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Biomedical subjects

E Hardy

Publications and source records attributed to E Hardy.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1996

HIV replication in CD4+ T cells of HIV-infected individuals is regulated by a balance between the viral suppressive effects of endogenous beta-chemokines and the viral inductive effects of other endogenous cytokines.

This study demonstrates that the beta-chemokines macrophage inflammatory proteins 1 alpha and 1 beta (MIP-1 alpha and MIP-1 beta) and, RANTES (regulated on activation, normally T-cell expressed and secreted) inhibit human immunodeficiency virus (HIV) replication in anti-CD3 or recall antigen-stimulated peripheral blood mononuclear cells (PBMCs) of asymptomatic HIV-infected subjects. Significant levels of beta-chemokines were produced by both CD4+ and CD8+ PBMC subsets from HIV-infected individuals. Neutralization of endogenous MIP-1 alpha, MIP-1 beta, and RANTES did not rescue HIV replication in cultures to which greater than 10% CD8+ T cells had been added, indicating that the HIV suppressor activity of CD8+ T cells cannot be explained entirely by the beta-chemokines. However, significant enhancement of viral replication was observed upon neutralization of endogenous beta-chemokines in CD8-depleted or CD4+ PBMCs from most donors, particularly in cultures with low inducible levels of HIV production. In contrast, certain endogenous proinflammatory cytokines induced HIV replication in these same cells. These data suggest that the levels of HIV replication in CD4+ PBMC reflect the balance of the opposing effects of endogenous suppressive factors, such as the beta-chemokines, and HIV-inducing cytokines, such as tumor necrosis factor alpha and interleukin 1 beta.

CD4-Positive T-Lymphocytes↗
PubMed1996

Expression and folding of an interleukin-2-proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C-peptide and the N-terminal fused sequence.

We report the expression in E. coli of a proinsulin fusion protein carrying a modified interleukin-2 N-terminal peptide linked to the N-terminus of proinsulin by a lysine residue. The key aspects investigated were: (a) the expression of the fused IL2-PI gene, (b) the folding efficiency of the insulin precursor when still carrying the N-fused peptide and (c) the selectivity of the enzymatic cleavage reaction with trypsin in order to remove simultaneously the C-peptide and the N-terminal extension. It was found that this construction expresses the chimeric proinsulin at high level (20%) as inclusion bodies; the fused protein was refolded at 100-200 micrograms/ml to yield about 80% of correctly folded proinsulin and then it was converted into insulin by prolonged reaction (5 h) with trypsin and carboxypeptidase B at a low enzyme/substrate rate (1:600). This approach is based on a single enzymatic reaction for the removal of both the N-terminal fused peptide and the C-peptide and avoids the use of toxic cyanogen bromide.

Amino Acid Sequence↗
PubMed1996

Molecular analysis of the TP53 gene in Barrett's adenocarcinoma.

The TP53 gene is the most frequently mutated gene in human cancers. Barrett's esophagus provides an excellent model by which to understand the genetic events that lead from dysplasia to cancer. We screened for mutations in the TP53 gene by a combination of denaturing gradient gel electrophoresis and DNA sequencing in ten cases of adenocarcinoma arising in Barrett's mucosa. We have identified missense mutations in five of the ten samples, three transitions (R282W, G245S, R248W) and two transversions (E286Q and C176F). In one case we have analyzed biopsy specimens taken from the same site, one year before the patient developed an intra mucosal carcinoma. The mutation that was identified in this high grade dysplastic area was identical to that detected in the cancer. This would suggest TP53 mutations occur as an early genetic event in the development of Barrett's adenocarcinoma.

Adenocarcinoma↗
PubMed1996

Zinc-imidazole positive: a new method for DNA detection after electrophoresis on agarose gels not interfering with DNA biological integrity.

Nucleic acids separated by gel electrophoresis are commonly detected within the gel matrix with ethidium bromide staining, followed by gel irradiation with ultraviolet (UV) light. When the separated nucleic acids are to be recovered for further characterization or use, this methodology is unsuitable (i) because a significant number of chemical lesions to the nucleic acid molecules are caused, heavily compromising their biological activity, and (ii) because of health hazards due to accumulative direct contact with ethidium bromide and exposure to UV-light. As an alternative, for preparative purposes, a new nontoxic detection method employing zinc and imidazole salts is described. After electrophoresis, the gel is first washed in distilled water to substantially remove remaining electrophoresis reagents, then incubated in 40 mM zinc sulfate for 10 min to allow binding of Zn2+ to the DNA, and subsequently washed with distilled water to remove unbound Zn2+ from gel regions devoid of DNA. On soaking in 0.2 M imidazole for a few minutes, zinc-DNA complexes are visualized as deep-white (positive) stained bands against a slightly opaque background. The sensitivity is similar to that of ethidium bromide. Gels can be kept in distilled water for months without loss of staining. After zinc chelation, e.g. with EDTA, it is feasible to quantitatively recover chemically intact and biologically active DNA from the gels, as shown by reelectrophoresis and transformation experiments.

DNA↗
PubMed1996

Negative staining with zinc-imidazole of gel electrophoresis-separated nucleic acids.

Zinc and imidazole salts were applied for the detection of nucleic acids on either polyacrylamide of agarose gels. After electrophoresis, polyacrylamide gels are washed in distilled water to remove most of the residual electrophoresis reagents, then incubated in 10 mM zinc sulfate for 10 min, and subsequently immersed in 0.2 M imidazole for 3 min. As a result, zinc salts precipitate on the gel surface, except in the positions occupied by nucleic acids, which appear as transparent, colorless bands. Staining of nucleic acids on agarose gels can be performed by incubation in 40 mM zinc sulfate for 10 min, followed by immersion in 0.2 M imidazole for 5 min to form a deep white-stained background. On soaking in 2 M imidazole for 45 min, the imidazole-induced zinc precipitate is removed from the positions were nucleic acids are located resulting in a negative image of colorless and transparent nucleic acid bands against a white background. The sensitivity of this stain ranges from 5 to 7 ng/band for small (from 1 to 0.2 kbp) DNA, from 7.8 to 13 ng/band for different 22-base oligonucleotides, from 62 to 125 ng/band for large (from 20 to 2 kbp) DNA, and is 1 microgram/band for human peripheral-blood monocyte RNA. After chelation of zinc with EDTA, the nucleic acids can be quantitatively recovered from the gel. The principal advantage of this technique over ethidium bromide staining is evident for preparative purposes. Using zinc-imidazole in the detection of purified pBACIB.1 (2.8 kbp) plasmid DNA and anti-HBsAg single chain Fv antibody fragment (0.7 kbp) DNA, followed by elution from gel slices, ligation and transformation of competent E. coli XL-1 Blue cells, the number of transformants notably increased from 280 (obtained with conventional ethidium bromide staining plus UV-irradiation at 312 nm) to 10,000.

Antibodies, Viral↗
PubMed1996

A procedure for protein elution from reverse-stained polyarcylamide gels applicable at the low picomole level: An alternative route to the preparation of low abundance proteins for microanalysis.

We developed a technique that allows rapid protein elution from polyacrylamide gel bands at room temperature into a detergent-free buffer (elution time 2 x 10 min, total working time about 30 min) with high yields (90-98%) even at a low picomole level (1 picomole per band). Its efficacy relies on the combination of protein detection by reverse staining with the enhancement of protein diffusion after gel crushing. Detection is accomplished by gel incubation in an imidazole solution, followed by incubation in a zinc salt solution to develop a negative stain pattern. Proteins are eluted by zinc complexation in Laemmli electrophoresis buffer (Tris + glycine), from which sodium dodecyl sulfate is omitted to allow direct subsequent microanalysis, e.g. high performance liquid chromatography (HPLC) and automatic sequencing. A variety of proteins were eluted efficiently (with no apparent restriction due to their intrinsic properties) as quantified with radioiodinated total E. coli proteins. Yields were independent of acrylamide concentration, protein molecular mass (from 10 to 100 kDa) and the amount (from 1 to 100 picomole) of protein in the band. This protocol was derived from a quantitative evaluation of the effect of protein staining and of sample reduction prior to electrophoresis on elution yields. For N-terminal sequencing, the protein eluate was automatically loaded on a polyvinylidene difluoride (PVDF) membrane with conventional HPLC equipment; both loading and membrane clean-up were monitored at 206 nm. By simultaneously processing several analytical bands, the procedure allowed trace enrichment of a natural scarce protein that was N-terminal sequenced.

Bacterial Proteins↗