional electrophoresis of the CNBr digests showed that three of the variants were modified in fragment CB4, whereas the fourth had an abnormal fragment CB1. These fragments were isolated, digested with trypsin and mapped by reverse-phase HPLC. Sequencing of altered tryptic peptides showed that the three variants modified in CB4 were caused by the same, previously unreported, amino acid substitution: Asp314-->Val (albumin Brest). The fourth, however, was a proalbumin variant with the change Arg-2-->Cys (albumin Ildut). Both amino acid substitutions can be explained by point mutations in the structural gene: GAT-->GTT (albumin Brest) and CGT-->TGT (albumin Ildut). The proalbumin Ildut is very unstable and already in vivo it is to a large extent cleaved posttranslationally to Arg-Albumin and normal albumin. Furthermore, we observed that during a lengthy isolation procedure the remaining proalbumin was changed to Arg-Albumin or proalbumin lacking Arg-6. In addition, part of normal albumin had lost Asp1. Gas chromatographic investigations using isolated proteins indicated that albumin Brest has improved in vivo fatty acid-binding properties, whereas the structural modification(s) of albumin Ildut does not affect fatty acid binding.

Fatty Acids↗
PubMed1999

Mice immunization with gel electrophoresis-micropurified bacterial lipopolysaccharides.

Some evidence on the possible use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to elicit antibodies against smooth- or rough-type bacterial lipopolysaccharides (LPS) is shown. Gel-separated LPS were negatively stained with zinc-imidazole to precisely localize the bands of interest under fully reversible conditions. Then the bands of interest were excised and the resulting gel slices washed in a solution of a zinc-complexing agent (e.g., 100 mM EDTA), after which they were extruded through a metal sieve of 32 microm average size contained in a 1 mL syringe, to generate homogeneous gel microparticles. The LPS-containing gel slurries were used directly to immunize female BALB/c mice. Using this procedure, positive mouse polyclonal antibody responses against gel-purified smooth- or rough-LPS forms from Escherichia coli K-235 or Bordetella pertussis were elicited, as tested by a dot-immunoblotting assay. Our results may encourage the use of SDS-PAGE-micropurified LPS to develop optimized immunization procedures for the generation of specific antibodies against LPS bands of defined sizes, and therefore they constitute an intermediate step toward that aim.

Animals↗
PubMed1998

Elution of lipopolysaccharides from polyacrylamide gels.

A simple procedure for elution in water of bacterial lipopolysaccharides (LPS) from sodium dodecyl sulfate-polyacrylamide gels is described. It consists of the combination of three principal steps: first, highly sensitive on-gel LPS detection (1-10 ng/band) with zinc-imidazole (negative or reverse staining); second, washing of the individual LPS band in a solution of a zinc-complexing agent (e.g., 100 mM EDTA); and finally, elution of the LPS (100-200 microliters water for a 0.5-microgram LPS band) from gel microparticles for 3 h at room temperature. Using this procedure, we have successfully eluted a variety of LPS forms from Bordetella pertussis, Escherichia coli 0111:B4, E. coli K-235, Salmonella enteritidis, and Pseudomonas aeruginosa. Elution recovery of rough or semismooth LPS was about 70-80%, while that of smooth LPS was only about 10%. Eluted LPS was biologically active as tested by limulus amebocyte lysate and TNF-alpha assays.

Acrylic Resins↗
PubMed1998

Understanding the mechanism of the zinc-ion stains of biomacromolecules in electrophoresis gels: generalization of the reverse-staining technique.

We have recently shown that a few nanograms of protein separated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels can be detected by reverse-staining, exploiting the precipitation reaction between zinc(II) and imidazole. Modifications of this method have also been generated to detect gel-isolated nucleic acids and bacterial glycolipids. Because there is no recourse to chemical modifiers, the reverse-staining technique has been valuable when micropreparing these biomacromolecules for later use or characterization. The mechanism underlying the reverse-staining effect, however, remains incompletely understood and this has prevented a further generalization of the technique. Here, we have conducted physicochemical experiments and identified zinc imidazolate (ZnIm2) as the main component of the precipitate that forms along the surface of zinc-imidazole reverse-stained gels. Many staining effects observed when gels containing electrophoretically separated biopolymers are subjected to zinc-imidazole stains have been rationalized. The reverse-staining method has been vastly generalized, now allowing the detection of proteins and glycolipids as well as complexes of these macromolecules in native gels. We demonstrate the application of the reverse-staining technique in situations where Coomassie blue or silver staining was inappropriate or failed to produce detection of the species of interest. The present generalization of the reverse-staining method facilitated the characterization of biomacromolecular interaction partners in mixtures of bacterial glycolipids and human tears.

Bacteria↗
PubMed1998

The risk of inadvertent intrauterine device insertion in women carriers of endocervical Chlamydia trachomatis.

The most important complication attributed to the use of intrauterine device (IUD) is pelvic inflammatory disease (PID), often associated with Neisseria or Chlamydia infection. Consequently, the IUD should not be inserted in women at risk of infection or with symptoms of endocervicitis. To evaluate the effectiveness of such a policy, a systematic investigation of Chlamydia and Neisseria was carried out among 407 contraceptive acceptors. Twenty-seven cases were positive for Chlamydia and none were positive for Neisseria. There were no statistical differences in the proportion of women with and without Chlamydia who had vulvovaginal or cervical signs or symptoms, although twice as many women had pain at pelvic exam in the Chlamydia-infected group. In 29 women in whom infection was clinically suspected, two were found to be infected with Chlamydia. IUD were not inserted in women suspected of having infection, but 19 of 327 IUD acceptors were subsequently found to have a positive Chlamydia test result. Two women returned with symptoms of PID and 17 were asymptomatic, but all were treated. PID was not suspected in any other subject. These results reinforce the need for careful selection of IUD acceptors and for thorough counseling for symptoms of PID and the need for immediate consultation.

Adult↗
PubMed1998

Women's preferences for vaginal antimicrobial contraceptives. II. Preferred characteristics according to women's age and socioeconomic status.

A study was carried out to identify characteristics that women would want for an idealized vaginal contraceptive, and the possible association of these characteristics with age and socioeconomic status. The study was done in Campinas, São Paulo State, Brazil. A total of 635 women were selected by age and socioeconomic status, using the "social network" technique. Almost half were adolescents (15-19 years old) and the rest were adults (20-45 years old). Half were of low socioeconomic status and the rest of medium-high status. The data were analyzed with SPSS-PC and EPI-INFO 6.0. Logistic regression and chi 2 were used for the analysis. Despite some differences found between age and socioeconomic status in regard to the characteristics desired for the idealized method, most of the participants expressed the same preferences. The results indicate that women would like the idealized method to be a cream, rather than a suppository, with no odor or flavor, to be colorless, to be placed in the vagina with an applicator well before coitus, and to offer protection against sexually transmitted diseases including AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Women's preferences for vaginal antimicrobial contraceptives. III. Choice of a formulation, applicator, and packaging.

Novel vaginal formulations are under development to combat the increasing incidence of sexually transmitted diseases, including AIDS, and also unplanned pregnancies. A study was performed to determine women's preferences for different dosage forms (gel, cream, ovule/suppository, film, foam, tablet), width, length, and color of an applicator, and various types of packages. The study was conducted in Campinas, Brazil. A total of 635 women were interviewed, including both adolescents and adults and low and middle-high socioeconomic groups. The large majority of the women preferred a gel over a cream; both were preferred over the other methods. When asked which method they would not use, the film was most frequently identified, followed by the tablet and ovule. The primary reasons for selecting a particular dosage form were ease of use, absence of odor or the presence of a pleasant one, absence of color, and insertion with an applicator. The major reasons for not using a method were discomfort, "plastic" appearance, distrust of effectiveness, difficulty with insertion, messiness, and rigidity/hardness. The majority of the women liked the applicator shown. The prefilled single dose applicator was by far the preferred packaging. This information should aid in the development of consumer-friendly, vaginal formulations.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Women's preferences for vaginal antimicrobial contraceptives. I. Methodology.

Sexually transmitted diseases, including AIDS, and unplanned pregnancies continue to be a serious worldwide problem. A number of organizations are developing woman-controlled vaginal formulations to prevent these problems. However, little information is available regarding the types of products women prefer even though such knowledge is essential to obtain widespread use. This is the first of several articles that describe the results of a consumer preference study for such vaginal formulations performed in Campinas, São Paulo State, Brazil. Because no published methodology was available, the instruments and interview techniques were developed first and procedures established for the identification and participation of research subjects. After preparation of a questionnaire, a pilot study was performed to evaluate it, to establish the interview technique, and to determine the optimal method for subject recruitment. Based on the results, the approach was selected and applied to 635 subjects from different age and socioeconomic groups. The developed methodology and questionnaire, the advantages and the problems encountered, are presented.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Women's preferences for vaginal antimicrobial contraceptives. IV. Attributes of a formulation that would protect from STD/AIDS.

Vaginal formulations may have "dual" protective activity, against sexually transmitted diseases/AIDS and unplanned pregnancy. The attributes that women find acceptable or unacceptable for such dual protective methods were investigated. More than 50% of the women would not accept messiness, but it was more accepted for dual protective methods than for contraceptives. Very few women would use a dual protective method if it caused vaginal irritation, itching, swelling, or burning, problems associated with presently marketed methods. More than half of the women would use it if it appeared on the penis of their partner or required refrigeration. Use of an applicator to insert the formulation was generally preferred over a manual method. Most women preferred the formulation to be colorless or white, about 16% liked light colors, and about 10% liked darker colors. Almost half of the women were willing to pay up to $5.00 per application of a dual protective formulation, about 15% $3.00, and 30% $1.00. Dual protective methods seem highly acceptable and women would pay much more for them than for condoms. However, these methods should be free of problems usually associated with presently marketed formulations.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Contraceptive use and pregnancy before and after introducing lactational amenorrhea (LAM) in a postpartum program.

There is good evidence that lactational amenorrhea (LAM) is an effective method of fertility regulation during the first 6 months postpartum, provided no other food is given to the baby and the mother remains amenorrheic. However, although breast-feeding is strongly promoted in many maternity hospitals that also run postpartum family planning programs, LAM is rarely included among the contraceptive options being offered. This paper presents the results of an operational study which compared the prevalence of contraceptive use and the cumulative pregnancy rate at 12-months postpartum among 350 women observed before and 348 women studied after introducing LAM as an alternative contraceptive option offered to women following delivery at the Instituto Materno Infantil de Pernambuco (IMIP), in Recife, Brazil. The percentage of women not using any contraceptive method was significantly lower (p<0.0001) after the intervention (7.4%) than before (17.7%). This difference remained statistically significant after controlling for age, number of living children, marital status and years of schooling. The proportion pregnant one year postpartum was also significantly lower (p<0.0001) after the introduction of LAM (7.4%) than before (14.3%), but the difference was no longer significant after controlling for the same variables. It is concluded that LAM is a useful addition to family planning postpartum programs.

Adolescent↗
PubMed1998

Extramedullary relapse after favorable molecular response to donor leukocyte infusions for recurring acute leukemia.

Donor leukocyte infusions (DLI) have turned out to be an efficient way to re-establish complete remission (CR) in chronic myeloid leukemia (CML) patients relapsing after allogeneic bone marrow transplantation (BMT). In these patients, absence of PCR bcr-abl fusion transcripts confirmed the potency of donor leukocytes to induce molecular response in relapsed CML. This ensured sustained remission and long-term survival. In this study, the capacity of DLI to induce molecular remission in acute leukemia relapse after BMT was analyzed. The results showed that following DLI, leukemic cell eradication gradually occurred over a prolonged time period. The time to complete disappearance of the molecular marker of the disease was 30 weeks in RT-PCR analysis. A sustained and persistent elimination of an AML1/ETO-positive leukemic clone in an AML-M2 patient was observed. In contrast, an AML-M5 with t(11;19) and an E2A/PBX1-positive ALL achieving cytogenetic and molecular bone marrow CR developed following DLI unusual sites of extramedullary leukemia relapse, despite continued bone marrow remission. This study adds further proof of the benefit of donor cell therapy in acute leukemia but shows that complete leukemic cell eradication appears to require a critical interval in order to establish effective immune responses at all sites where leukemic cells persist.

Adult↗
PubMed1998

Evaluation of a new Near Patient Test for the detection of Helicobacter pylori.

OBJECTIVE: Peptic ulcer disease is rare in the absence of Helicobacter pylori. Testing for H. pylori has recently become easier with the development of Near Patient Tests. However, the validity of many of these tests is unknown. The aim of this study was to assess the validity of the QuickVue one-step Near Patient Test for H. pylori in a group of patients attending for endoscopy at a tertiary referral hospital. METHODS: In a retrospective study the QuickVue one-step serum test (Quidel) was performed on the stored serum from 193 patients whose H. pylori status had been determined by a 'gold standard' using urease testing, histology, culture and 14C-urea breath testing. Twenty four other patients presenting to endoscopy were studied prospectively. For these patients QuickVue was performed on capillary and venous samples and the results were compared to a similar 'gold standard'. Enzyme immunoassay (Premier, H. pylori, Meridian Diagnostics, OH, USA) was used to define H. pylori status of patients with indeterminate H. pylori status by the above criteria. RESULTS: In the retrospective study the QuickVue one-step H. pylori test had a sensitivity (CI) of 82% (75-89) and specificity of 83% (74-92). In the prospective study on capillary blood its sensitivity (CI) was 89% (52-100) and its specificity 93% (68-100); and on venous blood, 78% (40-97) and 93% (68-100). Premier has previously been shown to have a sensitivity and specificity of 100%. CONCLUSION: In this group of patients the QuickVue one-step H. pylori Near Patient Test was less accurate than the Premier ELISA test, but its ease of use and availability in primary care make it useful for patient screening.

Adult↗
PubMed1997

Sensitive reverse staining of bacterial lipopolysaccharides on polyacrylamide gels by using zinc and imidazole salts.

We present a new method for visualizing bacterial lipopolysaccharides (LPS)/lipooligosaccharides (LOS) electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis, gels are washed in boiling water to appreciably remove remaining electrophoresis reagents, then incubated in 10 mM zinc sulfate for 15 min, and subsequently immersed in 0.2 M imidazole for 3 min. As a result, zinc salts precipitate all over the gel surface except in the zones occupied by LPS/LOS, which appear as transparent, colorless bands. Gels can be stored in distilled water for weeks without loss of the negative image. The sensitivity of this stain is similar to that of silver. We believe that zinc-imidazole may be a suitable nontoxic alternative to silver in the rapid analysis of LPS/LOS by polyacrylamide gel electrophoresis.

Coloring Agents↗
PubMed1997

Illegal abortion: consequences for women's health and the health care system.

Illegal abortion is responsible for up to half of maternal deaths and consumes a large proportion of health resources in many developing countries, particularly in Africa and Latin America. The legal situation of abortion in a country does not influence the abortion rate, but illegality is associated with a much greater risk of complications and death. To make abortion legal is not enough. Access to safe abortion strongly depends on the capacity and willingness of physicians and the health system to provide safe services, which sometimes are made available in spite of restrictive laws. The abortion rate will drop and the safety of the procedure will improve, parallel to the position women occupy in a given society, and to the level of recognition of their sexual and reproductive rights. The medical profession, and FIGO in particular, has a great role to play in implementing initiatives that will reduce the consequences of illegal abortion for women and society.

Abortion, Induced↗
PubMed1997

High yield elution of proteins from sodium dodecyl sulfate-polyacrylamide gels at the low-picomole level. Application to N-terminal sequencing of a scarce protein and to in-solution biological activity analysis of on-gel renatured proteins.

A simple, reliable procedure for practically quantitative (90-98%) and fast (< 30 min) elution of proteins from SDS-PA gels is described with reproducible recoveries in the range from 100 to 1 pmol per band, which does not require the inclusion of detergents in the elution buffer. It consists in the combination of (1) highly sensitive on-gel protein detection (50 mol per band) with imidazole-SDS-zinc (reverse staining), (2) crushing of the protein band to produce 32-micron gel particles, and (3) vortexing of the slurry in a solution of a zinc-complexing agent, e.g. glycine 0.5 M or EDTA 100 mM (100 microliters for a 100-pmol BSA band), at room temperature. Eluted proteins can be directly analyzed by RP-HPLC, quantitatively loaded onto a PVDF membrane, or, provided that they are previously renatured on-gel, analyzed by biological activity tests. The application of the procedure to in-solution enrichment of scarce proteins for N-terminal analysis is shown.

Acrylic Resins↗
PubMed1997

Effect of IL-1 beta on responses of cultured human airway smooth muscle cells to bronchodilator agonists.

Decreased beta-adrenergic responsiveness is a characteristic feature of asthma. In order to determine whether cytokines released in the asthmatic airway contribute to this phenomenon, we measured changes in stiffness of cultured human airway smooth muscle (HASM) cells induced by isoproterenol (ISO) in control HASM cells and HASM cells pretreated with IL-1 beta (20 ng/ml for approximately 42 h). Stiffness was measured by magnetic twisting cytometry. HASM cells were obtained from normal tracheal tissue obtained at lung transplant, and studied in passages 4-7. In control cells, ISO caused a dose-related decrease in cell stiffness. IL-1 beta had no effect on baseline cell stiffness. However, IL-1 beta caused a rightward shift in the concentration-response curve to ISO and decreased the maximal effectiveness of this agonist. Decreased responses to ISO were also obtained with 2 ng/ml IL-1 beta, or when cells were pretreated with IL-1 beta (20 ng/ml) for 22 h. This effect of IL-1 beta was not altered by pretreatment of the cells with pertussis toxin (100 ng/ml throughout the IL-1 beta exposure period). IL-1 beta also significantly attenuated the ability of prostaglandin E2 (PGE2) to decrease cell stiffness. In contrast, IL-1 beta had no effect on cell stiffness responses to dibutryl cAMP, a cell permeant analog of cAMP suggesting that the cytokine does not influence either the ability of cAMP to activate kinases, or the targets of these kinases which ultimately mediate cell relaxation. IL-1 beta (20 ng/ml for 40 h) caused a small (30%) but significant (P < 0.02) increase in basal cAMP, but also resulted in a 2-3-fold decrease in the changes in cAMP formation induced by either ISO or PGE2. In contrast, IL-1 beta had no effect on cAMP formation in response to forskolin, suggesting that IL-1 beta does not mediate its effects via changes in the expression or activity of adenylyl cyclase. Pretreatment with IL-1 beta had no significant effect on beta 2 adrenoceptor number assessed by [125I]-CYP binding in these cells, nor was there any significant effect of IL-1 beta on Gsa expression assessed by Western blot. In summary, our results indicate that IL-1 beta causes a concentration- and time-dependent decrease in responses of HASM cells to ISO and are consistent with the hypothesis that the effects of IL-1 beta are mediated by uncoupling of beta-receptors from Gs-induced activation of adenylyl cyclase.

Adenylyl Cyclases↗